Biomedical subjects
Publications and source records attributed to Mark R Parsons.
The de novo molecular design program SPROUT has been applied to the X-ray crystal structures of Plasmodium and human dihydroorotate dehydrogenase, respectively. The resulting design templates were used to prepare a series of molecules which, in keeping with predictions, showed useful levels of species-selective enzyme inhibition.
Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer.
In the Gram-positive bacterium Bacillus subtilis the concentration of the amino acid L-arginine is controlled by the transcriptional regulator AhrC. The hexameric AhrC protein binds in an L-arginine-dependent manner to pseudo-palindromic operators within the promoter regions of arginine biosynthetic and catabolic gene clusters. AhrC binding results in the repression of transcription of biosynthetic genes and in the activation of transcription of catabolic genes. The crystal structure of AhrC has been determined at 2.7 A resolution. Each subunit of the protein has two domains. The C-terminal domains are arranged with 32 point-group symmetry and mediate the major intersubunit interactions. The N-terminal domains are located around this core, where they lie in weakly associated pairs but do not obey strict symmetry. A structural comparison of AhrC with the arginine repressor from the thermophile B. stearothermophilus reveals close similarity in regions implicated in L-arginine binding and DNA recognition, but also reveals some striking sequence differences, especially within the C-terminal oligomerization domain, which may contribute to the different thermostabilities of the proteins. Comparison of the crystal structure of AhrC with a 30 A resolution model obtained by combining X-ray structure-factor amplitudes with phases derived from electron-microscopic analyses of AhrC crystals confirms the essential accuracy of the earlier model and suggests that such an approach may be more widely useful for obtaining low-resolution phase information.