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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.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2002 Mar 19; Vol. 99 (6), pp. 3452-7. - Publication Year :
- 2002
-
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. Third, truncated rhodopsin lacking the C-terminal sequence Asp-330-Ala-348 showed no signal, whereas the enzymatically produced peptide fragment containing the above sequence showed the single peak. The results indicate motion in the backbone amide groups of rhodopsin at time scales depending on their location in the sequence. At the C terminus, conformational averaging occurs at the nanosecond time scale but varies from microsecond to millisecond in other parts of the primary sequence. The motions reflecting conformational exchange may be general for membrane proteins containing transmembrane helical bundles.
- Subjects :
- Amino Acid Sequence
Animals
Antibodies, Monoclonal immunology
Cattle
Cell Line
Detergents
Disulfides metabolism
Humans
Light
Lysine chemistry
Micelles
Models, Molecular
Molecular Sequence Data
Mutation
Protein Structure, Secondary
Rhodopsin genetics
Rhodopsin immunology
Scattering, Radiation
Solutions
Spectrometry, Fluorescence
Temperature
Lysine metabolism
Magnetic Resonance Spectroscopy
Rhodopsin chemistry
Rhodopsin metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0027-8424
- Volume :
- 99
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 11904408
- Full Text :
- https://doi.org/10.1073/pnas.052713999