Fluorescence Spectroscopic Approaches to the Study of Three-dimensional Structure of Ribosomes

Abstract

INTRODUCTION
Fluorescence spectroscopy has a number of attractive features for the study of a system as large and complex as the ribosome. Because fluorescent probe molecules are individually or selectively introduced, one can concentrate attention on a small portion of the whole structure. The great sensitivity of the technique means that one can conveniently work with 10⁻⁶–10⁻⁷ molar solutions in sample volumes as small as 0.2 ml. At least three different kinds of information can be obtained from fluorescence studies:

(1) Environmentally sensitive probes such as ethidium bromide (EB) and anilinonaphthalene sulfonate (ANS) are especially useful for monitoring conformational changes.

(2) Polarization or decay of fluorescence anisotropy allow motions and flexibility to be examined.

(3) Energy transfer between sets of attached fluorescent dyes enable the measurement of distances between fixed points.

All of these techniques are well established and have been developed and tested on simpler biochemical systems (for recent reviews, see Steiner and Weinryb 1971; Cantor and Tao 1971; Cantor and Timasheff 1974; Stryer 1968; Brand and Gohlke 1972). A few additional complications arise when a system the size of a ribosome is involved. Here we shall examine these difficulties, survey some of the results of previous fluorescence studies on ribosomes, and attempt to chart the course of possible future studies.

Choice of Fluorescent Probes
All fluorescence studies demand the presence of fluorescent moieties. Their selection and specific introduction is frequently the major obstacle to meaningful experiments. The natural fluorescence of ribosomes arises principally from aromatic amino acid...