William E. Running

A Non-Nephrotoxic Gentamicin Congener That Retains Antimicrobial Efficacy

Aminoglycoside antibiotics, although of major clinical importance in the treatment of serious Gram- negative infections and a potential therapeutic agent in the amelioration of diseases that are characterized by premature stop mutations, are associated with a high incidence of acute renal failure. With the use of HPLC techniques, the four components (congeners) of gentamicin, the most commonly used aminoglycoside, were isolated and characterized. Described here is a congener with minimal cytotoxicity in cell culture and animal studies that retained normal bactericidal properties in both Bacillus subtilis and a multidrug-resistant form of Klebsiella pneumoniae. Furthermore, in animal studies, this congener failed to induce the functional and pathologic changes that are characteristic of gentamicin nephrotoxicity that is seen with the native compound. Finally, internalization of this non-nephrotoxic component was unaltered, but the subcellular distribution was different from native gentamicin or the other three cytotoxic congeners. These studies have identified a component of the native gentamicin congener mixture that retains its bactericidal properties with minimal or no apparent nephrotoxicity.

Ribosomal Proteins of Deinococcus radiodurans: Their Solvent Accessibility and Reactivity

The structure of proteins in native ribosomes from Deinococcus radiodurans R1 was probed by S-methylthioacetimidate (SMTA) modification of amino groups. The extent of protein labeling was quantified using top down methods, and modified positions were identified using bottom up experiments. Each proteins reactivity was predicted by examination of the crystal structures of the D. radiodurans 50S subunit and the T. thermophilus HB8 30S subunit. The close phylogenetic relation between D. radiodurans and T. thermophilus allowed the evaluation of D. radiodurans small subunit protein reactivity by alignment of homologous sequences. As a result, we were able to observe and characterize the reactivity of all of D. radiodurans ribosomal proteins. The extent of protein amidination was well correlated with the solvent-exposed surface area of each protein and even better correlated with the number of visible lysine residues. Lysine residues that are in close contact with rRNA structural features or buried in protein tertiary structure are nonreactive with SMTA, while those that are surface exposed are modified. Crystallographic disorder and post-translational modifications lead to differences between the observed and predicted extents of reactivity. Comparison of unmodified and disassembled amidinated protein mixtures also shows great promise for the quality control of the proteomic sequences and has facilitated the identification of four sequencing errors in the ribosomal proteome of D. radiodurans R1.

B. subtilis ribosomal proteins: structural homology and post-translational modifications.

Ribosomal proteins of the model gram-positive bacterium B. subtilis 168 were extensively characterized in a proteomic study. Mass spectra of the 52 proteins expected to be constitutive components of the 70S ribosome were recorded. Peptide MS/MS analysis with an average sequence coverage of 85% supported the identification of these proteins and facilitated the unambiguous assignment of post-translational modifications, including the methylation of S7, L11, and L16 and the N-terminal acetylation of S9. In addition, the high degree of structural homology between B. subtilis and other eubacterial ribosomal proteins was demonstrated through chemical labeling with S-methylthioacetimidate. One striking difference from previous characterizations of bacterial ribosomal proteins is that dozens of protein masses were found to be in error and not easily accounted for by post-translational modifications. This, in turn, led us to discover an inordinate number of sequencing errors in the reference genome of B. subtilis 168. We have found that these errors have been corrected in a recently revised version of the genome.

Ratiometric pulse-chase amidination mass spectrometry as a probe of biomolecular complex formation.

Selective chemical modification of protein side chains coupled with mass spectrometry is often most informative when used to compare residue-specific reactivities in a number of functional states or macromolecular complexes. Herein, we develop ratiometric pulse-chase amidination mass spectrometry (rPAm-MS) as a site-specific probe of lysine reactivities at equilibrium using the Cu(I)-sensing repressor CsoR from Bacillus subtilis as a model system. CsoR in various allosteric states was reacted with S-methyl thioacetimidate (SMTA) for pulse time, t, and chased with excess of S-methyl thiopropionimidate (SMTP) (Δ = 14 amu), quenched and digested with chymotrypsin or Glu-C protease, and peptides were quantified by high-resolution matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and/or liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). We show that the reactivities of individual lysines from peptides containing up to three Lys residues are readily quantified using this method. New insights into operator DNA binding and the Cu(I)-mediated structural transition in the tetrameric copper sensor CsoR are also obtained.

Chemical Reactivity of Brome Mosaic Virus Capsid Protein

Viral particles are biological machines that have evolved to package, protect, and deliver the viral genome into the host via regulated conformational changes of virions. We have developed a procedure to modify lysine residues with S-methylthioacetimidate across the pH range from 5.5 to 8.5. Lysine residues that are not completely modified are involved in tertiary or quaternary structural interactions, and their extent of modification can be quantified as a function of pH. This procedure was applied to the pH-dependent structural transitions of brome mosaic virus (BMV). As the reaction pH increases from 5.5 to 8.5, the average number of modified lysine residues in the BMV capsid protein increases from 6 to 12, correlating well with the known pH-dependent swelling behavior of BMV virions. The extent of reaction of each of the capsid proteins lysine residues has been quantified at eight pH values using coupled liquid chromatography-tandem mass spectrometry. Each lysine can be assigned to one of three structural classes identified by inspection of the BMV virion crystal structure. Several lysine residues display reactivity that indicates their involvement in dynamic interactions that are not obvious in the crystal structure. The influence of several capsid protein mutants on the pH-dependent structural transition of BMV has also been investigated. Mutant H75Q exhibits an altered swelling transition accompanying solution pH increases. The H75Q capsids show increased reactivity at lysine residues 64 and 130, residues distal from the dimer interface occupied by H75, across the entire pH range.

Variation of the chemical reactivity of Thermus thermophilus HB8 ribosomal proteins as a function of pH.

Ribosomes occupy a central position in cellular metabolism, converting stored genetic information into active cellular machinery. Ribosomal proteins modulate both the intrinsic function of the ribosome and its interaction with other cellular complexes, such as chaperonins or the signal recognition particle. Chemical modification of proteins combined with mass spectrometric detection of the extent and position of covalent modifications is a rapid, sensitive method for the study of protein structure and flexibility. By altering the pH of the solution, we have induced non‐denaturing changes in the structure of bacterial ribosomal proteins and detected these conformational changes by covalent labeling. Changes in ribosomal protein modification across a pH range from 6.6 to 8.3 are unique to each protein, and correlate with their structural environment in the ribosome. Lysine residues whose extent of modification increases as a function of increasing pH are on the surface of proteins, but in close proximity either to glutamate and aspartate residues, or to rRNA backbone phosphates. Increasing pH disrupts tertiary and quaternary interactions mediated by hydrogen bonding or ionic interactions, and regions of protein structure whose conformations are sensitive to these changes are of potential importance in modulating the flexibility of the ribosome or its interaction with other cellular complexes.

In Vitro and In Vivo Chemical Labeling of Ribosomal Proteins: A Quantitative Comparison

Thioimidates have emerged as reagents for probing the protein structure, folding, and interactions under physiological conditions. The same properties that give thioimidates biological relevance make these molecules ideal candidates for use in vivo. Through labeling of ribosomal proteins, we have quantified the in vivo and in vitro reactivity of two thioimidates: S-methylthioacetimidate (SMTA) and a novel, charge-carrying analogue, S-sulfethylthioacetimidate (SSETA). In vitro experiments demonstrate that both amidinating reagents can probe the protein structure. Under comparable in vivo conditions, SMTA is found to be membrane-permeable while SSETA is not. The use of mass spectrometry with permeant and impermeant thioimidates promises insights into the membrane topology and protein structure in the native environment.

Analysis of Methylation, Acetylation, and other Modifications in Bacterial Ribosomal Proteins

A wide variety of post-translational modifications of expressed proteins are known to occur in living organisms (1). Although their presence in an organism cannot be predicted from the genome, these modifications can play critical roles in protein structure and function. The identification of post-translational modifications can be critical in understanding the functions of proteins involved in important biological pathways and mass spectrometry offers a fast, accurate method for observing them. This chapter describes the procedure for analyzing ribosomal proteins of Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and Caulobacter crescentus ribosomal proteins by electrospray quadrupole time-of-flight (ESI-QTOF) mass spectrometry.