Skip Navigation

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by ARAI, K.-i.
Right arrow Articles by KAZIRO, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by ARAI, K.-i.
Right arrow Articles by KAZIRO, Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

J. Biochem, 1977, Vol. 81, No. 5 1335-1346
© 1977 Japanese Biochemical Society

research-article

Further Studies on the Properties of the Polypeptide Chain Elongation Factors Tu and Ts: Hydrogen-Tritium Exchange, Optical Rotatory Dispersion, and Intrinsic Fluorescence

Ken-ichi ARAI, Takao ARAI, Masao KAWAKITA and Yoshito KAZIRO

Institute of Medical Science, University of Tokyo Takanawa, Minato-ku, Tokyo 108

The conformation and conformational stability of the polypeptide chain elongation factors Tu (EF-Tu) and Ts (EF-Ts) have been investigated by means of hydrogen-tritium exchange, optical rotatory dispersion and fluorescence spectroscopy, and the following results were obtained.

  1. Free EF-Tu not liganded with guanine nucleotide rapidly exchanges its bound tritium atoms with hydrogen atoms in the medium, only 12 tritium atoms being retained in the protein after back-exchange for 10 h at 10°C. The rate of exchange was found to be markedly reduced by guanine nucleotides. Thus, in the presence of 20 µM GDP or 50 µM GTP, as many as 40 tritium atoms were retained under the same conditions. Even at 22°C, where the exchange in free EF-Tu becomes exceedingly rapid, about 25 tritium atoms remained associated with the protein after incubation for about 6 h. The kinetic distribution of very slowly exchanging hydrogen atoms, estimated by interrupted-flow gel filtration, was almost identical with both EF-Tu·GDP and EF-Tu·GTP.
  2. From the optical rotatory dispersion spectra, the {alpha}-helical contents of EF-Tu·GDP, EF-Ts, and EF-Tu·EF-Ts were estimated to be 24, 52, and 33%, respectively. The spectra of EF-Tu·GDP and EF-Tu·GTP were almost identical over the range of wavelength from 220 to 350 nm. The reduced mean residue rotation at 233 nm of free EF-Tu decreased in the presence of guanine nucleotide ligands.
  3. The intrinsic fluorescence of tryptophan is markedly quenched in EF-Tu·GDP and EF-Tu·EF-Ts in their native conformation. The fluorescence emission at 340 nm characteristic of tryptophan increased about 30 percent as a result of conversion of EF-Tu·GDP to EF-Tu·GTP or free EF-Tu without any accompanying change in fluorescence from tyrosine. Upon denaturation of the protein, fluorescence of tryptophan further increased with a shift of the emission maximum to 335 nm.

These results indicate that the binding of GDP or GTP causes a gross conformational change in EF-Tu, stabilizing its secondary and tertiary structure, increasing its {alpha}-helical content, and reducing the motility of the protein core. The shielding of tryptophan fluorescence is more marked in EF-Tu·GDP than in EF-Tu·GTP, indicating that the conformations of these two forms of EF-Tu are different.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.