Ligand-induced conformational changes in lactose repressor: a fluorescence study of single tryptophan mutants.
A key element in the ability of lac repressor protein to control transcription reversibly is the capacity to assume different conformations in response to ligand binding. To investigate regions of the protein involved in these conformational changes, mutant repressor proteins containing single tryptophans were created by mutating each of the two native tryptophan residues to tyrosine and changing the residue of interest to tryptophan. Tryptophans substituted in the following locations were highly accessible to quenchers with no changes in fluorescence or quenching properties in the presence of ligands: in the N-terminal helix-turn-helix for Y7, at the junction between the N-terminus and N-subdomain for L62, in the N-subdomain of the monomer-monomer interface for residue E100 or Q117, or at the C-terminal region for K325. Tryptophan at position F226 in the C-subdomain subunit interface was only moderately exposed to quenchers and unresponsive to ligands. In contrast, the fluorescence and quenching properties of single tryptophans placed in the central region of the protein were affected by ligands. Inducer binding altered the accessibility to quencher for tryptophan at H74 or F293, but no changes were detected upon binding operator. Exposure of tryptophan at the position occupied by Y273 was affected by both inducer and operator, indicating alterations in this region by both ligands. These results suggest that, in the areas of the lac repressor probed by these substitutions, the inducer-bound form differs from the conformation of the unliganded form.
Barry
J K
JK
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892, USA.
Matthews
K S
KS
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D007700:Kinetics; D056968:Lac Repressors; D008024:Ligands; D008958:Models, Molecular; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009875:Operator Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside; D014364:Tryptophan