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 acetylated in vivo. N-terminal acetylation has been confirmed by mass spectral analysis of intact and proteolytically digested protein. The histidine-heme loop-forming equilibrium, under denaturing conditions (3 M guanidine hydrochloride), has been measured by pH titration providing an observed pK(a), pK(a)(obs), for each variant. N-terminal acetylation prevents the N-terminal amino group-heme binding equilibrium from interfering with measurements of histidine-heme affinity. Significant deviation is observed from the linear 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 of -1.5 for a freely jointed random coil, which is commonly used to represent the conformational properties of protein denatured states. For loop sizes of nine to 22 monomers, chain stiffness is likely responsible for the decreases in histidine-heme affinity relative to a loop size of 37. The results are discussed in terms of residual structure and sequence composition effects on the conformational properties of the denatured states of proteins. Study holds ProTherm entries: 11565 Extra Details: variant of iso-1-cytochrome c with all surface histidine residues mutated with N-terminal acetylation protein folding; denatured state; random coil; cytochrome c;,guanidine hydrochloride
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:42 p.m.
|Number of data points||3|
|Proteins||Cytochrome c iso-1 ; Cytochrome c iso-1|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence TEFKAGSAKKGATLFKTRCLQCHTVEKGGPHKVGPNLHGIFGRHSGQAEGYSYTDANIKKNVLWDENNMSEYLTNPKKYIPGTKMAFGGLKKEKDRNDLITYLKKACE|