Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization–enhanced solid-state NMR

Proceedings of the National Academy of Sciences of the United States of America - Tập 106 Số 23 - Trang 9244-9249 - 2009
Vikram S. Bajaj1, Melody L. Mak–Jurkauskas1,2, Marina Belenky2, Judith Herzfeld2, Robert G. Griffin1
1Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139; and
2Department of Chemistry, Brandeis University, Waltham, MA 02454

Tóm tắt

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 ≈90. Multidimensional spectroscopy of U- 13 C, 15 N-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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Thông tin xuất bản

Proceedings of the National Academy of Sciences of the United States of America

Tập 106 Số 23

9244-9249

Thông tin tác giả