Half-of-the-sites reactivity of F235C lambda-repressor: implications for the structure of the whole repressor.
A site-directed mutation, F235C, was created at the penultimate residue of the lambda-repressor. Measurement of dimer-monomer dissociation constant suggested that dimer-monomer dissociation of the mutant repressor is similar to that of the wild-type. Affinity towards a single operator O(R)1 is also similar to that of the wild-type repressor. The mutant repressor gene in a multi-copy plasmid confers immunity towards infection by a cI(-) lambda phage, suggesting preservation of functional integrity. Far-UV circular dichroism spectra show no major change in the secondary structure. Fluorescence quenching experiments, however, suggest increased exposure of some tryptophan residues. The urea denaturation profile indicates decreased stability of a part of the C-terminal domain. Under non-denaturing conditions, cysteine-235 shows half-of-the-sites reactivity, i.e. on average only one out of two cysteine-235 residues in the dimer shows reactivity towards sulfhydryl reagents. Fluorescence energy transfer between randomly labeled donor and acceptor fluorescent probes indicates that only one sulfhydryl per dimer is reactive, suggesting true half-of-the-sites reactivity. The structural role of the C-terminal tail in the whole repressor dimer is discussed.
Bandyopadhyay
Sumita
S
Department of Biophysics, Bose Institute, P-1/12 C.I.T. Scheme VII M, Calcutta 700 054, India.
Deb
Sunanda
S
Bose
Sudeep
S
Roy
Siddhartha
S
D020106:Acrylamide; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014364:Tryptophan; D014508:Urea / Q000378:metabolism; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D012097:Repressor Proteins; D013438:Sulfhydryl Compounds; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D020106:Acrylamide; D014364:Tryptophan; D014508:Urea; D003545:Cysteine