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National Biomedical Center
for Advanced Electron Spin Resonance Technology

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Distance Distributions in Protein Folding as Studied by Pulsed ESR: Cytochrome C

One of the major issues in structural biology is to understand the dynamic conformations of proteins as they fold into or unfold from their tertiary and secondary structures. A complete study of dynamic protein conformation consists of the kinetics of folding/unfolding and the conformations of possible intermediates. The former has been studied by Scholes et al using rapid-mix flow ESR techniques on the heme-containing protein iso-1-cytochrome c. We have employed pulsed ESR methods to study the pair distributions in its mutant that is double labeled at sites S47C and K79C. The pair distributions were obtained for the folded protein and for partially unfolded states induced by variation in the reagent GdnHCl concentrations. These experiments were performed using ACERT's 17.3 GHz pulse spectrometer configured for four pulse DEER. The time evolution data were then inverted to yield the distance distributions, P(r) utilizing the method of Tikhonov regularization with the L-curve criterion, developed at ACERT. As a complement to this procedure, the method of Maximum Entropy regularization, also developed at ACERT, was also used to obtain P(r); this approach implicitly guarantees a non-negative P(r) and minimum bias in its determination. We show below the distance distributions obtained for iso-cyt-c in its completely folded state (left figure), and in two different partially unfolded states, corresponding to [GdnHCL] of 0.7 M (middle figure) and 1.5 M (right figure). (Note that iso-cyt-c is completely unfolded in 2.0 M GdnHCl). The folded state shows the narrowest distribution with an average of 1.96 nm, whereas those of the partially unfolded states are much broader. For the 0.7 M GdnHCl case, P(r) is much broader and the average distance of 2.78 nm is much larger, in good agreement with expectations. For the even more unfolded case of 1.5 M GdnHCl, the average distance between the labels is even greater, 3.66 nm and the distribution takes on a bimodal appearance with a strong narrow component centered at 4.35 nm and a broader one centered at 2.74 nm. These results, obtained directly from inversion of the DEER dipolar signals, are consistent with the more indirectly obtained distributions from previous FRET measurements. The ESR method used in this study could be combined with rapid freeze methods to provide distance distribution maps of protein unfolding as a function of the temporal stage of unfolding.

Y.-W. Chiang, P. P. Borbat, J. H. Freed (ACERT)
V. Grigoryantz, C. P. Scholes (Department of Chemistry, SUNY Albany).
June, 2005