Results are presented showing how photoaffinity labeling can be used to identify ribosomal components at functional sites within the Escherichia coli ribosome. Following brief overviews of ribosomal structure and function and of the use of photoaffinity labeling to study complex biological macrostructures, results obtained in the authors' laboratory, principally with the ribosomal antibiotic puromycin and with an aryl azide derivative of puromycin, are presented to illustrate procedures used to identify photoaffinity-labeled ribosomal components, to determine whether such labeling occurs at a functionally important site, and to localize sites of labeling in a three-dimensional sense.

A convenient method for protein estimation is described, making use of uv detectors and peak integrators that are standard equipment on modern high-performance liquid chromatographs to determine the product of integrated peak area and flow rate of eluting protein at 214 nm (AF214). We demonstrate that AF214 is proportional to the amount of eluted protein and describe two approaches for calibrating the integrator, by quantitative amino acid analysis and by determining the elution yield of a known amount of applied protein, allowing direct estimation of protein from AF214. Both approaches yield similar results. The basis for the method is that, for virtually all proteins, absorbance at 214 nm is dominated by the summed contributions from the peptide groups. More accurate estimates can be made when the amino acid composition of the eluting protein is known, since this permits a correction to be made for contributions of amino acid side chains to absorbance at 214 nm. Comparison of AF214 estimates for proteins from the small (30 S) subunit of the Escherichia coli ribosome with those obtained by Bradford analysis shows the latter to give somewhat higher values.

Publisher Summary This chapter focuses on the reversed-phase high-performance liquid chromatography (RP-HPLC) of ribosomal proteins. Higher resolution and sensitivity are obtainable with RP-HPLC, but ion-exchange high-performance liquid chromatography (IE-HPLC) provides higher capacity. Because the bases for separation are quite different, the order of ribosomal protein elution differs markedly for RP-HPLC vs. IE-HPLC. As a result, the availability of both kinds of column allows straightforward solutions to problems that can arise with the use of a single column, such as the separation of coeluting proteins, or the definitive identification of a modified protein that elutes slightly differently than the unmodified protein. RP-HPLC has proved to be extraordinarily useful for both the analysis and preparation of ribosomal proteins. Analytically, it has been used to compare the protein composition of native and reconstituted ribosomal subunits to compare ribosomal proteins obtained from different bacterial strains, and to identify covalently modified proteins, derivatized either in an enzyme-catalyzed process (methylation or phosphorylation) or via affinity labeling.

We are currently utilizing reversed-phase high-performance liquid chromatography (RP-HPLC) in reconstitution experiments designed to study the structure and functions of Escherichia coli ribosomes. The applications of RP-HPLC in these experiments include: (a) preparation of individual proteins or groups of proteins on a milligram scale for reconstitution pools, (b) analysis of the protein stoichiometry of reconstituted subunits, (c) determination of the extent and specificity of modification of proteins extracted from ribosomal subunits which have been subjected to chemical modification, and (d) resolution of modified forms of proteins S14 and L23 from the corresponding unmodified proteins. Proteins prepared by RP-HPLC from 30S and 50S ribosomal subunits were found to reconstitute into 30S and 50S subunits respectively, as well as into slower sedimenting particles. The reconstituted subunits contain a full complement of proteins and are active in ribosomal function assays, whereas the slower sedimenting particles lack several proteins and have little or no activity.

We have previously reported the application of reversed-phase high-performance liquid chromatography (RP-HPLC) to the separation of Escherichia coli ribosomal proteins (A. R. Kerlavage, L. Kahan and B. S. Cooperman, AnaL Biochem., 123 (1982) 342-348; A. R. Kerlavage, T. Hasan and 13. S. Cooperman, J. Biol. Chem., in press). In the present studies RP-HPLC is shown to yield much greater resolution of these proteins than does size-exclusion HPLC. In addition, we report on various aspects of RP-HPLC of ribosomal proteins including column capacity, resolution, reproducibility, recovery, separation of irreversibly denatured protein, and analysis of affinity-labeled ribosomal protein. The capacity of analytical columns was found to range from several micrograms to several milligrams with minimal loss in resolution and highly reproducible retention values. Recovery varied from protein to protein and ranged from 27 % to 91 %, with an average total protein recovery of 70 %. The partitioning of several proteins between two peaks was shown to be due to irreversible denaturation of a small fraction. Finally, the utility of RP-HPLC in the studv of the ribosome was demonstrated by analyses of [3H]puromyein-labeled ribosomal proteins, and the demonstration that labeling slightly alters protein elution.