Substrate-induced changes in domain interaction of vacuolar H⁺-pyrophosphatase.
Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H(+)-pyrophosphatase (EC 3.6.1.1) from Vigna radiata. H(+)-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PPi hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H(+)-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H(+)-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was ∼4 nm in length, ∼2 nm in width, and ∼1 nm in protrusion height. Upon extracting the H(+)-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H(+)-pyrophosphatase in the presence and absence of substrate and its analog.
['Hsu|Shen-Hsing|SH|', 'Lo|Yueh-Yu|YY|', 'Liu|Tseng-Huang|TH|', 'Pan|Yih-Jiuan|YJ|', 'Huang|Yun-Tzu|YT|', 'Sun|Yuh-Ju|YJ|', 'Hung|Cheng-Chieh|CC|', 'Tseng|Fan-Gang|FG|', 'Yang|Chih-Wei|CW|', 'Pan|Rong-Long|RL|']
D007887:Fabaceae D006868:Hydrolysis D043564:Inorganic Pyrophosphatase D017136:Ion Transport D007700:Kinetics D008051:Lipid Bilayers D018625:Microscopy, Atomic Force D017434:Protein Structure, Tertiary D011522:Protons D013379:Substrate Specificity D014617:Vacuoles
Force-Distance Curve Atomic Force Microscopy (AFM) Bioenergetics Membrane Transporter Reconstitution Protein-Protein Interaction Proton Pump Vacuolar H+-pyrophosphatase