Conformational Substates of Calmodulin Revealed by Single-Pair Fluorescence Resonance Energy Transfer: Influence of Solution Conditions and Oxidative Modification

摘要

A calmodulin(CaM)mutant(T34,110C-CaM)doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains(CaM-DA)has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer(spFRET).Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains of CaM-DA.We recently reported distinct conformational substates of Ca~(2+)-CaM-DA and apoCaM-DA,with peaks in the distance distributions centered at ~28 A,34-38 A,and 55 A [Slaughter et al.(2004)J.Phys.Chem.B 108,10388-10397].In the present study,shifts in the amplitudes and center distances of the conformational substates were detected with variation in solution conditions.The amplitude of an extended conformation was observed to change as a function of Ca~(2+)over a free Ca~(2+)range that is consistent with binding to the high affinity,C-terminal Ca~(2+)binding sites,suggesting the existence of communication between lobes of CaM.Lowering pH shifted the relative amplitudes of the conformations,with a marked increase in the presence of the compact conformations and an almost complete absence of the extended conformation.In addition,the single-molecule distance distribution of apoCaM-DA at reduced ionic strength was shifted to longer distance and showed evidence of an increase in conformational heterogeneity relative to apoCaM-DA at physiological ionic strength.Oxidation of methionine residues in CaM-DA produced a substantial increase in the amplitude of the extended conformation relative to the more compact conformation.The results are considered in light of a hypothesis that suggests that electrostatic interactions between charged amino acid side chains play an important role in determining the most stable CaM conformation under varying solution conditions.