Intact Escherichia coli ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass͞charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg 2؉ concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 ؎ 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components. In recent years mass spectrometry has become the method of choice for a number of important aspects of experimental structural biology. These include: the characterization of the stability and folding behavior of proteins under a wide range of conditions (1-3), the identification of subpicomole quantities of proteins from two-dimensional gels (4), the de novo sequencing of peptides and subsequent cloning of novel proteins (5), and the analysis of the components of single vesicles (6). These applications have driven mass spectrometry to new levels of detection from a range of complex biological matrices. In addition, highresolution ion cyclotron resonance mass spectrometry has enabled the isolation of individual ions from polyethylene glycol (7) and DNA (8), with masses in excess of 10 8 Da. As well as being able to characterize highly charged polymers, it has been possible to detect signals from noncovalent complexes of small molecule ligands bound to proteins and of multiprotein complexes (9, 10). As the size of such assemblies increases, however, the number of charges acquired during the electrospray process increases less rapidly than the total mass. This phenomenon has been attributed to ionic interactions in the intermolecular interfaces and the appropriation of negatively charged counterions from the volatile buffers used in the analysis of such species (11). The net result of these effects is that large multimolecular complexes, such as the 2.3-MDa ribosome, have been outside the mass range of conventional mass spectrometers. In previous mass spectrometry experiments disruption of the ribosome enabled the identification of the contact sites between ribosomal proteins and RNA (12) and a novel protein component from the Saccharomyces cerevsiae ribosome (13). In add...
A new approach is described to gain further information concerning the ribosomal components involved in the peptidyltransferase (PTF) activity exerted by Escherichia coli 50S subunits. A particle is reconstituted from highly purified proteins and RNA under modified incubation conditions. This particle contains only 16 out of the 34 distinct components constituting the native subunit, and yet still exhibits significant PTF activity. Single omission tests at the level of this “minimal ribosomal particle” indicate the limits set on a further reduction of the components, and in particular reveal that protein L18 can be excluded from the set of proteins which are essential for PTF activity, thus leaving L2, L3, L4, L15, and L16 as primary candidates for this function. 5S RNA is not needed for PTF activity of the “minimal ribosomal particle”. Furthermore, a buffer condition is described which drastically improves the stability of total protein preparations and facilitates the isolation of individual proteins.
1.The 43 S precursor of the 50 S ribosomal subunit shows a protein pattern very similar to that of a "core" particle derived from 50 S subunits on CsCl gradients.2. A comparison of the protein patterns of "core" particles from 50 S subunits after CsCl equilibration runs or incubation with increasing LiCl concentrations over the molarity range 0.4-6 M revealed that the proteins are successively released from the "core" in five distinct groups.3. The protein pattern of the 21 S precursor of 30 S ribosomal subunits was compared with that of the reconstituted intermediate particle and LiC1-treated "core" particles of 30 S subunits. The 21 S is similar to both the reconstituted intermediate and the "core" particles derived by a 1 M LiCl treatment, whereas the reconstituted intermediate and this core particle differ rather more. Those proteins which are needed to form the complete 30 S from the 21 S precursor appear to be very similar to those which are split from the mature 30 S in high salt [8-lo]. When 50 S ribosomes are treated with CsCl [8,11,12] or LiCl [13][14][15][16], the proteins are removed in discrete stages. It was suggested that this cooperative dissociation reflects the mechanism of assembly in vivo [15,16].We have used the two-dimensional polyacrylamide electrophoresis system [I71 to separate the protein components of various artificial and naturally occurring ribosomal intermediates. I n this paper we present our results, comparing (a) the 21 S biosynthetic precursor, the reconstituted intermediate particle, and the 30 S LiCl core particles; and (b) the 43 S biosynthetic precursor with the LiCl and CsCl core particles from 50 S.
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