Role of the C-terminal tail region in the self-assembly of lambda-repressor.
Acrylamide quenching of the tryptophan fluorescence of the lambda-repressor at different protein concentrations indicates that one of the three tryptophan residues, W129, W142, and W230, undergoes a change in environment upon self-assembly, from dimer to associated species. Quenching data suggest that this tryptophan residue is inaccessible to low concentrations of acrylamide and is blue-shifted in the associated form. In the dimer, this tryptophan residue is highly accessible to acrylamide and is red-shifted. NBS oxidation, at protein concentrations which favor the associated form, showed that this tryptophan is also significantly protected from NBS oxidation. HPLC peptide mapping of NBS-oxidized lambda-repressor, amino acid analysis, and sequencing indicate that the protected, blue-shifted tryptophan is tryptophan 230. A mutant repressor (F235C) was specifically labeled at Cys 235 with an environment-sensitive probe, acrylodan. The acrylodan fluorescence of the labeled F235C lambda-repressor undergoes a significant blue-shift, accompanied by fluorescence enhancement, upon protein association. Along with other genetic evidence, these results suggest involvement of the C-terminal tail region in the self-assembly of the lambda-repressor.
Bandyopadhyay
S
S
Department of Biophysics, Bose Institute, Calcutta, India.
Banik
U
U
Bhattacharyya
B
B
Mandal
N C
NC
Roy
S
S
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D000596:Amino Acids / Q000032:analysis; D004268:DNA-Binding Proteins; D006863:Hydrogen-Ion Concentration; D010449:Peptide Mapping; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D000178:Acrylamides; D000596:Amino Acids; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D020106:Acrylamide; D014364:Tryptophan