Time-Resolved Fluorescence Studies On Components Of The Prokaryotic Protein Elongation System

Theodore L Hazlett; David M Jameson

doi:10.1117/12.945421

24 June 1988 Time-Resolved Fluorescence Studies On Components Of The Prokaryotic Protein Elongation System

Theodore L Hazlett, David M Jameson

Proceedings Volume 0909, Time-Resolved Laser Spectroscopy in Biochemistry; (1988) https://doi.org/10.1117/12.945421
Event: 1988 Los Angeles Symposium: O-E/LASE '88, 1988, Los Angeles, CA, United States

Abstract

The hydrodynamics of aminoacyl-tRNA free and bound to protein elongation factor Tu (EF-Tu) were examined using multifrequency phase and modulation fluorometry. The probes fluorescein, covalently linked to Phe-tRNAphe (Phe-tRNAphe-F8), and ethidium bromide, noncovalently bound to Phe-tRNAphe, were utilized. At 5°C the Debye rotational relaxation times found for ethidium bromide bound to tRNA and to the complex, EF-TuPhe-tRNAphe, were 136 ns and 195 ns, respectively. Both values are longer than would be expected for spheres of equal volume and reflect the irregular shapes of the tRNA and ternary complexes. Global rotational relaxation times (5°C) for Phe-tRNAphe-F8 free and bound to EF-TuGTP were 103 ns and 206 ns, respectively, consistent with the ethidium bromide results. A second, faster rotational relaxation time of approximately 4.7 ns, presumably due to local structural motion of the fluorescein, was unchanged upon ternary complex formation. This result suggests that the local mobility of the bound fluorescein is not hindered by the presence of the protein, and it is therefore unlikely that the region of the tRNA which includes the fluorescein-labeled residue S4-U-8, interacts closely with EF-Tu in the ternary complex. The dynamic polarization measurements on the ethidium bromide/tRNA system also demonstrated the interesting phenomenon of anomalous phase delays, an effect which is quite sensitive to small amounts of free or bound probe.

Citation Download Citation

Theodore L Hazlett and David M Jameson "Time-Resolved Fluorescence Studies On Components Of The Prokaryotic Protein Elongation System", Proc. SPIE 0909, Time-Resolved Laser Spectroscopy in Biochemistry, (24 June 1988); https://doi.org/10.1117/12.945421

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available