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

Abstract

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.

Chemical Reactivity of Brome Mosaic Virus Capsid Protein

“…33–34,36,38 The average extent of modification of proteins shows excellent agreement with predictions based on crystal structures, indicating minimal effects of native biomolecular structure. 33–38 …”

“…34–38 In order to extend this technique to different solution conditions, a procedure for the amidination of lysine residues that mitigated the effect of changes in solution pH on labeling efficiency was optimized using RNase A. When subjected to 10 cycles of SMTA modification, the SMTA reactivity of monomeric RNase A is nearly pH independent (Fig.…”

“…The localized molecular interactions responsible for the differential reactivity of K64 and K130 are probably hydrogen bonds or salt bridges, as observed in previous experiments involving SMTA modification of bacterial ribosomal proteins. 34–36,38 Inspection of the BMV capsid protein crystal structure (PDB:1JS9) reveals that the ε-amino group of K64 (red) is within 4.4 Å of one of the carboxyl oxygens of E160 (orange). If allowance is made for conformational flexibility of lysine and glutamate side chains (Fig.…”

“…We have used thioimidate reagents such as S-methylthioacetimidate (SMTA) to probe the structure of soluble proteins and the proteins of the bacterial ribosome. 32–38 Lysines that are protected from SMTA modification are buried at the interface of protein-protein or protein-nucleic acid interactions, or are involved in ionic orhydrogen bonding interactions with other amino acid residues. 33–34,36,38 The average extent of modification of proteins shows excellent agreement with predictions based on crystal structures, indicating minimal effects of native biomolecular structure.…”

Mapping Protein Surface Accessibility via an Electron Transfer Dissociation Selectively Cleavable Hydrazone Probe

“…These interactions can be studied through this covalent modification strategy based on monitoring the differential reactivity of selected residues in the presence/absence of the interacting protein or ligand, indicating their involvement in a binding interaction. Several different Lys-specific chemical modification methods have been reported such as aminoacetylation (8 -13), amidination (14,15), and many using biotin labeled reagents (16 -20). These modifications, coupled with mass spectrometric analysis, have been used to characterize the topology of multiple proteins, protein-ligand complexes and protein-protein interactions (8 -20).…”