期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 106, 期 23, 页码 9244-9249出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0900908106
关键词
magic-angle spinning; photocycle intermediate; retinal protein; ion transport; DNP
资金
- NIBIB NIH HHS [R01 EB001960, R01 EB001035, R01 EB002804, P41 EB002026, EB-001960, EB002026, EB-002804] Funding Source: Medline
Observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. Here, we use a 250 GHz gyrotron ( cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (DNP) NMR experiments that enhance sensitivity in magic-angle spinning NMR spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (bR) by a factor of approximate to 90. Multidimensional spectroscopy of U-C-13, N-15-labeled samples resolved coexisting states and allowed chemical shift assignments in the retinylidene chromophore for several intermediates not observed previously. The correlation spectra reveal unexpected heterogeneity in dark-adapted bR, distortion in the K state, and, most importantly, 4 discrete L substates. Thermal relaxation of the mixture of L's showed that 3 of these substates revert to bR(568) and that only the 1 substate with both the strongest counterion and a fully relaxed 13-cis bond is functional. These definitive observations of functional and shunt states in the bR photocycle provide a preview of the mechanistic insights that will be accessible in membrane proteins via sensitivity-enhanced DNP NMR. These observations would have not been possible absent the signal enhancement available from DNP.
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