Structure-fluorescence correlations in a single @entity2340 mutant of XXXX parvalbumin: solution structure, backbone and side-chain dynamics.

@entity104 @entity2340 @entity460 @entity7136 @entity72386 @entity13140
Heterogeneous fluorescence intensity decays of @entity2340 in proteins are often rationalized using a model which proposes that different rotameric states of the @entity104 alanyl side-chain are responsible for the observed fluorescence lifetime heterogeneity. We present here the study of a mutant of @entity7136 parvalbumin bearing a single @entity2340 residue at position 102 ( @entity72386 ) whose fluorescence intensity decay is heterogeneous and assess the applicability of a rotamer model to describe the fluorescence decay data. We have determined the solution structure of @entity72386 in the @entity460 ligated state using multi-dimensional nuclear magnetic resonance (NMR) and have used the minimum perturbation mapping technique to explore the possible existence of multiple conformations of the @entity104 moiety of Trp102 of @entity72386 and, for comparison, Trp48 of holo-azurin. The maps for parvalbumin suggest two potential conformations of the @entity104 side-chain. The high energy barrier for rotational isomerization between these conformers implies that interwell rotation would occur on time-scales of milliseconds or greater and suggests a rotamer basis for the heterogeneous fluorescence. However, the absence of alternate Trp102 conformers in the NMR data (to within 3 % of the dominant species) suggests that the heterogeneous fluorescence of Trp102 may arise from mechanisms independent of rotameric states of the Trp side-chain. The map for holo-azurin has only one conformation, and suggests a rotamer model may not be required to explain its heterogeneous fluorescence intensity decay. The backbone and Trp102 side-chain dynamics at 30 degrees C of @entity72386 has been characterized based on an analysis of @entity13140 NMR relaxation data which we have interpreted using the Lipari-Szabo formalism. High order parameter (S(2)) values were obtained for both the helical and loop regions. Additionally, the S(2) values imply that the @entity460 binding CD and EF loops are not strictly equivalent. The S(2) value for the @entity104 side-chain of Trp102 obtained from the fluorescence, NMR relaxation and minimum perturbation data are consistent with a Trp moiety whose motion is restricted.
