Surface plasmon resonance studies of wild‐type and AV77 tryptophan repressor resolve ambiguities in super‐repressor activity

The interactions of wild‐type (WT) and AV77 tryptophan repressor (TR) with several operators have been studied using surface plasmon resonance. The use of this real‐time method has been able to settle several outstanding issues in the field, in a way that has heretofore not been possible. We resolve the issue of the super‐repressor status of the AV77 aporepressor and find that in contrast to early studies, which found no significant difference in the binding constants in vitro to those of the WT, that there is indeed a clear difference in the binding constant that can simply account for the phenotype. Accordingly, there is no need for alternative proposals invoking complex equilibria with in vivo components not found in the in vitro experiments. In addition, we find that the AV77 holorepressor–DNA complex is much more stable than the equivalent WT complex, which has not been apparent from either in vitro or equilibrium binding experiments.

[1]  P. Stockley,et al.  Probing the molecular mechanism of action of co-repressor in the E. coli methionine repressor-operator complex using surface plasmon resonance (SPR). , 1995, Nucleic acids research.

[2]  R. Gunsalus,et al.  Escherichia coli tryptophan repressor binds multiple sites within the aroH and trp operators. , 1987, Genes & development.

[3]  Mengsu Yang,et al.  Kinetic study of various binding modes between human DNA polymerase beta and different DNA substrates by surface-plasmon-resonance biosensor. , 2002, The Biochemical journal.

[4]  C. Yanofsky,et al.  High level production and rapid purification of the E. coli trp repressor. , 1986, Nucleic acids research.

[5]  R. Parslow,et al.  Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences. , 2001, European journal of biochemistry.

[6]  P. Youderian,et al.  The challenge-phage assay reveals differences in the binding equilibria of mutant Escherichia coli Trp super-repressors in vivo. , 1993, Nucleic acids research.

[7]  V. LeTilly,et al.  Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding. , 1993, Biochemistry.

[8]  J Yang,et al.  Thermodynamics of ligand binding to trp repressor. , 1993, Biochemistry.

[9]  D. Myszka,et al.  Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. , 1997, Current opinion in biotechnology.

[10]  K Furukawa,et al.  Kinetic analysis of DNA binding by the c‐Myb DNA‐binding domain using surface plasmon resonance , 1999, FEBS letters.

[11]  J. Carey,et al.  Gel retardation at low pH resolves trp repressor-DNA complexes for quantitative study. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[12]  K. Matthews,et al.  trp repressor mutations alter DNA complex stoichiometry. , 1994, The Journal of biological chemistry.

[13]  K. Matthews,et al.  Dependence of trp repressor-operator affinity, stoichiometry, and apparent cooperativity on DNA sequence and size. , 1993, The Journal of biological chemistry.

[14]  C. Royer,et al.  The basis for the super-repressor phenotypes of the AV77 and EK18 mutants of trp repressor. , 2000, Journal of molecular biology.

[15]  J. Carey,et al.  How does trp repressor bind to its operator? , 1991, The Journal of biological chemistry.

[16]  R. Gunsalus,et al.  Interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan. , 1986, The Journal of biological chemistry.

[17]  P. Sigler,et al.  An alkaline phosphatase protection assay to investigate trp repressor/operator interactions. , 1991, Biochemistry.

[18]  J. Cann.  Phenomenological theory of gel electrophoresis of protein-nucleic acid complexes. , 1989, The Journal of biological chemistry.

[19]  C. Yanofsky,et al.  Increased binding of operator DNA by trp superrepressor EK49. , 1988, The Journal of biological chemistry.

[20]  C. Yanofsky,et al.  Enhanced operator binding by trp superrepressors of Escherichia coli. , 1990, The Journal of biological chemistry.

[21]  C A Royer,et al.  Affinity and specificity of trp repressor-DNA interactions studied with fluorescent oligonucleotides. , 1997, Journal of molecular biology.

[22]  L. S. Klig,et al.  Analysis of trp repressor-operator interaction by filter binding , 1987, Nucleic Acids Res..

[23]  L. S. Klig,et al.  Flexibility of dna binding domain of trp repressor required for recognition of different operator sequences , 1996, Protein science : a publication of the Protein Society.

[24]  C. Yanofsky,et al.  Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA. , 1985, Proceedings of the National Academy of Sciences of the United States of America.