Stability and folding kinetics of structurally characterized cytochrome c-b562.
The four-helix-bundle protein fold can be constructed from a wide variety of primary amino acid sequences. Proteins with this structure are excellent candidates for investigations of the relationship between folding mechanism and topology. The folding of cytochrome b(562), a four-helix-bundle heme protein, is hampered by heme dissociation. To overcome this complication, we have engineered a variant of cytochrome b(562) (cyt c-b(562)) featuring a c-type linkage between the heme and the polypeptide chain. The replacement of the native cyt b(562) leader sequence in this protein with that of a c-type cytochrome (cyt c(556)) led to high yields of fully matured and correctly folded cyt c-b(562). We have determined the X-ray crystal structure of cyt c-b(562) at 2.25 A and characterized its physical, chemical, and folding properties. These measurements reveal that the c-type linkage does not perturb the protein fold or reduction potential of the heme group. The covalent attachment of the porphyrin to the polypeptide does, however, produce a substantial change in protein stability and folding kinetics.
Faraone-Mennella
Jasmin
J
Beckman Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA.
Tezcan
F Akif
FA
Gray
Harry B
HB
Winkler
Jay R
JR
D018360:Crystallography, X-Ray; D003573:Cytochrome b Group / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D003573:Cytochrome b Group; D006418:Heme