| Autor: |
Sanjay M. Sonar, Cheryl F.C. Ludlam, Uttam L. RajBhandary, Kenneth J. Rothschild, Matthew A. Coleman, T Marti, Chan Ping Lee, Xiao Mei Liu |
| Rok vydání: |
1996 |
| Předmět: |
|
| DOI: |
10.1016/s1080-8914(96)80019-6 |
| Popis: |
Publisher Summary This chapter introduces a method for assigning bands in Fourier transform infrared (FTIR)-difference spectra based on a technique termed as site-directed isotope labeling (SDIL). The key element in SDIL is the use of a suppressor tRNA aminoacylated with an isotopically labeled amino acid. This tRNA is targeted to insert the isotopic amino acid at the proper position in the nascent protein by using an amber codon at the corresponding position in the gene. Cell-free synthesis (in vitro translation) and exogenous addition of the aminoacylated suppressor tRNA prevent aminoacylation of non-suppressor tRNAs with the isotopic amino acid, similar to the approach used for site-directed non-native amino acid replacement. The integral membrane protein bacteriorhodopsin (bR) was chosen as a model system for demonstrating the application of SDIL-FTIR. Studies on bacteriorhodopsin demonstrate that the FTIR-SDIL approach can probe the local environment and structural changes of specific residues and backbone carbonyl groups in a protein. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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