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

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 polarizat...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2009-06, Vol.106 (23), p.9244-9249
Main Authors: Bajaj, Vikram S, Mak-Jurkauskas, Melody L, Belenky, Marina, Herzfeld, Judith, Griffin, Robert G
Format: Article
Language:English
Subjects:
Antimony
Bacteria
Bacterial proteins
Bacteriorhodopsins
Bacteriorhodopsins - chemistry
Bacteriorhodopsins - metabolism
Biochemistry
Biological Sciences
Carbon Isotopes - metabolism
Chemical equilibrium
Chemical mixtures
Chromophores
Light
Membranes
Microwaves
Nitrogen Isotopes - metabolism
NMR
Nuclear magnetic resonance
Nuclear Magnetic Resonance, Biomolecular - methods
Protons
Retinaldehyde - metabolism
Spectroscopy
Spectrum analysis
Studies
Temperature
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Summary: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 [almost equal to]90. Multidimensional spectroscopy of U-¹³C,¹⁵N-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 bR₅₆₈ 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0900908106