Rate of intrachain contact formation in an unfolded protein: Temperature and denaturant effects

We have measured the effect of temperature and denaturant concentration on the rate of intrachain diffusion in an unfolded protein. After photodissoci ating a ligand from the heme iron of unfolded horse cytochrome c, we use tr ansient optical absorption spectroscopy to measure the time scale of the di ffusive motions that bring the heme, located at His18, into contact with it s native ligand, Met80. Measuring the rate at which this 62 residue intrach ain loop forms under both folding and unfolding conditions, we find a signi ficant effect of denaturant on the chain dynamics. The diffusion of the cha in accelerates as denaturant concentration decreases, with the contact form ation rate approaching a value near similar to6 x 10(5) s(-1) in the absenc e of denaturant. This result agrees well with an extrapolation from recent loop formation measurements in short synthetic peptides. The temperature de pendence of the rate of contact formation indicates an Arrhenius activation barrier, E-a similar to 20 kJ/mol, at high denaturant concentrations, comp arable to what is expected from solvent viscosity effects alone. Although E -a increases by several k(B)T as denaturant concentration decreases, the ov erall rate of diffusion nevertheless increases. These results indicate that inter-residue energetic interactions do not control conformational diffusi on in unfolded states, even under folding conditions. (C) 2001 Academic Pre ss.