Denatured state thermodynamics: Residual structure, chain stiffness and scaling factors

A set of nine variants of yeast iso-1-cytochrome c with zero or one surface histidine have been engineered such that the N-terminal amino group is ace tylated in vivo. N-terminal acetylation has been confirmed by mass spectral analysis of intact and proteolytically digested protein. The histidine-hem e loop-forming equilibrium, under denaturing conditions (3 M guanidine hydr ochloride), has been measured by pH titration providing an observed pK(a), pK(a)(obs), for each variant. N-terminal acetylation prevents the N-termina l amino group-heme binding equilibrium from interfering with measurements o f histidine-heme affinity. Significant deviation is observed from the linea r dependence of pK(a)(obs) on the log of the number of monomers in the loop formed, expected for a random coil denatured state. The maximum. histidine -heme affinity occurs for a loop size of 37 monomers. For loop sizes of 37- 83 monomers, histidine-heme pK(a)(obs) values are consistent with a scaling factor of -4.2 +/-0.3. This value is much larger than the scaling factor o f -1.5 for a freely jointed random coil, which is commonly used to represen t the conformational properties of protein denatured states. For loop sizes of nine to 22 monomers, chain stiffness is likely responsible for the decr eases in histidine-heme affinity relative to a loop size of 37. The results are discussed in terms of residual structure and sequence composition effe cts on the conformational properties of the denatured states of proteins. ( C) 2001 Academic Press.