Solution NMR spectroscopy of [alpha -15N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence

Proceedings of the National Academy of Sciences of the United States of America
Judith Klein-SeetharamanH Gobind Khorana

Abstract

[alpha-(15)N]Lysine-labeled rhodopsin, prepared by expression of a synthetic gene in HEK293 cells, was investigated both by conventional and transverse relaxation optimized spectroscopy-type heteronuclear single quantum correlation spectroscopy. Whereas rhodopsin contains 11 lysines, 8 in cytoplasmic loops and 1 each in the C-terminal peptide sequence and the intradiscal and transmembrane domains, only a single sharp peak was observed in dodecyl maltoside micelles. This result did not change when dodecyl maltoside was replaced by octyl glucoside or octyl glucoside-phospholipid-mixed micelles. Additional signals of much lower and variable intensity appeared at temperatures above 20 degrees C and under denaturing conditions. Application of the transverse relaxation optimized spectroscopy sequence resulted in sharpening of resonances but also losses of signal intensity. The single peak observed has been assigned to the C-terminal Lys-339 from the following lines of evidence. First, the signal is observed in HNCO spectra of rhodopsin, containing the labeled [(13)C]Ser-338/[(15)N]Lys-339 dipeptide. Second, addition of a monoclonal anti-rhodopsin antibody that binds to the C-terminal 8 aa of rhodopsin caused disappearance of the peak...Continue Reading

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Citations

Feb 24, 2007·Journal of Biomolecular NMR·Karla WernerH Gobind Khorana
May 10, 2007·Journal of Biomolecular NMR·Lianmei FengJames H Prestegard
Sep 30, 2009·Journal of Biomolecular NMR·Hideo Takahashi, Ichio Shimada
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Apr 13, 2013·Annual Review of Analytical Chemistry·Stanley J Opella
Mar 3, 2004·Proceedings of the National Academy of Sciences of the United States of America·Judith Klein-SeetharamanH Gobind Khorana
Oct 9, 2002·Proceedings of the National Academy of Sciences of the United States of America·Philip J ReevesH Gobind Khorana
Oct 9, 2002·Proceedings of the National Academy of Sciences of the United States of America·Li NiuH Gobind Khorana
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