A stable, molten-globule-like cytochrome c.
Expression of cytochrome c from Thermus thermophilus in Escherichia coli (E. coli) leads to a protein with characteristics of a molten globule. Unfolding induced by guanidine hydrochloride (GdHCl) shows that E. coli-expressed cytochrome c has lower stability (and less cooperativity of unfolding) compared to the protein extracted from Thermus thermophilus, even though the two proteins have identical amino-acid sequences. Moreover, Soret and far-UV circular dichroism signals differ for the two proteins, suggesting a distorted heme environment and more side-chain dynamics of E. coli-expressed cytochrome c. Still, tryptophan fluorescence in E. coli-expressed cytochrome c is quenched as in native protein, and the iron coordinates in a low-spin form. Amino-acid sequencing indicates the presence of only one covalent cysteine-linkage to the heme in E. coli-expressed cytochrome c (normally, there are two linkages), a possible explanation for the trapped, molten-globule-like structure. The features of this non-native protein may be of interest for interpretation of cytochrome c folding kinetics in vitro, since a molten globule may be an intermediate on the folding pathway.
Wittung-Stafshede
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Beckman Institute, California Institute of Technology, Pasadena 91125, USA. pernilla@cco.caltech.edu
D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D006418:Heme / Q000737:chemistry; D010084:Oxidation-Reduction; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis; D016963:Thermus thermophilus / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
D003574:Cytochrome c Group; D011994:Recombinant Proteins; D006418:Heme; D014364:Tryptophan; D019791:Guanidine