Diffusional barrier in the unfolding of a small protein.
To determine how the dynamics of the polypeptide chain in a protein molecule are coupled to the bulk solvent viscosity, the unfolding by urea of the small protein barstar was studied in the presence of two viscogens, xylose and glycerol. Thermodynamic studies of unfolding show that both viscogens stabilize barstar by a preferential hydration mechanism, and that viscogen and urea act independently on protein stability. Kinetic studies of unfolding show that while the rate-limiting conformational change during unfolding is dependent on the bulk solvent viscosity, eta, its rate does not show an inverse dependence on eta, as expected by Kramers' theory. Instead, the rate is found to be inversely proportional to an effective viscosity, eta + xi, where xi is an adjustable parameter which needs to be included in the rate equation. xi is found to have a value of -0.7 cP in xylose and -0.5 cP in glycerol, in the case of unfolding, at constant urea concentration as well as under isostability conditions. Hence, the unfolding protein chain does not experience the bulk solvent viscosity, but instead an effective solvent viscosity, which is lower than the bulk solvent viscosity by either 0.7 cP or 0.5 cP. A second important result is the validation of the isostability assumption, commonly used in protein folding studies but hitherto untested, according to which if a certain concentration of urea can nullify the effect of a certain concentration of viscogen on stability, then the same concentrations of urea and viscogen will also not perturb the free energy of activation of the unfolding of the protein.
Pradeep
Lovy
L
National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore, India.
Udgaonkar
Jayant B
JB
D001407:Bacillus / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D004058:Diffusion / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology; D017510:Protein Folding; D012997:Solvents; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology; D014783:Viscosity / Q000187:drug effects; D014867:Water / Q000378:metabolism; D014994:Xylose / Q000494:pharmacology
D001426:Bacterial Proteins; D012997:Solvents; D014867:Water; C057438:barstar protein, Bacillus amyloliquefaciens; D014508:Urea; D014994:Xylose; D005990:Glycerol