Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron-electron resonance. Proc Natl Acad Sci U S A 2014 Apr 01;111(13):E1201-10
Date
04/08/2014Pubmed ID
24707053Pubmed Central ID
PMC3977274DOI
10.1073/pnas.1403179111Scopus ID
2-s2.0-84897536162 (requires institutional sign-in at Scopus site) 39 CitationsAbstract
The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higher-energy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Site-directed spin labeling in combination with double electron-electron resonance (DEER) provides long-range (20-80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the high-pressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0-3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure- and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter.
Author List
Lerch MT, Yang Z, Brooks EK, Hubbell WLAuthor
Michael Lerch PhD Associate Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsApoproteins
Electron Spin Resonance Spectroscopy
Electrons
Freezing
Hydrogen-Ion Concentration
Hydrostatic Pressure
Models, Molecular
Myoglobin
Protein Structure, Secondary
Sperm Whale
Spin Labels
