Osmolyte perturbation reveals conformational equilibria in spin-labeled proteins.
Recent evidence suggests that proteins at equilibrium can exist in a manifold of conformational substates, and that these substates play important roles in protein function. Therefore, there is great interest in identifying regions in proteins that are in conformational exchange. Electron paramagnetic resonance spectra of spin-labeled proteins containing the nitroxide side chain (R1) often consist of two (or more) components that may arise from slow exchange between conformational substates (lifetimes > 100 ns). However, crystal structures of proteins containing R1 have shown that multicomponent spectra can also arise from equilibria between rotamers of the side chain itself. In this report, it is shown that these scenarios can be distinguished by the response of the system to solvent perturbation with stabilizing osmolytes such as sucrose. Thus, site-directed spin labeling (SDSL) emerges as a new tool to explore slow conformational exchange in proteins of arbitrary size, including membrane proteins in a native-like environment. Moreover, equilibrium between substates with even modest differences in conformation is revealed, and the simplicity of the method makes it suitable for facile screening of multiple proteins. Together with previously developed strategies for monitoring picosecond to millisecond backbone dynamics, the results presented here expand the timescale over which SDSL can be used to explore protein flexibility.
López
Carlos J
CJ
Department of Chemistry and Biochemistry, Jules Stein Eye Institute, University of California, Los Angeles, California 90095-7008, USA.
Fleissner
Mark R
MR
Guo
Zhefeng
Z
Kusnetzow
Ana K
AK
Hubbell
Wayne L
WL
D000818:Animals; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry*; D005362:Ficoll / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry*; D011487:Protein Conformation; D051381:Rats; D012243:Rhodopsin / Q000737:chemistry*; D050661:Sperm Whale; D013113:Spin Labels; D013395:Sucrose / Q000737:chemistry
D050556:Fatty Acid-Binding Proteins; D009211:Myoglobin; D013113:Spin Labels; D005362:Ficoll; D013395:Sucrose; D012243:Rhodopsin; D009113:Muramidase