Characterization of charge change super-repressor mutants of trp repressor: effects on oligomerization conformation, ligation and stability.
We have carried out a physical characterization of mutant repressor proteins of the trp repressor system of Escherichia coli by circular dichroism, chemical denaturation, and 8-anilino-1-naphthalenesulfonate binding. We have also probed the protein-protein interactions via fluorescence anisotropy and lifetime measurements and measured the thermodynamics of ligand (L-tryptophan) binding by isothermal titration calorimetry. Here, we present investigations of four charge change super-repressor mutants: EK13, EK18, DN46 and EK49, and compare these results with those previously obtained for wild-type trp repressor and the AV77 super-repressor mutant. These studies demonstrate that super-repressor phenotypes may result from changes in operator affinity (DN46, EK49), protein-protein interactions (EK18), as well as the coupling of folding to ligand binding (AV77, EK13, EK18). Correlations between the oligomerization behavior and cooperativity of DNA binding for some of these mutants indicate that coupling of oligomerization to DNA binding modulates operator site occupation giving rise to the super-repressor phenotype. The present results underscore the complex interplay between the multiple equilibria in this system. Moreover, they provide insights into the structural basis for the mutational perturbation of the energetics of this classical allosterically controlled transcriptional regulator.
Reedstrom
R J
RJ
School of Pharmacy University of Wisconsin-Madison 53706, USA.
Martin
K S
KS
Vangala
S
S
Mahoney
S
S
Wilker
E W
EW
Royer
C A
CA
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism; D014508:Urea; D029968:Escherichia coli Proteins
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D008024:Ligands; D012097:Repressor Proteins; D014364:Tryptophan; D014508:Urea; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins; C027132:1-anilino-8-naphthalenesulfonate