| Abstract
| - A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probesAlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformationalheterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integratedFRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance betweendomains of CaM-DA. We recently reported distinct conformational substates of Ca2+-CaM-DA andapoCaM-DA, with peaks in the distance distributions centered at ∼28 Å, 34−38 Å, and 55 Å [Slaughteret al. (2004) J. Phys. Chem. B108, 10388−10397]. In the present study, shifts in the amplitudes andcenter distances of the conformational substates were detected with variation in solution conditions. Theamplitude of an extended conformation was observed to change as a function of Ca2+ over a free Ca2+range that is consistent with binding to the high affinity, C-terminal Ca2+ binding sites, suggesting theexistence of communication between lobes of CaM. Lowering pH shifted the relative amplitudes of theconformations, with a marked increase in the presence of the compact conformations and an almost completeabsence 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 inconformational heterogeneity relative to apoCaM-DA at physiological ionic strength. Oxidation ofmethionine residues in CaM-DA produced a substantial increase in the amplitude of the extendedconformation relative to the more compact conformation. The results are considered in light of a hypothesisthat suggests that electrostatic interactions between charged amino acid side chains play an importantrole in determining the most stable CaM conformation under varying solution conditions.
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