Molecular absorption analysis confirmed the electronic excitation in the open isomers compared to closed isomers. The results show that reversible thermochromic compounds act as excellent NLO molecular switches and can be used to design advanced electronics.Switchable nonlinear optical (NLO) materials have widespread applications in electronics and optoelectronics. Thermo-switches generate many times higher NLO responses as compared to photo-switches. Herein, we have investigated the geometric, electronic, and nonlinear optical properties of spiropyranes thermochromes via DFT methods. The stabilities of close and open isomers of selected spiropyranes are investigated through relative energies. Electronic properties are studied through frontier molecular orbitals (FMOs) analysis. The lower HOMO-LUMO energy gap and lower excitation energy are observed for open isomers of spiropyranes, which imparts the large first hyperpolarizability value. The delocalization of π-electrons, asymmetric distribution and elongated conjugation system are dominant factors for high hyperpolarizability values of open isomers. For deep understanding, we also analyzed the frequency-dependent hyperpolarizability and refractive index of considered thermochromes. The NLO response increased significantly with increasing frequency. Among all those compounds, the highest refractive index value is observed for the open isomer of the spiropyran  Since the 1970s, the designing and synthesis of thermochromic materials have been an important interdisciplinary research field because of their excellent efficiency in changing their characteristics concerning temperature . SeveralThermochromes are classified as reversible or irreversible . Irrever2) has a pronounced effect on the first hyperpolarizability compared to the electron donating group (NH2) [N-(4-hydroxy)-salicylidene-amino-4-(methylbenzoate) and N--4-aminopyridine. Upon irradiation with UV light using TD-DFT analysis, they change color and possess better NLO response than standard urea [The literature revealed the generation of optical and NLO properties of compounds if electronic excitations occur ,17,18,19up (NH2) . Previtaup (NH2) . Sliwa eard urea . Photochard urea ,24.Le-Bozec and co-workers thoroughly studied the photochromic transformation of 4,4′-bis(ethenyl)-2,2′-bipyridine ligands functionalized by a dimethylaminophenyl–dithienylethene group and the corresponding zinc (II) complex. The compound efficiently undergoes reversible interconversion into open and closed forms, where the closed form shows outstanding NLO response due to π-conjugation (which is absent in open form) . They alAll calculations were performed by using Gaussian-09 software . The selαo) and nonlinear optical properties (static first hyperpolarizability (βo), frequency-dependent first, second hyperpolarizability and reflective index were calculated at the above-mentioned level of theory.NBO charge analysis is also performed to know the charge transfer in molecules . Frontieαo) and static first hyperpolarizability (βo) are given below: Mathematical expressions for polarizability (14 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au). Frequency-dependent hyperpolarizability contains the approximation of electro-optic Pockel’s effect β and second harmonic generation β. For estimation of frequency-dependent second hyperpolarizability (γ), dc-Kerr effect (ω) = γ and electric field induced second harmonic generation (ω) = γ and degenerate four-wave mixing are calculated. These values were also used to estimate the refractive index of the respective compounds.The frequency-dependent nonlinear optical analysis was investigated at 5.63 × 101 show a clear change in bond distance between the ring closing atoms (C-O). In the close isomer of spiropyran 1, the ring-closing carbon and oxygen atoms (C-O) bond length is 1.43 Å. This bond distance increases as the close structure is converted to an open form. The C-O bond length of ring closing atoms is increased to 4.11 Å, which confirms the breakage of the C-O bond. Similar behavior is observed for spiropyran 2, where the bond length between ring closing C and O atoms is increased from closed isomer to open isomer. The optimized structure of the close isomer of spiropyran 2 shows terminal C-O atoms bond length of 1.36 Å. In contrast, this terminal C-O atoms bond length in an open isomer increases to 4.30 Å. These results are comparable to the X-rays data of the C-O bond length of spyropyran reported by Ozhogin et al. [The geometric stability of close and open isomers of spiropyranes is estimated from their geometric properties. The optimized geometries are given in n et al. .1 & 2) and are given in 1, the close isomer is thermodynamically more stable than the open isomer of the spiropyran. The open isomer is 5.78 kcal mol−1 less stable than the closed isomer. Menzonatto and Lopes studied the mechanistic pathway of spiropyrane close-form conversion to open-form, and they observed more stability for close compared to open-form [2, the open isomer is thermodynamically more stable  than the close isomer of spiropyran 2  are carried out to estimate the acceptor and donor sites of the closing terminal carbon and oxygen (C-O) atoms, and the results are given in 1, the NBO charge value obtained for the carbon atom is 0.64 |e| and for the oxygen atom is −0.63 |e|. In spiropyran 2, the charge on terminal carbon is −0.05 |e| and −0.65 |e| is observed on the oxygen atom in open form. The opposite charges on terminal carbon and oxygen atoms in open isomers of spiropyranes 1 & 2 depict the possibility of electrostatic interactions. In a close isomer of 1, the charge on the ring closing oxygen atom is −0.59 |e| and on the carbon atom is 0.75 |e|, respectively. In the close isomer of spiropyran 2, ring-closing oxygen and carbon atoms have −0.57 |e| and 0.37 |e| charges, respectively. Variations in charges of terminal (ring closing) oxygen and carbon atoms indicate the electrostatic interaction that results in bond formation between terminal carbon and oxygen atoms in individual spiropyranes (1 & 2). These opposite charges on terminal carbon and oxygen atoms in close isomers represent the polarity of the bonds. Loopes and coworkers also theoretically designed spiropyranes derivatives, and their NBO data is similar to our results [The NBO charges of close and open isomers of spiropyranes ( results . 1 & 2). The open forms of both spiropyranes (1 & 2) have a higher dipole moment than the close form. In spiropyran 1, the close isomer has a lower dipole moment of 6.49 D than the open isomer (8.78 D). In the case of spiropyran 2, the open isomer has a higher dipole moment of 10.23 D than 6.26 D of close form. The variation in dipole moments is due to the larger internuclear distance between oppositely charged carbon and oxygen, along with large quantities of charges on these terminal atoms (C and O), as discussed vide supra. The difference in charges of spiro-carbon and oxygen is more for open isomers than closed isomers, and distances are also more for open isomers. As a result (open isomers of spiropyranes 1 & 2) dipole moment is also higher. Dipole moment values for spiropyranes are reported in 1 & 2) illustrates that these could be good candidates for generating high NLO response.The separation of charges in a system is defined as the dipole moment. More separation of charges results in a higher dipole moment. The NBO results indicate a similar trend of increasing dipole moment for both spiropyran isomers (1 & 2). The energies of HOMOs (EH), the energies of LUMOs (EL) and the HOMO-LUMO gap (EH-L) of spiropyranes (1 & 2) are given in H-L of a close isomer of spiropyran 1 is 8.36 eV which is noticeably higher than the open isomer (6.34 eV). Similarly, the close isomer of spiropyran 2 has an EH-L of 7.51 eV, more than the open isomer of spiropyran 2 (4.26 eV). The reduction of EH-L in open isomers spiropyras (1 & 2) illustrates the shifting of electronic density in these conjugated systems, which is related to the conductivity. The lower EH-L of a system has more charge transfer, generating conductive properties. The close isomers of both spiropyranes 1 & 2 indicate insulator properties due to large EH-L converted to semi-conductors in open isomer. The isodensities of HOMOs, LUMOs and their energy gaps (EH-L) of close and open isomers of spiropyranes are shown in H-L values was observed experimentally by Kudernac et al. during their work on nano-electronic switches [After calculating the geometric parameters, FMOs analysis is executed to observe the electronic stability and reactivity of open and closed isomers of spiropyranes  and the first hyperpolarizability (βo) of close and open isomers of spiropyranes 1 & 2 are given in Linear and nonlinear optical parameters are calculated after geometric and electronic analyses. Polarizability  results of both isomers show a prominent increase in hyperpolarizability value from close to open isomers of both 1 and 2. The βo of close and open isomers of the spiropyrane 1 is 231.89 au and 2381.11 au, respectively. Bond length alternation and asymmetric electronic density distribution affect βo of an open isomer of spiropyran 1 absent in a closed isomer. The βo value for the close isomer of spiropyran 2 is 1424.51 au and the open isomer has βo value is 3594.49 au. The hyperpolarizability response of the open isomer of spiropyran 2 is larger due to extended conjugation , which is absent in the close isomer. The polarizability value of close and open isomers of spiropyran 1 & 2 are also observed in acetonitrile solvent, and their results are given in 1 & 2, we performed NLO calculations in this solvent. As the previous reports illustrate that reversible spiropyranes show high thermal stability in acetonitrile we select this solvent in our study [βo of close and open isomers of the spiropyran 1 are 1242.20 au and 4876.61 au, respectively. The βo values increase in the solvent phase.The NLO response of close and open isomers of spiropyranes ur study . The βo βo of close and open isomers of the spiropyran 2 are 2844.07 au and 3578.72 au, respectively. The βo values increase in the solvent phase for the close isomer, but for the open isomer, it is a little bit decreased. Similar to spiropyrane 1, the βo values of open isomers are higher in comparison to close isomers. The increasing trend of βo values from close to open form of both spiropyranes 1 & 2 is similar to gas phase calculations. Following is an increasing trend of βo values; open isomer-spiropyran 1 (4876.61 au) > open isomer-spiropyran 2 (3578.72 au) > close isomer-spiropyran 2 (2844.07 au) > close isomer-spiropyran 1 (1242.20 au). The increase in hyperpolarizability is increased in the dipole moment and polarizability of these isomers, as given in The 1 & 2. These parameters include excitation energy (ΔE), oscillating strength (ƒo) and excited dipole moment (Δµ), and their results are summarized in 1 & 2 is 6.40 eV and 4.62 eV, respectively. The ΔE value is lower for open isomers of both spiropyranes 1 (2.97 eV) & 2 (4.55 eV). The trend of decreasing ΔE values from close to open isomers is inconsistent with an increase in βo values. The βo values of open isomers 1 & 2 are higher, and their excitation energies are also lower. Moreover, the ƒo and Δµ values also show a similar increasing trend compared to βo values moving from close to open isomers of spiropyran 1 & 2. The ƒo (Δµ) values of open isomers of spiropyranes 1 and 2 are 0.68 (8.78 Debye) and 0.53 (10.22 Debye), respectively. The ƒo (Δµ) values of close isomers of spiropyranes 1 and 2 are 0.54 (6.49 Debye) and 0.41 (6.26 Debye), respectively. In addition, it is confirmed that the dipole moment values do not show any change between the ground and the excited states in the gas phase. The variational trend of βTLM values is comparable to the trend of increasing βo values. So, ΔE, ƒo and Δµ are the decisive factors for the enhancement of NLO response in these isomers.Two level model is implemented to investigate the internal parameters responsible for the increase in the first hyperpolarizability value of closed and open isomers of spiropyranes 1 & 2, and their results are described in the vide infra). The ΔE and Δµ of spiropyranes 1 & 2 in the solvent phase are similar to gas phase results. The ΔE value (5.03 eV) of the open isomer is lower, and Δµ value (11.74) is higher. The βo value (2381.11 au) of this isomer is also higher. The ΔE value (5.26 eV) of the close isomer is lower, and Δµ value (11.74 Debye) is higher and βo value of this isomer is also low (231.89 au). The trend of increasing ƒo is opposite to the βo value for closed and open isomers. Similar results are obtained for spiropyran 2 as obtained for spiropyran 1. The ΔE value (4.60 eV) of the open isomer is lower, and ƒo (0.89) & Δµ value (13.91 Debye) are higher, which is according to the two-level model. Alternatively, the ΔE value (4.57 eV) of the open isomer is lower, and ƒo (0.62) & Δµ value (8.36 Debye) are higher. Overall, excitation energy and variational dipole moment are decisive factors in describing the NLO response of these systems.The internal parameters are also observed in the presence of acetonitrile solvent for closed and open isomers of spiropyranes 1 & 2, and results are given in 14 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au) [β values for close and open isomers of spiropyran 1 are in the range of 255.9 to 6324.6 au at a wavelength of 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The close and open isomers of spiropyran 1 showed the most prominent second harmonic generation effect β values in the range of 317.9–6698.7 au at 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The higher electro-pockel effect value (6324.6 au) and second harmonic generation effect value (6698.7 au) are obtained for the open isomer of spiropyran 1 at 5.63 × 1014 Hz (0.09 au). These results are similar to the work of Tonnele and Castet [Frequency-dependent first hyperpolarizability is the fundamental parameter in the NLO phenomenon. Experimentalists required frequency-dependent first hyperpolarizability information for the development of optical devices. These properties give guidelines for the behavior of NLO materials when they are treated with light beams of different frequencies in practice. The second harmonic generation is one of the effects governed by hyperpolarizabilities . Here, t0.04 au) . The eled Castet .β values for close and open isomers of spiropyran 2 range from 1565.4 au to 22,805.2 au at a wavelength of 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au). Again, the close and open isomers of spiropyran 2 showed prominent second harmonic generation effect β values from 1893.9–54,124.8 au at 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The higher electro-optical pockel effect value  and second harmonic generation effect value  are obtained for the open isomer of spiropyran 2 at 5.63 × 1014 Hz (0.09 au). The electro-optical Pockel effect and second harmonic generation are highly affected at the wavelength of 5.63 × 1014 Hz (0.09 au) for both open and closed isomers of spiropyran 2. Among spiropyranes 1 & 2, the highest electro-optical pockel effect and second harmonic generation effect values are seen for the open isomer of spiropyran 2. These results are comparable to frequency-dependent work on other photoswitches [The electro pockel effect switches .1 & 2 are given in 14 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au) [γ) values for close and open isomers of spiropyran 1 at static frequency are 3564.7 au and 6907.9 au, respectively. The large second hyperpolarizability (6907.9 au) value is obtained for the open isomer of the spiropyran 1. The dc-Kerr effect values for close isomer of the spiropyran 1 are 5095.1 au and 3909.5 au at 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The EFSHG values for close isomers are 3037.8 au and 4636.6 au at frequencies of 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The dc-Kerr effect values for an open isomer of the spiropyran 1 are 52,842.8 au and 9030.3 au at a wavelength of 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The open isomer of spiropyran 1 also shows the prominent electric field-induced second harmonic generation effect values of 2398.2 au and 23,299.4 au at 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The high values of the dc-Kerr effect coefficient are observed for open isomers of the spiropyran 1  & 2  at 5.63 × 1014 Hz (0.09 au). The higher values of EFSHG coefficient are seen for open isomers of the spiropyran 1  & 2  at 2.82 × 1014 Hz. The highest frequency-dependent dc-Kerr effect coefficient value  is obtained for open isomers of the spiropyran 1 at 5.63 × 1014 Hz (0.09 au). These results are similar to the reported data for acid-controlled second-order nonlinear optical switches [1 & 2.The second-order electric susceptibility per unit volume is known as the second hyperpolarizability. Experimentalists required frequency-dependent second hyperpolarizability for practical applications where the hyperpolarizability is calculated at different wavelengths . The fre0.04 au) . It is oswitches . We obseγ) values for close and open isomers of spiropyran 2 are 11,533.7 au and 27,098.8 au, respectively. The large γo value is obtained for the open isomer of the spiropyran 2. The dc-Kerr effect values for close isomer of spiropyran 2 are 20,011.3 au and 13,033.1 au at 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The EFSHG values for close isomers are 1027.6 au and 1713.9 au. At 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), the dc-Kerr effect values for an open isomer of spiropyran 2 are 126,583.9 au and 120,760.7 au, respectively. The open isomer of spiropyran 2 also shows the noticeable electric field-induced second harmonic generation effect values, which are 112,874.0 au and 33,722.1 au at 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The higher dc-Kerr effect value  is detected for both isomers of spiropyran 2 at 5.63 × 1014 Hz (0.09 au). The high EFSHG effect value  is detected for both isomers of spiropyran 2 at 2.82 × 1014 Hz.Static second hyperpolarizability (n2) values obtained for a close isomer of the spiropyran 1 are 3.23 × 10−18 cm2/W and 1.01 × 10−18 cm2/W at 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The open isomer of the spiropyran 1 has refractive index values of 1.99 × 10−17 cm2/W and 3.25 × 10−18 cm2/W at a wavelength of 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. Among both isomers (close and open) of spiropyran 1, the open isomer has the large n2 (1.99 × 10−17 cm2/W) value at the wavelength of 5.63 × 1014 Hz (0.09 au). The refractive index values found for close isomer of spiropyran 2 are 1.11 × 10−17 cm2/W and 3.45 × 10−18 cm2/W. At 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), the open isomer of spiropyran 2 has refractive index values of 2.21 × 10−17 cm2/W and 1.50 × 10−17 cm2/W at 5.63 × 1014 Hz (0.09 au) and 2.82 × 1014 Hz (0.04 au), respectively. The highest refractive index value (2.21 × 10−17 cm2/W) is observed at the wavelength of 5.63 × 1014 Hz (0.09 au) for open isomer of spiropyran 2 at 5.63 × 1014 Hz (0.09 au).The refractive index  of spiropyran 1 & 2 show transparency in the deep UV region (below 200 nm), except for the close isomer of spiropyran 1. The increase in λmax values for open isomers supports the NLO response. As the electronic excitation increases in a system, the NLO response also increases. Such behavior is previously seen by Bibi et al. for on-off switches based on cobalt and iron-containing coordinate complexes [The absorption spectra of both isomers of spiropyranes omplexes , which j1 & 2 are also performed in acetonitrile solvent, and their results of λmax are given in max for the open isomer (246 nm) is higher than the closed isomer (236 nm) of spiropyran 1. In continuation to this result, the λmax of the close isomer (271 nm) is higher than the open isomer (270 nm) of spiropyran 2. Although compared to gas phase calculations, the λmax of closed isomer is increased, and for open isomer, this value is decreased significantly. The λmax values in acetonitrile are comparable to already reported λmax values reported by spiropyranes in the same solvent [The UV-Vis analysis of close and open isomers of spiropyranes  solvent .1 & 2 isomers are calculated with Multiwfn software [1 & 2 to five excited states are given in 1, excited states 1 (D/S = 1.57/0.21) and 5 (D/S = 2.07/0.17), have high D-values and lower S values, showing CT-type excitations. The rest of the excited states 2–4 must have LE-type excitations. The open isomer of spiropyran 1, excited states 2 (D/S = 2.36/0.15) and 5 (D/S = 2.84/0.27) have high D-values and lower S values, showing CT-type excitations. The rest of the excited states 1,3, and 4 have LE-type excitations. The close isomer of spiropyran 2, excited states 1 (D/S = 1.94/0.26) and 4 (D/S = 2.51/0.20), have high D and lower S values, showing CT-type excitations. The rest of the excited states 2, 3, and 5 have LE-type excitations. The open isomer of spiropyran 2, excited states 3 (D/S = 2.43/0.25) and 4 (D/S = 2.98/0.34), have high values of D values and lower S values, so they show CT-type excitations. The rest of the excited states, 1 and 5, have LE-type excitations. Compared to other states, the excited state 2 shows Rydberg-type excitation as both D (1.73 Å) and S (0.21) values are small.The charge transfer distance and charge density difference of both spiropyranes software . Figure 1 & 2 are studied through FMOs analysis. It is observed that the lower HOMO-LUMO gap with lower excitation energy in open isomers of both spiropyranes 1 & 2 imparts large hyperpolarizability values. The extended electronic delocalization and unsymmetrical electronic distribution are responsible for the decrease in the HOMO-LUMO gap. Comparatively, the first hyperpolarizability values of open isomers are more than the closed isomers. The delocalization of π-electrons, asymmetric distribution of electrons (in spiropyran 1) and elongated conjugation system (in spiropyran 2) are dominant factors for the increase of hyperpolarizability values of open isomers (1 & 2). In both spiropyranes, the highest βo (3594.49 au) is observed for the open isomer of spiropyran 2. To get a deep understanding of our studied reversible thermochromic spiropyranes (1 & 2), we also analyzed the frequency-dependent hyperpolarizability. The more prominent frequency-dependent NLO response is at 5.63 × 1014 Hz (0.09 au). To further observe their NLO response, we also calculated the refractive index of these thermochromic compounds. Among all those compounds, the highest refractive index value is observed for the open isomer of the spiropyran 1 (1.99 × 10−17 cm2/W). Molecular absorption analysis confirmed electronic excitation in open and closed isomers, where the effect is more pronounced for open isomers of thermochromic spiropyranes (1 & 2). Therefore, the calculated values reveal reversible thermochromic compounds as excellent NLO molecular switches for future applications.We have investigated the geometric, electronic, optical, and nonlinear optical properties of reversible thermochromic spiropyranes at the DFT method. Electronic properties of these close and open isomers of spiropyranes


We examine Pr4+-doped perovskite oxide lattices(BaSnO3 and BaZrO3). These systems are definedby IC which quenches orbital angular momentum. Therefore, the single-ionspin–orbit coupled states in Pr4+ can be chemicallytuned. We demonstrate a relatively large hyperfine interaction of Aiso = 1800 MHz for Pr4+, coherentmanipulation of the spin with QM = 2ΩRTm, reaching up to ∼400for 0.1Pr:BSO at T = 5 K, and significantimprovement of the temperature at which Tm is limited by T1 (T* = 60 K) compared to other RE ion qubits.Crystal field (CF)control of rare-earth (RE) ions hasbeen employedto minimize decoherence in qubits and to enhance the effective barrierof single-molecule magnets. The CF approach has been focused on theeffects of symmetry on dynamic magnetic properties. Herein, the magnitudeof the CF is increased via control of the RE oxidation state. Theenhanced 4f metal–ligand covalency in Pr This electronicstructure results in rich physics and has been used to design newquantum materials with emergent phenomena.14 Therefore,in RE ions, the CF states can act as qubit states.11 Among the RE ions, Pr3+,15 Nd3+,16 Er3+,11 and Yb3+18 have been of primary interest. Recently, synthetic chemistry hasbeen proposed to offer tunability of quantum states by engineeringthe ligand field experienced by the electron spin in the form of molecularqubits.21 Besides tuning the ligand field, synthetic chemistryalso offers the ability to engineer the electronic structure of theRE ion by providing control over the formal oxidation state of themetal center, as evidenced in recently explored reduced RE molecularcomplexes of La2+ and Lu2+. In these systems,a single unpaired electron resides in an orbital with a mixed 5d/6scharacter rather than the 4f orbital, giving rise to a clock transitionand enhanced coherence.23Paramagnetic RE ions exhibit unquenchedorbital angular momentumfrom the atomic-like 4f states possessing electron and nuclear spinsand accessible optical transitions. These properties make them attractivematerials to generate a versatile quantum interface by bringing togetheroptical and microwave addressability. As a result, a hybrid quantumsystem can be achieved to develop efficient and faithful microwave-opticalconversion, entanglement storage, and light–matter teleportationin the telecom wavelength. In the trivalent oxidation state  are modulated.31 Suppressing theefficacy of the Raman and Orbach processes driven by acoustic andoptical phonons, respectively, is key to establishing long coherencetimes. The intuitive approach to minimize spin–phonon couplingis engineering of lattice vibrational modes and can be achieved byjudicious choice of the host lattice/ligand architecture.34 However, the chemical properties that drive T1 remain an open question and demonstrate that there is a richchemical space still to be explored for QIS applications.A key ingredient in determiningthe coherence timeof an electronspin is the spin–lattice relaxation time, orb/μspin ≈ 0) suppresses spin–phonon coupling. Within thisframework, Pr4+ ions are advantageous, given that the firstCF excited state is at ∼2000 cm–1 comparedto a few hundreds of cm–1 observed for other Ln3+ ions.35 Furthermore, the largeCF energy scale competes with spin–orbit coupling (SOC), whichmixes the excited-state SOC multiplet into the GS and thereby minimizesthe orbital momentum (as observed from X-ray magnetic circular dichroismmeasurements: μorb/μspin ≈1.8 for Pr4+ compared to μorb/μspin ≈ 8 for Ce3+).35 Pr4+ also has a very small g value (gav < 0.8), one of the smallest among theRE ions, which offers the ability to suppress decoherence from spectraldiffusion (SD) due to magnetic dipolar interactions. As a bonus, Prhas a very large nuclear spin, 141Pr , which can further be used for coherentmanipulation via hyperfine interactions.Alternative strategies for RE ions includeCF control usually achieved by careful choice and control of symmetryand use of ions with an S-like electronic GS, where the vanishingorbital angular momentum  and pulsed X-band EPR measurements lends credibilityto our design strategy and establishes Pr4+ as a potentialcandidate for QIS applications.In this work, we use the anomalouslylarge CF splittingof Pr3, Pr:BaSnO3 , and Pr:BaZrO3  were synthesized using traditionalsolid-state reactions (see Supporting Information), and phase purity was confirmed using powder X-ray diffraction. The parent compound, BaPrO3, crystallizes in an orthorhombic Pnma spacegroup37 different from the host materials  which crystallize in a cubic, ideal perovskite Pm3̅m structure, as shown in 38 The orthorhombic distortion in BaPrO3 is dueto cooperative buckling of the corner sharing octahedra with respectto each other, resulting in reduction of local symmetry at the B sitefrom m3̅m (Oh) in the host materials to 1̅ in the parent compound.However, the PrO6 octahedra in BaPrO3 are veryclose to a perfect Oh with only smallchanges in the nearest-neighbor oxygen coordination. Therefore, BaPrO3 is an ideal model compound to study the single-ion electronicstructure of Pr4+ in the PrO6 moiety and tounderstand the microscopic origins of coherent spin dynamics of 141Pr4+ ions in BaMO3  hostlattices. It should be noted here that BaMO3 host latticeswere chosen to provide stabilization of the 4+ oxidation state andto obtain phase pure compounds. Experiments to design host latticeswith minimal nuclear spins in the surrounding bath ) were notfruitful as phase segregation was observed.Crystalline powder samples ofBaPrOfractionS1. The p39 Pr4+ exhibits an unusually large CF splitting which competeswith the SOC, yielding drastically different single-ion propertiesthan expected in the ζSOC ≫ VCF limit as shown in 40 Pr4+ is a Kramers ion with a4f1 electron configuration and couples the electron spin, S = 1/2, and orbital angular momentum, L = 3, to give rise to a J = 5/2 GS (2F5/2) and a J = 7/2 excited state (2F7/2) in the |j, mj⟩ basis. The GS Kramers doublet(KD) is given by 2 ∼ 1/6.In this framework, the CF Hamiltonian (ĤCF) is diagonalized only within the 2F5/2 SOC manifold as is the case for traditional trivalent Ce3+ systems.As reported by our group and others,j is nota good quantumnumber in the ICregime, the |ml, ms⟩ basis can be usedto describe the electronic structure of Pr4+. In the |ml, ms⟩ basis,the Oh CF splits the seven 4f orbitalsto GS a2u and excited triply degenerate t1u andt2u states. In the presence of SOC, the seven 4f orbitalsmix, yielding seven KD. In the |ml, ms⟩ basis, the nature of the GS KD isgiven as 7LS KD are derived from j, mj⟩ basis, while the last components are derived from ĤCF is diagonalized using the entire setof 14 |ml, ms⟩ states.Since 4+ is studied in themodel Hamiltonian:Bnm are the fourth- and sixth-orderterms and Ônm are the correspondingStevensoperator equivalents,41 constrained bythe Oh symmetry of an isolated PrO6 unit. The eigen energies, GS wavefunction composition, and gav as a function of B40 for fixed valuesof B60 are calculated as shown in Supporting Information). With B60 = −0.004, for small values of B40, the system can be considered as a traditional trivalent lanthanidewhere the ζSOC ≫ VCF limit still applies. The eigen states split as expected for the Oh CF , as shownin 37 Curie–Weiss analysis in the10 < T < 40 K range yields θCW ∼ −35 K and μeffCW = 0.75(2) μB, which is significantlylower than the expected value for a free f1 ion (2.54 μB). All CF excitations for Pr4+ in BaPrO3  havebeen reported previously with E1 = 0, E2 ≈ 249.5, E3 ≈ 252, E4 ≈ 389, E5 ≈ 655, E6 ≈ 657, and E7 ≈ 818 meV.47 As expected, Pr4+ exhibits an unusuallylarge VCF energy scale comparable to theζSOC ≈ 112 meV,48 and therefore, the single-ion properties must be modeled in theIC regime as described earlier. The single-ion CF Hamiltonian forPr4+ can be written in a truncated symmetry basis, as shownin ĤCFPr, is then fit to the observed eigen energies from opticalmeasurements, their corresponding degeneracies, and magnetic susceptibilitydata at μoH = 0.1 T above 40 K (T > 40 K was chosen to avoid short-range correlations)asshown in SOC ≈ 112 meV. Thisanalysis yields a set of new KD’s with the GS wavefunctionexpressed as gavCF = 0.68 comparable to the valueextracted from the CW fits. The resulting model reproduces magnetizationat T = 50 K, as shown in 4+ deviates significantly from the expected VCF ≪ ζSOC.BaPrO4+ in a perovskite ABO3 lattice, the relaxationand coherent spin dynamics of electron and nuclear spins of 141Pr4+:BaMO3  were investigated usingCW and pulsed EPR at the X-band (f = 9.4 GHz and B0 < 1.8 T). 2Pr:BSO and 2Pr:BZO measured at T = 5 K, revealinga six-line pattern due to the unpaired electron and its hyperfinecoupling with the I = 5/2 141Pr isotope.The EPR spectrum was analyzed using an effective spin Hamiltonianof S = 1/2 electron coupled to an I = 5/2 nuclear spin:g̃ is the g-tensor, the third term represents theelectron–nuclear hyperfine interaction, and Ã is the hyperfine coupling tensor. The X-band EPR simulations (usingthe MATLAB toolbox EasySpin)49 also shownin gisoEPR ≈ 0.57 comparable to gisoCF and a largehyperfine interaction of Aiso ≈1771 MHz. At zero field, where the hyperfine interaction is the strongest,the nuclear spin and electron spin couple, yielding two states withtotal angular momentum F = I ± S = 5/2 ± 1/2 = 2 and 3 separated by 141Pr4+ and the corresponding forbiddentransitions (gray lines) calculated with the parameters extractedfor 2Pr:BSO. 2Pr:BZO yields very similarresults with gisoEPR≈ 0.63 and Aiso ≈ 1789 MHz as shown in Table S6.Having established the single-ion electronic structureof Pr141Pr4+ withpulsed EPR methods, the echo-detected field-swept (EDFS) spectrumof 2Pr:BSO was recorded by monitoring the integratedspin echo intensity as a function of applied dc field using the two-pulseecho sequence (π/2 – τ – π –τ – echo) with τ = 120 ns andis shown in 2Pr:BZO yields very similarresults in good agreement with CW data as shown in Table S6. Electron spin relaxation, characterizedby the spin–lattice relaxation time constant, T1, is commonly caused by spin–phonon coupling and,in most cases, limits the coherence times of the electron spin. Furthermore, T1 also affects decoherence indirectly, wherethe spin flips of neighboring electron spins lead to SD of the centralspin.18 Therefore, the T1 of 141Pr4+ ions in the temperaturerange 5–60 K was studied using the inversion-recovery method(π – τr – π/2 –τe – π – τe – echo), where τr is swept, as shown in B0 = 592.7 mT (2Pr:BSO) and 530.8 mT (2Pr:BZO), focus on thefield of the largest intensity echo. The resulting saturation recoverytraces were fit with a standard stretched mono-exponential function(see Supporting Information), and the extracted T1 values are plotted in T12 Pr:BSO shows weak temperaturedependence, reaching a maximum of ∼13 ms at 5 K, while T12 Pr:BZO shows a strong temperature dependence,reaching a maximum of ∼0.7 ms at 5 K, comparable to other oxidehost lattices.18 In either case, the electronspin–lattice relaxation rate in the low-temperature regimeis inversely proportional to temperature which can be attributed toa direct one-phonon process.18 At hightemperatures (>12 K), T1 begins toprecipitouslydecrease, reaching a value of T12 Pr:BSO = 4.65μs and T12 Pr:BZO = 0.31 μs at T = 30 K. In order to understand the effects of dipolarmagnetic interactions, further diluted samples with a Pr4+ concentration of ∼0.1% were analyzed. Dilution to ∼0.1%improves T1, reaching a maximum of T10.1 Pr:BSO ≈ 33 and T10.1 Pr:BZO ≈ 16 ms for the BSO and BZO host lattices at T = 5 K, respectively. 0.1% dilution improved T1 only for T < 12 K, as shown in T >12 K, T1 for 0.1% dilutions overlaps with2% dilutions for both host lattices, indicating that decoherence isnot limited by dipolar interactions with additional decoherence mechanismscoming in to play.Probing the spin dynamics of 50 The Orbach process dominateswhen the temperature is sufficient to excite phonons that resonatewith an excited state (in this case CF states). Given the first CFexcited state is ∼2000 cm–1, Orbach processesshould have little effect on the relaxation. Therefore, the temperaturedependence of T10.1 Pr:BSO for T > 10 K was fit to a combination of Raman and local modes basedona general description of the two-phonon process which takes into accountthe maximum phonon energy kbθD, where θD is the characteristic Debye temperature(Supporting Information). Thedata suggests a θD = 180 K consistent with the IR-phononspectra of BaSnO3 which identifies the lowest optical phononmode at 135 cm–1 (195 K).51In this higher temperature regime,two-phonon processescharacterizedvia a combination of resonant (Orbach), nonresonant (Raman), and localmodes dominate.Tm, were measured through two-pulse Hahn echo measurements.The echo intensity was measured as a function of 2τ as shownin Tm was extracted by fitting the data to a standard mono-exponentialfunction, yielding Tm2 Pr:BSO = 3.1 and Tm2 Pr:BZO = 2.3 μs at T =5 K. Tm2 Pr:BSO follows T12 Pr:BSO with weak temperature dependence, while Tm2 Pr:BZO shows weaker temperature dependence compared to T12 Pr:BZO in the temperature range T < 12 K. Similarto T1, Tm improvedon dilution to 0.1%, reaching a maximum of Tm0.1 Pr:BSO = 18 and Tm0.1 Pr:BZO = 2.5 μsat T = 5 K for both host lattices and begins to decreaseat higher temperatures, reaching Tm0.1 Pr:BSO =0.26 and Tm0.1 Pr:BZO = 0.1 μsat T = 60 K with minimal effects of dilution for T ≳ 12. Tm is bound by T1 above T* = 40 and 60 K for 0.1Pr:BZO and 0.1Pr:BSO, respectively. Note thatat low temperatures, the echo decay curves show strong electron spin-echoenvelope modulation (ESEEM) from Ba nuclear spins (vide infra). Themaximum Tm value extracted for 141Pr4+ is one of the largest among the RE ions .Having now established the bounds on spin coherence lifetimesfromspin–lattice relaxation, the lifetimes of the coherent superpositionstate of the qubit, parameterized by phase memory time  RE ions 1. Furthe derived 1. We notT1 relaxation rates—particularlyin the T < 12 K regime, indicating that an additionaldecoherence mechanism besides the SD from neighboring spin-flips isactive. In order to understand the decoherence mechanism, a three-pulsestimulated echo technique (π/2 – τ – π/2– TW –π/2 – τ – echo) and T = 5 K is used. Fitting the echo decay to a combinationof SD linewidth (ΓSD) and relaxation time, a T1SD = 0.7 and 1.8 ms are obtained for 2Pr:BSO and 2Pr:BZO, respectively. The extracted T1SD values are halfof those obtained from the inversion recovery measurements. This differenceindicates that besides the spin-flip process, spin-flip flops fromneighboring nuclear spins are active as well. The fast Fourier transform(FFT) of the time domain data of 2Pr:BSO measured withτ = 120 ns clearly shows a peak ≈2.8 MHz, correspondingto the Larmour frequency of 135Ba (I =3/2) in the field measured  nuclei, yielding A∥Ba = 0.8MHz and A⊥Ba = 1.8 MHz for both nuclei and quadrupolarcontributions of QBa = 3.5 for both nucleias shown in 117,119Sn nuclei (I = 1/2) in BSO and 91Zr nuclei (I = 5/2)in BZO in the surrounding bath of 141Pr4+. Itis possible that the large nuclear spin of 91Zr explainsthe faster T1 relaxation rate for BZOcompared to that for BSO, leading to a faster decoherence.Additionally, Hyperfine Sublevel Correlations (HYSCORE) spectroscopyfurther resolves the coupling to the surrounding Ba nuclei. Both hostlattices show two sets of two sharp peaks, which can be simulatedby coupling to both 141Pr4+ is theunusually large hyperfine interaction of ∼2000 MHz. Among theRE elements, holmium metal exhibits the largest hyperfine interactionwith AJHo = 6500 MHz, followed by praseodymiummetal with AJPr = 4500 MHz.52 Such large hyperfine coupling interactions have been attributedto the polarization of conduction electrons by s–d mixing minimizingSOC.52 A similar argument has been invokedfor a very large hyperfine interaction of Aiso2+Lu ≈ 3500 MHz observed in Lu2+ molecular complexeswhere the spin bearing d orbitals undergoes symmetry-allowed mixingwith s orbitals minimizing SOC.22 However,in the solid-state, RE ions, when doped in wide band-gap host lattices,exhibit a significantly reduced hyperfine interaction, as evidencedfor Ho3+:LiYF4 with Aiso ≈ 800 MHz53 or in molecules likeHo3+ polyoxometalates with Aiso ≈ 700 MHz.55 This large reduction of the hyperfineinteraction necessitates the need to understand the origin of thevery large hyperfine coupling in 141Pr4+ anda comparison of the hyperfine interaction of 4f1 141Pr4+ with 4f2 141Pr3+.One of the key properties of 3+ is a non-Kramers ion, it is oftenEPR silent, at least in the X-band. Therefore, specific-heat measurementswere employed. The heat-capacity CN arisingfrom a discrete set of 2I + 1 hyperfine energy levels Wm  occurs as a Schottkyanomaly with a peak or maximum at a temperature T ≃ ⟨ΔW⟩av/k, where ⟨ΔW⟩av is the mean spacing of the energy levels and k isthe Boltzmann constant. The Hamiltonian for the hyperfine Schottkycontribution can be written as56A isthe hyperfine interaction constant, Iz is the expectation values of I,μeff is the saturated magnetic moment, gJ is the landau g-factor,and P is the quadrupolar contribution. P for 141Pr is usually three to four orders of magnitudesmaller than A and therefore can be neglected. Simply, A and μeff. By fitting the observedSchottky anomaly in the specific heat data arising from thermal depopulationof the hyperfine spin levels, experimental values of A can be extracted.However, since Pr3+:LnCl3 yield A3+ : LnCl3Pr ≃ 1089 MHz, significantly less than the Pr metalas expected.57 Following a similar approach,the heat capacityof 2Pr:BSO at μ0H =7 and 14 T was measured . The A4+Pr value obtained is almost twofold greater than the value reportedfor Pr3+ : LnCl3 or other oxide host latticeslike Pr3+:Y2O315. Onincreasing the oxidation state from Pr3+ to Pr4+, two antagonistic effects compete to drive the observed hyperfineinteraction with the increased nuclear charge, leading to a largervalue which is diminished by the enhanced 4f metal–ligand covalency.In this system, the increase in nuclear charge appears to dominateand lead to the significant enhancement in hyperfine interaction.58Heat-capacity measurements on Prmeasured 5e. BelowTm, coherentspin manipulations can be performed, as demonstrated by the observationof Rabi oscillations for 0.1Pr:BSO in transient nutationexperiments (R isthe damping time and ΩR is the Rabi frequency. Thecorresponding fits are shown in R ≈ 0.2 μsat 0 dB microwave power (which is significantly less than the phasememory time Tm0.1 Pr:BSO due to homogeneousand inhomogeneous broadening mechanisms). The FFT of the time domaindata yields ΩR, consistent with the values obtainedby fitting to R and relative amplitude B1 as shown in Nc = τR(c)ΩR with Nc0.1 Pr:BSO ≈ 15 compares well with the valuereported for 167Er3+ doped in CaWO4. The qubit figure of merit given by QM = 2ΩRTm reaches upto ∼400 for 0.1Pr:BSO at T =5 K, the same order of magnitude as other RE qubits except for Er3+ which is in the order of 104.Given the relatively long eriments 7a. The d4+ offers the abilityto chemically tune the spin–orbit coupled single-ion statesas the paradigm shifts from ζSOC ≫ VCF limit to ζSOC ≪ VCF limit. CW X-band EPR measurements of Pr:BSO and Pr:BZO and CF analysis of the parentmaterial establish the unique single-ion electronic structure of Pr4+ with a very small gav ≈0.6 and a large hyperfine interaction of Aiso ≈ 1800 MHz. Building on these results, pulsed X-band measurementsshow the possibility for coherent manipulation of the Pr4+ ion with coherence times, reaching a maximum of T1 = 33 ms and Tm = 18 μs,with spin dynamics detectable up to T* = 60 K. Therefore,in this tetravalent RE qubit, we have demonstrated long phase memorytimes exceeding most trivalent RE qubit systems via an alternativeapproach by employing the large CF energy scale of Pr4+ with a vanishing orbital angular momentum via control of the metaloxidation state. Additionally, our work establishes the IC regimeas a potential avenue for designing new RE, actinide, heavy (4d and5d) transition-metal, and main-group60-based quantummaterials, both in solid-state and molecular systems.In conclusion, we showthat Pr


It is a dodecameric serine/threonine kinase that has been highly conserved across metazoans for over a million years. Despite the extensive knowledge of the mechanisms underlying CaMKII activation, its behavior at the molecular level has remained unobserved. In this study, we used high-speed atomic force microscopy to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII with nanometer resolution. Our imaging results revealed that the dynamic behavior is dependent on CaM binding and subsequent pT286 phosphorylation. Among the species studies, only rat CaMKIIα with pT286/pT305/pT306 exhibited kinase domain oligomerization. Furthermore, we revealed that the sensitivity of CaMKII to PP2A in the three species differs, with rat, C. elegans, and hydra being less dephosphorylated in that order. The evolutionarily acquired features of mammalian CaMKIIα-specific structural arrangement and phosphatase tolerance may differentiate neuronal function between mammals and other species.Ca High-speed atomic force microscopy reveals the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII S1A). In contrast, the linker region is relatively variable among these three species, implying that the linker is possibly involved in distinct activation mechanisms and functions among species.CaMKIIα consists of four components: a kinase domain, a regulatory segment with a Cab domain  (4). The2+/CaM) to the regulatory segment, leading to kinase activation (fig. S2B). The adjacent activated kinase domains autophosphorylate each other at Thr286 (pT286). Even after Ca2+/CaM dissociation, the CaMKIIα subunit maintains pT286 and functions in adjacent kinase phosphorylation. This CaMKIIα autonomy has been hypothesized to be a form of molecular memory (as autonomy enables CaMKIIα to “remember” past Ca2+ stimuli by sustained activity)   are exposed and autophosphorylated by both inter- (trans) and intramolecular (cis) subunits (fig. S2B) (305/306 (pT305/pT306) inhibits subsequent Ca2+/CaM binding  (286 and Thr305/306 was confirmed by Western blotting (fig. S4). The purified proteins were immobilized on a positively charged mica surface modified with pillar[5]arene (n = 96); Dh-k) and the radius of gyration Rg. In particular, Rg was calculated as the root mean square displacement of the kinase domain position from its average position, indicating the compactness of the movement range of the individual kinase domains . The results showed that each kinase domain was located at an average distance of 10.57 ± 0.36 nm from the center of the hub assembly with a large distribution of Rg . This result is consistent with the CaM-trapping model (2 and 1 mM ATP), Ca2+/CaM can continue to bind to CaMKIIα virtually indefinitely (2+/CaM was released from CaMKIIα during observation. It may be due to the invasive interaction between an AFM tip and Ca2+/CaM. Similar to CaMKIIα (Ca2+/CaM) (2+/CaM and pT286) showed a structure extended in the radial direction by 3.3 nm. One of the remarkable differences is the reappearance of high Rg . The reaction was initiated by the addition of 1 μl of PP2A. After 3 min, the amount of p-nitrophenol was determined by reading the absorbance at 405 nm . This method gives PP2A activity in units per microliter, and our purified PP2A typically had the activity of ~0.5 unit/μl.One unit is defined as the amount of PP2A that hydrolyzes 1 nmol of 50 mM 2, and 1 mM ATP in a reaction buffer . To release CaM from CaMKII, 2 mM EGTA was added and incubated for 5 min at 30°C and an additional 10 min at 25°C to match the conditions for AFM observation . For substrate phosphorylation, sfGFP-Syn2 was added at 1 μM and incubated for 7 min at 25°C. For CaMKII dephosphorylation by PP2A, phosphorylated CaMKII proteins were incubated with the indicated concentration of PP2A for 10 min at 25°C. The reactions were stopped by adding SDS sample buffer and then analyzed by Western blotting.Standard kinase assays were performed at 30°C for 5-min incubation with 50 nM purified CaMKII proteins, 800 nM CaM, 1 mM CaCl286) , anti-pT305 , anti–phospho-CaMKII (Thr306) , anti–phospho-PKA substrate (RRXS*/T*) , anti–His-tag , and HRP–anti-rabbit/mouse .Western blotting was performed with the following antibodies: anti–phospho-CaMKII  by means of Avalanche-Everyday transfection reagent , followed by incubation for 16 to 20 hours. FLIM-FRET imaging was conducted in a solution containing Hepes –buffered artificial cerebrospinal fluid  at room temperature (23° to 25°C).HeLa cells were cultured in DMEM supplemented with 5% fetal bovine serum at 37°C and 5% CODetails of the 2pFLIM-FRET imaging are described elsewhere  to change the surface charge from negative to positive , 150 mM KCl, and 10 mM MgCl2] with 0.1 mM EGTA. Then, HS-AFM observations were performed in buffer A with 0.1 mM EGTA and 50 μM bosutinib.CaMKII holoenzymes in the basal state were observed in buffer A [50 mM tris-HCl (pH 7.4), 15 mM KCl, 10 mM MgCl2+/CaM-bound CaMKII holoenzymes, 50 nM CaMKII and 800 nM CaM were premixed in buffer B with 1 mM CaCl2 and incubated at 30°C for 5 min. Then, HS-AFM observations were performed in buffer A with 1 mM CaCl2.For Ca2+/CaM-bound CaMKII holoenzymes in the presence of ATP, we premixed 50 nM CaMKII and 800 nM CaM in buffer B with 1 mM CaCl2 and 1 mM ATP and incubated the mixture at 30°C for 5 min. Then, HS-AFM observations were performed in buffer A with 1 mM CaCl2 and 1 mM ATP.For Ca2 and 1 mM ATP and incubated at 30°C for 5 min. After that, to dissociate Ca2+/CaM from CaMKII, we added 2 mM EGTA and incubated at 30°C for an additional 5 min. Then, HS-AFM observations were performed in buffer A with 2 mM EGTA and 1 mM ATP. All HS-AFM experiments were performed at room temperature (24° to 26°C).For CaMKII holoenzymes in EGTA and ATP, we premixed 50 nM CaMKII and 800 nM CaM in buffer B with 1 mM CaClHS-AFM images were processed using Fiji (ImageJ) software  , rat CaMKIIα + bosutinib , rat CaMKIIα + ADP , rat CaMKIIα + ATP , rat CaMKIIα in EGTA/ADP after Ca2+/CaM/ADP stimulation , rat CaMKIIαT286A , rat CaMKIIαT286A in EGTA/ATP after Ca2+/CaM/ATP stimulation , rat CaMKIIαT305A/T306V , rat CaMKIIαT305A/T306V in EGTA/ATP after Ca2+/CaM/ATP stimulation , hydra CaMKIIα , hydra CaMKIIα in EGTA/ATP after Ca2+/CaM/ATP stimulation , C. elegans CaMKII , and C. elegans CaMKII in EGTA/ATP after Ca2+/CaM/ATP stimulation  were analyzed. The motion of Ca2+/CaM binding kinase domains in rat CaMKIIα without ATP (4557 points of 1199 frames of 41 kinase domains in 9 oligomers), rat CaMKIIα + ATP (8757 points of 1604 frames of 71 kinase domains in 11 oligomers), rat CaMKIIαT286A + ATP , rat CaMKIIαT305A/T306V + ATP , hydra CaMKIIα +ATP , and C. elegans CaMKII + ATP  were analyzed. HS-AFM experiments were repeated at least three times independently with similar results.The motions of each kinase domain of rat CaMKIIα , rat nlCaMKIIα , rat nlCaMKIIαDh-k) was computed as followsn and n.To quantify the trajectory of a single kinase domain, the distance from the center of the hub assembly to the kinase domains , defined as followsx and y positions of the highest pixel for an individual kinase domain and angular brackets represent the time average. By diagonalizing C, eigenvectors were computed.To visualize the coupled motions of the kinase domains, PCA was performed on their trajectories. Vectors on the kinase domains show the coupled motions along the vectors, and the lengths of the vectors indicate the degree of coupling. PCA was applied using the conventional method  and the Kruskal-Wallis test were used for the analysis of multiple groups with a single independent variable. The Dunnett’s and Dunn’s tests were used as follow-up tests to the Kruskal-Wallis test, where Dunn’s test was used to compare every mean with every other mean, and the Dunnett’s test was used to compare every mean to a control mean. The F test was used to compare the variance of two groups: P > 0.05, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant. Data are represented as the means ± SDs.All statistical analyses were performed using Igor Pro 9 software , OriginPro 2023 , or GraphPad Prism . A significance level of α = 0.05 was used for all analyses, and


Citric acid was also used in low concentration for the hydrolysis reaction. A sol-gel method was used to prepare bioactive glass (BG) specimens of H2O2-untreated BG, 1M, 2M, and 3M H2O2-treated BGs. X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), energy-dispersive spectroscopy (EDS), and nitrogen adsorption/desorption isotherm with the Brunauer–Emmett–Teller (BET) method were used for analyzing the samples' phase, surface morphology, chemical composition, constituent composition, pore size, and specific surface area respectively. In vitro bioactivity, as well as biodegradability tests, was performed on samples by immersing them in simulated body fluid (SBF) solution. According to the results, BG particles treated with 2 M H2O2 exhibited higher specific surface area (SSA), which is 189.55 cc/g, and better in vitro bioactivity and biodegradability.In this study, carbon-free and completely soluble hydrogen peroxide (H The body's musculoskeletal system is essential in that it is constantly susceptible to injury or damage by illnesses, pathologies, trauma, infections, physiological resorption, and age . A lot o2 in melted BGs will lead to a decrease in the glass' bioavailability. Even after being subjected to simulated bodily fluid (SBF) for several weeks, the BGs that contain more than 60% SiO2 are unable to develop a carbonated hydroxyapatite layer and do not bind to soft or bone tissues [The earliest and most thoroughly studied BG, called “45S5 Bioglass®,” is regarded as the industry standard because it can chemically adhere to bone tissue despite developing non-adherent fibrous capsules . However tissues .Thus, the BGs were prepared by another processing method like the sol-gel technique, which allows a greater surface area through a porous microstructure, enabling a wider range of applications , 19, 20.2O2) are employed as a pore-forming agent [2O2) as a novel pore-forming agent and BG with high SSA resulting from its decomposition behavior at 180°C (2H2O2 ⟶ 2H2O + O2) produces significant amounts of oxygen gas to build the pore structures [2O2 has no carboxyl groups and can be entirely soluble in water, allowing no carbon contamination as well as aggregation effects. Hence, H2O2 can substitute traditional surfactants in the role of pore formation.Cetyltrimethylammonium bromide (CTAB), poly(ethylene glycol) (PEG), polyurethane (PU), and Pluronics  are the typical surfactants used as pore-forming agents for synthesizing porous BGs –27. Howeng agent . Using hructures , 30. ImpThe acidic or basic conditions used for the catalysis of hydrolysis during the sol-gel technique have a considerable impact on the properties of BG . Inorgan2, 36mol% CaO, and 4mol% P2O5) was synthesized using the sol-gel synthesis method. Precursors used as Si, Ca, and P sources include tetraethyl orthosilicate 4), calcium nitrate tetrahydrate 2.4H2O), and phosphoric acid (H3PO4), respectively. 0.5 mM citric acid was added in 50 mL distilled water and ethanol alcohol (1 : 1) solvents and stirred for 15 minutes at room temperature. The two solvents were used together because of solubility differences between the precursors. TEOS is highly soluble in ethanol, whereas CN and H3PO4 are soluble in water. Additionally, the citric acid solution is used to catalyze the hydrolysis of silicon alkoxide (TEOS) and plays a great role in the morphology of bioactive glass that enhances the bioactivity and biodegradability properties. When ethanol and water are mixed together, they form an alcohol-water mixture which can effectively dissolve organic compounds including citric acid. The solubility of citric acid or other solutes in the alcohol-water solution depends on the proportion of these solvents. Using equal amounts of these solvents promote enhanced solubility and versatile solvent system which allow solute dissolution while maintaining polar properties of water and organic properties of ethanol. Then, TEOS, phosphoric acid, calcium nitrate tetrahydrate, and hydrogen peroxide were added in to the solution sequentially. The precursor solution was stirred on a hot plate with a magnetic stirrer for 2 hours at room temperature in order to create a homogeneous solution. The prepared sol was aged and gelled for 24 hours at 60°C. Then, the obtained gel was dried at 100°C for 3 hr to remove solvents. The dried specimens were calcined for 2 hr at 600°C to remove the remaining precursor residues and solvents. The sample without using any H2O2 was denoted as BG0. 1M, 2M, and 3M H2O2-treated BG were denoted as BG1, BG2, and BG3, respectively.In this study, BG powder with a 58S composition , scanning electron microscopy (SEM), Fourier-transform infrared microscopy (FTIR), energy-dispersive x-ray spectroscopy (EDS), and Brunauer–Emmett–Teller (BET) surface analysis were used to obtain details of phase composition, surface morphologies of the specimens, chemical identification, identify the constituent composition, and the specific surface area of each specimen, respectively.In vitro bioactivity studies have previously used Kokubo's simulated bodily fluid (SBF) test solution [in vitro bioactivity of all BGs. Each of the specimens was then submerged in SBF and kept in a water bath that was maintained at a constant 37°C for 3 days. HAp layer formation on soaked samples was characterized by XRD and FTIR for phase and composition analysis after rinsing the soaked samples with distilled water three times and dried at 80°C for 3 days. The Debye-Scherer equation  of the diffraction peak, λ is the X-ray wavelength (1.54178Å), D is the crystallite size, and K (=0.94) is the Scherer's constant.solution . All BG equation ) is usedBG0, BG1, BG2, and BG3 specimens in pellet form were examined for in vitro biodegradability in SBF solution. For each specimen, three BG pellets were utilized to calculate the weight loss's average and standard deviation. The ratio of 1 gm to 10 mL of SBF was used to submerge the measured and produced BG powder pellets. Every day, the solutions were discarded, and the pellets were dried and weighed once more. Every day, the entire assessment was repeated for 30 days, replacing the SBF.θ = 20° to 30° suggests that the BG specimens were effectively synthesized without a sharp X-ray diffraction peak, indicating an amorphous phase, i.e., the absence of ordered crystal structure.2O2 exhibited porous morphologies. 2O2-treated BG particles exhibited aggregation and some porous particles. For the BG2 particles with 2M H2O2, more uniform and narrower pore distributions are seen from 2O2, relatively larger holes with irregular distributions were seen as illustrated in 2O2 gathering into the larger clusters, creating wider pores.2O2 induces the formation of oxygen for pore formation, its full elimination shows the successful pore formation process enhancing the specific surface area of the particles.The EDS spectra in 2/g, respectively. According to the BET results, which are shown in 2O2 concentration from 0 to 2M, the specific surface area were increased. However, in 3M H2O2-treated BG powder, the specific surface area were decreased due to the H2O2 molecules being accumulated into larger clusters, leading to larger pores.Specific surface areas and pore size of all BG specimens were analyzed using the BET measurements as shown in 2O2 (BG3) shows the highest pore size due to the highest concentration of the H2O2 molecules being aggregated into larger clusters that created larger pores [On the other hand pore size of BG0, BG1, BG2, and BG3 powders were 1.65 ± 0.05, 1.63 ± 0.071, 1.52 ± 0.053, and 2.51 ± 0.046 nm, respectively, as shown on er pores .n = 3 in which every sample reveals p <  0.05 [p value shows there is an important distinction between the BG specimens. <  0.05 –40. The −1, 800 cm−1, and 482 cm−1 exhibit only Si-O-Si bands. After BG powders were soaked in SBF for 3 days, all specimens showed a new peak that occurred at 566 cm−1 and was assigned to the P-O band [FTIR spectra are shown in P-O band , as show2O2 produces superior crystallite sizes than the other BG specimens, which suggests a higher formation of HAp and higher bioactivity.The XRD pattern of BG0, BG1, BG2, and BG3 powders that were soaked in SBF for 3 days is shown in in vitro bioactivity analyses were obtained from SEM images after soaking in SBF for 3 days. They illustrate that smaller crystallites are formed and distributed on the surface of the BG particles which demonstrate the formation of HAp layers on BG particles.As shown on 2O2 in SBF solution. The graph demonstrates that the weight loss of the BG0, BG1, BG2, and BG3 samples was 6.37 ± 0.386%, 8.43 ± 0.271%, 16.04 ± 0.315%, and 9.93 ± 0.540%, respectively, after being soaked for 30 days in SBF solution. In addition, the degradation rate of BG0, BG1, BG2, and BG3 specimens were 0.21 ± 0.013%, 0.28 ± 0.009%, 0.53 ± 0.011%, and 0.33 ± 0.018%, respectively. The results shown that 2M H2O2-treated BG had the highest weight loss.2O2 concentration from which the bioactivity and biodegradation of BGs are determined. However, for 3M H2O2-treated BG, specific surface area shows decrement. Thus, it leads to reduce the bioactivity as well as biodegradability of the specimens.According to 2O2 ⟶ 2H2O + O2 decomposition characteristic, BG treated with H2O2 has a higher specific surface area [2O2. This is mainly due to H2O2 molecules may have aggregated into larger clusters which will hinder the formation of HAp as well as the degradation of the BG specimens [2O2 concentration. It demonstrates that H2O2-treated BG specimens show better bioactivity than the untreated one. As the H2O2 concentration increases up to 2M, bioactivity also increases. But raising the concentration to 3M results the bioactivity to decrease.Because of its rapid ability to produce oxygen through its 2Hace area . Therefopecimens .  Figure2O2 as a pore-forming agent enhance BG's specific surface area, bioactivity, and biodegradability. The formation of BG particles with a high specific surface area has been made possible by the high solubility and rapid oxygen producing capabilities of H2O2 through its 2H2O2 ⟶ 2H2O + O2 decomposition. According to the BET analysis, the BG particles with high specific surface area were obtained for 2M H2O2 concentrations. The in vitro bioactivity test was done by SEM, XRD, as well as FTIR analysis and reveals crystal phase (HAp) formation. Thus, it would enhance bioactivity and biodegradability properties of BG.In this study, the bioactive glass  powders were successfully synthesized using the sol-gel method. The results of this study generally indicate that utilizing H


SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine , have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases. Glutamate is the major excitatory neurotransmitter in the mammalian brain. After release from the presynaptic nerve terminal excitatory amino acid transporters (EAATs) quickly take up glutamate into surrounding glial and neuronal cells . EAAT-meSLC1A3, the gene encoding the glial glutamate transporter EAAT1 , and shown to be caused by mutations in er EAAT1 . Later, epilepsy , as wellepilepsy .Slc1a3P290R/+mouse, increased EAAT1/GLAST-mediated chloride efflux causes apoptosis of Bergmann glia in early infancy, resulting in cerebellar dysfunction via impaired glutamate reuptake and cerebellar network formation  to the 5´ end of the EAAT4 coding region using PCR-based strategies (3(PO4)2 technique as described (pcDNA3.1(−) rategies . Point mrategies . For traescribed . For WT 3, 2 MgCl2, 5 EGTA, 10 HEPES, pH 7.4; or 115 NaNO3, 2 MgCl2, 5 EGTA, 10 HEPES, ±0.1 L-glutamate, pH 7.4. Bath solution contained: 140 NaNO3 or CholineNO3, 5 TEA-Cl, 4 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, ±0.1 L-glutamate, pH 7.4. Anion currents were determined without subtraction procedure. For rapid solution exchange to solutions, in which 140 NaNO3 was substituted with KNO3, or 140 CholineNO3 by 140 NaNO3 + 1 L-glutamate or in which 0.1 L-glutamate was added to 140 NaNO3, a self-assembled piezo-driven system with a dual-channel theta glass tubing was used (n = 56).Standard whole-cell patch-clamp recordings were performed using an Axopatch 200B amplifier , with borosilicate pipettes with resistances between 1.0 to 2.0 MΩ . To reduwas used . After p2, 5 EGTA, 10 HEPES, pH 7.4. Cells were subsequently perfused with solutions containing 140 Na-D-gluconate, 5 TEA-D-gluconate, 4 K-D-gluconate, 2 Ca-D-gluconate2, 1 Mg-D-gluconate2, 5 HEPES, pH 7.4, with or without 0.1 L-glutamate. Transport currents were determined as L-glutamate-sensitive currents by subtracting currents obtained in the absence of glutamate from currents measured in the presence of glutamate. For all experiments, Ag/AgCl electrodes were connected via external and/or internal agar salt bridges, made from plastic tubing filled with 3 M KCl in 1% agar. Offset potentials were determined at the end of each experiment, and junction potentials were corrected using the JPCalc software .Glutamate transport currents were measured in cells dialyzed with (in mM) 115 K-D-gluconate, 2 Mg-D-gluconatex/1.40 NA oil immersion objective. mYFP was excited at 488 nm (argon laser), and emission was imaged between 543–549 nm, and fluorescences were analyzed with Fiji image analysis software (NIH). Transfection rates were quantified as ratios of transfected cells by the total number of cells  with a 20x/1.4 NA oil immersion objective for three independent cell transfections for each construct.HEK293T cells were plated on poly-L-lysine coated coverslips 48 h after cell transfection and imaged with an inverted microscope  using a 63For SDS-PAGEs, HEK293T cells were harvested 18 h after transfection with EAAT4-mYFP fusion proteins and lysed in phosphate buffer, supplemented with 0.4% dodecyl-maltoside for 30′ on ice. Lysates were then centrifuged for 35′  and equal amounts of whole cell lysates were analyzed with SDS-PAGEs (12%) and fluorescence scanning . Each variant (WT/P312R/P312N) was tested in three independent transfections. Complex glycosylation was tested with recombinant PNGase F  at a concentration of 16.6 U/μL sample volume for 30´ at 37°C.Simulations of EAAT4 anion channel open probabilities were performed by solving differential equations on the basis of a modified EAAT2/Glt-1 model . Transitt the time, V the voltage, i(V) the single channel amplitude and p(V) the absolute open probability. Dividing the current time course by current amplitudes at the end of the application providesWe initially calculated the steady-state distribution of all transporter states for each conditions before substrate application; these values subsequently serve as initial values to numerically solve a linear system of differential equations and to provide time and voltage-dependent absolute open probability ductance .It,V=N∙ISS) at −150 mV 0) between experimentally determined Our fitting procedure aimed at minimizing the sum of squared residuals  or as box-whisker plots drawn between the first and third quartiles with whiskers providing the upper and lower 95% of the data range. Fluorescence levels of confocal images were analyzed with 2-way ANOVA with WT as controls, and Holm-Sidak post hoc testing, protein amounts and transfection rates were compared with one-way ANOVA, and Holm-Sidak post hoc testing. For comparison of dissociation constants and glutamate transport values, Kruskal–Wallis and Dunn’s post hoc tests were used.Data were analyzed using a combination of Clampfit , Origin , SigmaPlot , Excel  and Libre office Calc  software. All summary data are given as means ± +, one glutamate and one H+ in exchange with one K+  both strongly decrease L-glutamate KMs. Increased L-glutamate affinity was already described for P290R EAAT1 , 4.4 ± 0.6 pA , and 1.8 ± 0.1 pA  at −120 mV  to fast solution exchanges. Cells were held at 0 mV, and voltage steps between −150 mV and +150 mV were applied. After reaching steady-state current amplitudes under the original ionic condition the external solution was quickly changed using a two-barreled application pipette attached to a piezoelectric bimorph  were assumed to be accessible from intermediate conformations with bound Na+ and L-glutamate or bound K+, in agreement with recent MD results demonstrating that anion channels with functional properties that resemble experimental results open during lateral movements of the transport domain from intermediate positions . apo states in P312R EAAT4  We chose EAAT4 because of its robust and well described anion currents in transfected mammalian cells . UnexpecTk, carrying the homologous P208R mutation  on the human glutamate transporter EAAT3 . EAAT3—ee of HP2 .+-based solutions identified increased activity of an anion channel that is accessible in the outward facing apo state as main reason of gain-of-anion channel function in P312R EAAT4. Since the mechanisms of K+ coupling are conserved in all EAATs, and since P290R EAAT1 and P289R EAAT2 share the unique K+ dependence with P312R EAAT4 (We here studied the functional consequences of disease-causing point mutations not in the affected glial EAAT isoforms, EAAT1 and EAAT2, but rather in the neuronal transporter EAAT4. This approach raises the question how our findings can help explaining the pathophysiology of episodic ataxia and epilepsy. Experiments with K2R EAAT4 , this re− efflux (In recent years, an increasing number of genetic diseases has been identified that are caused by dysfunctional ion channels and were dubbed “channelopathies”. In many cases, the linkage of human diseases helped identification of cellular roles of the affected ion channels . Disease− efflux . We here


The interaction of the 19S regulatory particle of the 26S proteasome with ubiquitylated proteins leads to gate opening of the 20S core particle and increases its proteolytic activity by binding of the ubiquitin chain to the inhibitory deubiquitylation enzyme USP14 on the 19S regulatory subunit RPN1. Covalent modification of proteins with the cytokine inducible ubiquitin-like modifier FAT10 is an alternative signal for proteasomal degradation. Here, we report that FAT10 and its interaction partner NUB1L facilitate the gate opening of the 20S proteasome in an ubiquitin- and USP14-independent manner. We also show that FAT10 is capable to activate all peptidolytic activities of the 26S proteasome, however only together with NUB1L, by binding to the UBA domains of NUB1L and thereby interfering with NUB1L dimerization. The binding of FAT10 to NUB1L leads to an increased affinity of NUB1L for the subunit RPN1. In conclusion, the herein described cooperation of FAT10 and NUB1L is a substrate-induced mechanism to activate the 26S proteasome. Saccharomyces cerevisiae, rescues poly-ubiquitylated substrate proteins from degradation. UBP6 binds via its N-terminal ubiquitin-like (UBL) domain at the “T2” site of RPN1 next to the “T1” site, where poly-ubiquitin conjugates and substrate shuttle factors, like Rad23, bind  and one or two copies of the 19S regulator . The thr23, bind . USP14/U23, bind . Apart f23, bind . In cont23, bind . It has 23, bind . More re23, bind . In thesAnother targeting pathway leading to proteasomal degradation of proteins is the conjugation of them to the UBL modifier HLA-F adjacent transcript 10 (FAT10). Unlike ubiquitin, it consists of two UBL domains which are connected by a short and flexible linker . FAT10 iUb)  . To ensuUb) . Such anUb) . These rUb)  which miBecause these findings suggested a gate opening of the 26S proteasome by NUB1L and FAT10, we performed a cycloheximide chase in WT baker’s yeast and its open-gate mutant α3∆N in which the N-termini of α3 were deleted  Fig 2A)Fig 2A). Investigation of the different peptidase specificities of the 20S proteasome showed the highest increase of the chymotrypsin-like activity, whereas trypsin- and caspase-like activities were only slightly less increased after activation of the 26S proteasome by FAT10 and NUB1L . To distHaving shown an activation of the 26S proteasome by NUB1L and monomeric FAT10, we aimed to investigate whether FAT10ylated proteins can activate the 26S proteasome as well. Therefore, we used the UBA6-specific E2 enzyme USE1, the FAT10ylation of which has been documented in detail , 2014. AAs we demonstrated, FAT10 and NUB1L can activate the 26S proteasome only as full-length proteins, giving first indications to a possible mechanism. USP14 serves as a receptor protein for ubiquitylated proteins at the 19S regulator, leading to an open-gate state of the 26S proteasome , either To investigate a possible competition between USP14 and NUB1L binding to RPN1, we performed another pull-down experiment. We used GST–RPN1 bound to GSH beads and performed a pulldown with NUB1L alone . The purified 26S proteasomes were tested for the presence of USP14, which is commonly co-purified with the 26S proteasome because of its non-covalent binding to the 26S complex. 26S proteasome affinity purified from human erythrocytes and from C4 WT control cells contained USP14, whereas USP14 was not detectable in the purified 26S proteasome from USP14−/− cells  supplemented with 10% fetal calf serum (Gibco/Thermo Fisher Scientific), 1% stable glutamine , and 1% penicillin/streptomycin (100×) (both from Biowest/VWR). The C4 murine fibroblast line was derived from embryonic BALB/c mice by SV40 infection in vitro and is described in reference  according to manufacturer’s instructions. The bacteria were grown at 37°C to an OD600nm of 0.6 and induced for 3 h with 1 mM IPTG (Roth). Pellets of 1 liter bacteria culture were then lysed in 25 ml GSH-binding buffer (GBB)  by sonication. Afterwards, the bacteria were centrifuged at 100,000g for 1 h. 500 μl glutathione-Sepharose  was added to the supernatant for batch purification. After washing with 75 ml GBB, the beads were stored for further use.pDEST15–UBL–hHR23B was transformed into B834 E. coli (Novagen) according to the manufacturer’s instructions. The bacteria were grown at 37°C to an OD600 of 0.6 and induced for 3 h with 1 mM IPTG (Roth). Pellets corresponding to 2 liters bacteria culture were lysed in 25 ml lysis buffer  by sonication. Thereafter, the lysed bacteria were centrifuged at 100,000g for 1 h. Afterwards, 0.5 ml of Ni–IDA resin (Protino) were added to the supernatant for batch purification. After washing with 75 ml of lysis buffer, the protein was eluted by adding 2 ml elution buffer  for 2 h hours at 4°C while rotating. The imidazole concentration was then reduced to 0.1 mM by ultrafiltration . The protein was then concentrated to ∼2 mg/ml and stored at −20°C for further use.pET26b–His10–UIM2 was transformed into B834 2, 1 mM ATP, 1 mM DTT) left on ice for 1 h and then sonicated. After a centrifugation at 100,000g for 1 h, the supernatant was added to GST–UBL bound to GSH beads. After 2 h at 4°C with rotation, the beads were washed with 100 ml affinity purification buffer. The 26S proteasome was then eluted by adding 250 μl HIS–UIM (∼2 mg/ml) and rotated 4°C for 20 min. Remaining unbound HIS–UIM was removed by incubation of the supernatant of the previous step with Ni–IDA resin for additional 20 min. The 26S proteasome was then used or stored at −20°C for further use.10 ml of human erythrocytes were mixed with 25 ml affinity purification buffer  . 5 nM 26S proteasome was measured in presence of 500 nM FAT10 or/and 500 nM NUB1L for 60 min at 37°C. The reaction mixture contained 25 mM HEPES/KOH (pH 8.0), 2.5 mM MgClescribed .Native gels with the 26S proteasome were prepared as described by The overlay assay was exactly performed as described before .For SDS–PAGE, a 12% separation and stacking gel was used. After adding fourfold SDS sample buffer to the samples, the samples were boiled at 95°C for 5 min. Afterwards, the samples and the protein marker were applied, the gel was run for 30 min with 45 V and afterwards for 1.5 h with 110 V. The gel was stained with Coomassie dye afterwards.After performing a SDS–PAGE, a Western blot was performed using 0.2 μm nitrocellulose membrane. The blot was run for 1 h 10 min with 110 V. To stain the bands two antibodies were used. The two first antibodies were reactive to β5c to stain the PSMB5 subunit and reactive to αHis6 to stain the His tag. The first antibodies were incubated overnight and washed 3x with TBS-Tween. The two secondary antibodies were anti-rabbit IgG 800 and anti-mouse IgG 680. They were incubated for 2 h, again washed 3x and the blot was imaged and quantitatively evaluated with the LI-COR Odyssey imager and the Image Studio Lite Version 5.2.The radioactive protein was mixed with substrate buffer and 6.32 μg 26S proteasome and in the positive control with 9.75 μg FAT10 and 34.56 μg NUB1L. The reaction was then performed at 37°C, and samples were taken at 0, 5, 10, and 20 min for poly-ubiquitylated p53 and at 0, 15, 30, and 60 min for poly-ubiquitylated E6AP. The samples were immediately boiled at 95°C with SDS–PAGE sample buffer to stop the reaction. After Western blotting, the radioactivity was measured with a radio imager .The vector pTYP2–HA–FAT10 containing human HA–tagged FAT10 as a fusion protein with intein and a chitin-binding domain was a gift from Benedict Kessler (Oxford University). All cysteines except for cysteine 134 of FAT10 were mutated to serines, Cys134 was mutated to leucine using the primers: PR3-06 SDM FAT10 INT-CBD C7S + C9S (t19a_g26c): 5′-CTCCCAATGCTTCCAGCCTCTCTGTGCATGTCCGTT-3′; PR3-08 SDM FAT10 INT-CBD C163S (t485a): 5′-CTCTTCCTGGCATCTTATTCTATTGGATGCTTTGCC-3′; PR3-35 SDM FAT10 INT-CBD C134L (t400c_g401 t_c402g): 5′-CCCTGAAACCCAGATTGTGACTCTGAATGGAAAGAGACTGGAAGATG-3′. FAT10 C(0)C134L protein was expressed and purified as described in the manual provided by the supplier of the chitin beads . Cleavage from the column was prepared as previously described with 100 mM cystamin over night at room temperature to allow for the S to N shift. Like this, a modified FAT10 with a single SH group at the C-terminus was generated. After desalting of 500 μg of FAT10 via a PD10 column  in ligation buffer , a 10-fold molar excess of 5,5′- dithio-bis-(2-nitrobenzoic acid) (DTNB) was added and incubated at room temperature for 30 min. After gel filtration via a PD10 column in ligation buffer, the activated FAT10 was concentrated to 250 μl via an Amicon Ultra-0.5 filtration unit. Expression and purification of His6-tagged USE1 was performed as described in the study by The generation of tagless human FAT10 via a pSUMO vector and ULP1 digestion was performed as described before . Human NTo express only the UBL domain of NUB1L as a fusion protein with GFP, or with a GFP and cytB fusion, we used pGEM-vectors provided by Andreas Matouschek . We replE. coli BL21 as detailed elsewhere (2) and concentrated with Amicon Ultra-0.5 filtration units. The reaction was incubated with 50 ng of UBE1, 50 ng of UbcH7, and 20 μg of ubiquitin (Sigma-Aldrich) in 120 μl with 2 mM ATP final concentration  for 30 s. This process was repeated three times. Nickel sensors were loaded with 20 mM NiCl for 60 s after the last regeneration step. GST served as negative control on the second sensor, when GST-tagged proteins were used, the second sensor was equilibrated in buffer when hexa-histidine–tagged proteins were used. The assay consisted of the steps baseline recording (in Octet buffer for 60 s), binding of 0.1 mg/ml GST, or hexa-histidine–tagged proteins to the sensor in 300 s, baseline 2 determination (Octet buffer for 60 s), association (varying concentrations of the ligand protein in Octet buffer for 300 s), dissociation (300 s in Octet buffer), and regeneration. All experiments were repeated at least three times with different protein preparations. For analysis, the curves were fitted, and dissociation constants were calculated using the 1:1 binding model . Recombinant human His6–NEDD8 was purchased from R&D systems (UL-813-500).Statistical analysis was performed in the programs Excel and GraphPad 6. The fluorescence intensity per min was calculated as mean between the time points 30–60 min. The error bars define the SD calculated by GraphPad. Calculation of the percentage was performed by this formula:This study includes no data deposited in external repositories. Supplementary data for this article are available.


Fourier-transform infrared and thermogravimetric analysis measurements supported the characterization of the PRs. Interestingly, tensile testing demonstrated that coverage ratio affected the mechanical properties of the PRATs more strongly than molecular weight. The insights obtained in this study would facilitate the development of soft materials based on atelocollagens and PRs.Preparing strong and flexible atelocollagen-based materials for biomedical applications is still a challenging task. To address this challenge, this study describes the synthesis and characterization of water-soluble polyrotaxanes (PRs) with different coverage ratios and molecular weights of axle polymers, and their potential applications for PR-reinforced atelocollagen threads (PRATs). A novel method was established for the syntheses of PRs with relatively low coverage ratio at the sub-gram scale, in which the aldehyde groups were employed as crosslinking sites for preparing the PRATs via reductive amination. The aldehyde groups were successfully quantified by  Polyrotaxanes (PRs) are essential components of composite materials ,3,4,5,6.Collagen is a natural protein and major component of mammalian organs and tissues ,32. OwinHerein, we report the synthesis, characterization, and potential applications of CD-threaded PRs for reinforced atelocollagen threads. Two different types of PRs were synthesized and crosslinked to investigate the effects of their molecular weight and coverage ratio on the mechanical properties of the PR-reinforced atelocollagen threads (PRATs) .10k–OH and Peg20k–OH), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and βCD were purchased from FUJIFILM Wako Pure Chemical Corporation . αCD, iodobenzene diacetate (PhI(OAc)2), N,N-dimethylformamide (DMF), dichloromethane, diethyl ether, dry acetonitrile (MeCN), and 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) were procured from Kanto Chemical Co., Inc . DMF was distilled under reduced pressure and stored with a molecular sieve prior to use. CD monoaldehydes  were synthesized according to the previous method benzene (PhI(OAcTf)2), 1,1-dimethylhydrazine (DMHZ), and sodium cyanoborohydride (NaBH3CN) were obtained from Tokyo Chemical Industry Co., Ltd . Amino group-terminated Pegs (Peg10k–NH2 and Peg20k–NH2) were synthesized from Peg10k–OH and Peg20k-OH, respectively, via a typical Gabriel amine synthesis procedure described elsewhere [10kPRαCD1 and Peg20kPRαCD1). αCD-threaded pseudo-PR (PegPRαCD3) and adamantane end-capped PR (PegPRαCD2) were prepared according to a previously developed method [9kPRβCD1 and Plu15kPRβCD1). Hydroxyl group-terminated Pluronic reagents (Plu9k–OH and Plu15k–OH) were purchased from Sigma–Aldrich . Carboxyl group-terminated Pluronic compounds (Plu9k–COOH and Plu15k–COOH) were synthesized via bleach oxidation as described previously [Hydroxyl group-terminated polyethylene glycols  and a catalytic amount of TEMPO  were added to the obtained solution. The resulting mixture was stirred at 4 °C for 10 days and then added dropwise to diethyl ether. The obtained precipitate was repeatedly centrifuged in cold MeCN and dialyzed in deionized water. The dialyzed aqueous solution was freeze-dried to produce Peg10kPRαCD1 or Peg20kPRαCD1 with a nearly quantitative yield. IR (KBr) ṽ = 3377, 2911, 1645, 1152, 1082, and 1032 cm−1. See 1H NMR measurement.Synthesis of PegPRαCD1 from PegPRαCD2 is described in g, 20 min) and washed with cold water. Freeze drying the resulting precipitate produced PluPRβCD3 as a white powder. The further addition of βCD (0.36 g) to the supernatant and repetition of the above-mentioned procedure generated more PluPRβCD3. In total, approximately 0.7 g of Plu9kPRβCD3 and Plu15kPRβCD3 was obtained from one gram of Plu–COOH. δH (DMSO-d6): 1.01–1.05 , 3.30–3.38 , 3.41–3.67 , 3.94 , 4.37–4.40 , 4.83–4.84 , 5.63–5.68  (Plu–COOH (1 g) and βCD  were dissolved in deionized water (56 mL) and stirred at 25 °C for 7 days. A white precipitate was gradually formed during the reaction, which was thoroughly centrifuged  .2 , and DIPEA  were dissolved in dry MeCN (3 mL). Powdered PluPRCD3 (0.5 g) was added to the resulting solution under vigorous stirring. The obtained reaction mixture was stirred at 25 °C for 2 days. Subsequently, it was repeatedly centrifuged  in cold MeCN until the supernatant became colorless. The precipitate was thoroughly washed with dichloromethane and filtered. The dialysis of the solid residue followed by freeze drying produced PluPRβCD2 as a white powder. The yields of Plu9kPRβCD2 and Plu15kPRβCD2 were 0.24 g (48%) and 0.20 g (40%), respectively. δH (DMSO-d6): 1.03–1.05 , 3.28–3.38 , 3.42–3.66 , 4.45–4.47 , 4.82–4.83 , 5.68–5.75 , 7.16–7.32  , Trt–NHl group) . IR  and a catalytic amount of TEMPO  were added to the prepared solution, and the resulting mixture was stirred at 4 °C for 10 days. The reaction mixture was added dropwise to diethyl ether, and the obtained precipitate was centrifuged . Subsequently, the precipitate was washed and centrifuged using cold MeCN and dialyzed in deionized water. Freeze drying the inner dialysis solution produced Plu9kPRβCD1 or Plu15kPRβCD1 with a nearly quantitative yield. IR (KBr) ṽ = 3389, 2934, 1649, 1456, 1155, 1082, and 1030 cm−1. See the section below regarding the aldehyde marking with DMHZ for 1H NMR measurement.PluPRβCD2 (0.12 g) was dissolved in HMPA (3 mL) and cooled to 4 °C. PhI(OAc)3CN was added to the obtained solution at a final concentration of 0.2 M. The resulting mixture was stirred at 37 °C for another 48 h and subsequently dialyzed in deionized water for 24 h. Freeze drying the inner dialysis solution and thoroughly washing with ethanol produced the DMHZ-labeled PegPRαCD1 or PluPRβCD1 with a nearly quantitative yield. For PegPRαCD1-DMHZ δH (DMSO-d6): 2.54 , 3.24 , 3.40–3.52 , 3.59–3.81 , 4.31 , 4.80–4.81 , 5.51  (PegPRαCD1 or PluPRβCD1 (10 mg) was dissolved in DMHZ-buffered water  and stirred at 37 °C for 48 h. NaBHh’,i,i’) a.H (DMSO-d6): 1.04–1.05 , 2.54 , 3.25 , 3.40–3.52 , 3.64–3.82 , 4.34 , 4.83 , 5.59   b.PRATs were fabricated according to a procedure described in our previous study . Brieflyp < 0.01 and p < 0.05 were considered significant.A Micro Autograph MST-X HS/HR (SHIMADZU) was employed for the tensile testing. The fabricated thread samples were immersed in a 50 mM phosphate buffer (pH = 7) for 3 min before tensile testing under wet conditions. After the thread samples affixed to the polypropylene sheet were secured onto the measuring apparatus, the frame was severed, initiating the measurement process. The thread was pulled upward under a mist of water at the rate of 2 mm/min using the top jig connected to the 10 N load cell. The stress–strain curve of the sample was monitored in real time until the thread sample was broken. Statistical significance of the experimental data was assessed by Tukey’s test, where 1H NMR measurement was performed on an ECS-400 spectrometer (JEOL). The powder PR sample (~5 mg) was dissolved in DMSO-d6 and employed for the 1D measurement at 293 or 318 K. The 1H NMR spectrum was obtained using 160 scans with a relaxation delay of 5 s.−1).Fourier-transform infrared (FT–IR) measurements of the synthesized PRs were performed using an IRAffinity-1S spectrometer (SHIMADZU). Powder PR samples were mixed and milled with potassium bromide (KBr), and the resulting powder mixture was fabricated into a pellet. The pellet was subjected to a transmittance measurement in the infrared region  was utilized to monitor the thermal decomposition of the samples during thermogravimetric analysis (TGA). The powder sample (~10 mg) was weighed on a platinum pan and placed in the sample chamber. The residual weight was monitored in real time while the sample was heated from 25 to 700 °C at the heating rate of 10 °C/min under N10kPRαCD1 and Peg20kPRαCD1 were synthesized from the hydroxyl group-terminated Peg via a typical Gabriel amine synthesis process, pseudo-PR formation, terminal amide coupling by adamantane carboxylic acid, and catalytic oxidation of the primary alcohol groups of αCD (see 2. Previous research used Dess–Martin Periodinane (DMP) to introduce the aldehyde groups into CDs of PRs, although the DMP potentially produces ketones besides aldehydes [2 redox couple is more suitable than the DMP for the selective oxidation of hydroxymethyl groups of CDs [1H NMR spectroscopy [1H NMR spectrum of PegPRαCD1-DMHZ is shown in 3 protons (k) of DMHZ were detected at 2.54 ppm, while the N–H peak (j) likely overlapped with the peaks of the protons at the Cb, Cb’, Cd, and Cd’ positions. The coverage ratio of 20 mol% was calculated from the ratio between the peaks obtained for the Peg units and the methyl groups. Moreover, the number of DMHZ molecules was consistent with that of αCD species, indicating that one aldehyde group was introduced per αCD molecule.Pegldehydes ,28,29,30s of CDs . To calctroscopy . Howeverpseudo-PR formation originates from the complexation between polypropylene glycol (Ppg) units and βCD [9kPRβCD1 and Plu15kPRβCD1, we initially attempted to prepare the corresponding pseudo-PRs  from Plu–COOH according to a previously developed procedure [pseudo-PR, the corresponding procedure was not reproduced under the same experimental conditions . Therefore, the preparation conditions were reinvestigated using Plu–OH as a model compound. One gram of Plu–OH was dissolved in a saturated aqueous solution of βCD (56 mL) and stirred at 25 °C for 7 days. A white suspension was obtained during stirring owing to the formation of pseudo-PR. The product was collected via centrifugation and freeze-dried. Further addition of βCD to the supernatant enabled the preparation of more pseudo-PR. The same procedure was applied for Plu–COOH. In total, approximately 0.7 g of PluPRβCD3 was obtained from the initial amount of Plu–COOH. The corresponding 1H NMR spectra were used to calculate the coverage ratio of βCD with respect to the total number of Peg and Ppg units, which was equal to 6 mol%  [9kPRβCD1 and Plu15kPRβCD1 significantly enhance the mechanical properties of the PRATs. Plu9kPRβCD1 and Plu15kPRβCD1 further increased the fracture stress by approximately 2.3 times as compared with that of PegPRαCD1, maintaining the fracture strain  . Both the., Peg) . Note the., Peg) . Therefoe strain b. Conseq1H NMR, FT–IR, and TGA. Interestingly, the results of tensile testing demonstrated that coverage ratio was the main factor affecting the mechanical properties of the PRATs, whereas molecular weight produced a negligible effect on these properties. It is noteworthy that the effects of PRs on the PRAT properties potentially depend on the alignment of atelocollagen molecules. Although further investigations are required to support our conclusions and to further improve the mechanical properties for practical use in humans, the results of this study offer insights into the fundamental properties and applications of PR-reinforced biomaterials.In this study, we investigated the effects of the molecular weight and coverage ratio of PRs on the mechanical properties of the produced PRATs. A novel method was established for the syntheses of PRs with relatively low coverage ratios. The obtained PRs were successfully characterized by