Equilibrium unfolding of dimeric and engineered monomeric forms of lambda Cro (F58W) repressor and the effect of added salts: evidence for the formation of folded monomer induced by sodium perchlorate.
The equilibrium unfolding transitions of Cro repressor variants, dimeric variant Cro F58W and monomer Cro K56[DGEVK]F58W, have been studied by urea and guanidine hydrochloride to probe the folding mechanism. The unfolding transitions of a dimeric variant are well described by a two state process involving native dimer and unfolded monomer with a free energy of unfolding, DeltaG(0,un)(0), of approximately 10-11 kcal/mol. The midpoint of transition curves is dependent on total protein concentration and DeltaG(0,un)(0) is independent of protein concentration, as expected for this model. Unfolding of Cro monomer is well described by the standard two state model. The stability of both forms of protein increases in the presence of salt but decreases with the decrease in pH. Because of the suggested importance of a N2<-->2F dimerization process in DNA binding, we have also studied the effect of sodium perchlorate, containing the chaotropic perchlorate anion, on the conformational transition of Cro dimer by CD, fluorescence and NMR (in addition to urea and guanidine hydrochloride) in an attempt both to characterize the thermodynamics of the process and to identify conditions that lead to an increase in the population of the folded monomers. Data suggest that sodium perchlorate stabilizes the protein at low concentration (<1.5 M) and destabilizes the protein at higher perchlorate concentration with the formation of a "significantly folded" monomer. The tryptophan residue in the "significantly folded" monomer induced by perchlorate is more exposed to the solvent than in native dimer.
Maity
Haripada
H
Department of Chemistry and Biochemistry, University of Mississippi, Coulter Hall University, MS 38677, USA.
Mossing
Michael C
MC
Eftink
Maurice R
MR
D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D019281:Dimerization; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010472:Perchlorates / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics*; D012965:Sodium Chloride; D017670:Sodium Compounds / Q000494:pharmacology; D055672:Static Electricity; D013816:Thermodynamics; D014508:Urea; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D010472:Perchlorates; D011994:Recombinant Proteins; D012097:Repressor Proteins; D017670:Sodium Compounds; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D012965:Sodium Chloride; D014508:Urea; C031068:sodium perchlorate; D019791:Guanidine