Folding energy landscape of cytochrome cb562.
Cytochrome cb(562) is a variant of an Escherichia coli four-helix bundle b-type heme protein in which the porphyrin prosthetic group is covalently ligated to the polypeptide near the terminus of helix 4. Studies from other laboratories have shown that the apoprotein folds rapidly without the formation of intermediates, whereas the holoprotein loses heme before native structure can be attained. Time-resolved fluorescence energy transfer (TRFET) measurements of cytochrome cb(562) refolding triggered using an ultrafast continuous-flow mixer (150 micros dead time) reveal that heme attachment to the polypeptide does not interfere with rapid formation of the native structure. Analyses of the TRFET data produce distributions of Trp-59-heme distances in the protein before, during, and after refolding. Characterization of the moments and time evolution of these distributions provides compelling evidence for a refolding mechanism that does not involve significant populations of intermediates. These observations suggest that the cytochrome b(562) folding energy landscape is minimally frustrated and able to tolerate the introduction of substantial perturbations (i.e., the heme prosthetic group) without the formation of deep misfolded traps.
Beckman Institute, California Institute of Technology, Pasadena, CA 91125-7400, USA.
Tetsunari
T
Kimura
Jennifer C
JC
Lee
Harry B
HB
Gray
Jay R
JR
Winkler
D001671:Biochemistry / Q000379:methods; D003573:Cytochrome b Group / Q000737:chemistry*; D030562:Databases, Protein; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D019791:Guanidine / Q000494:pharmacology; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D015233:Models, Statistical; D008968:Molecular Conformation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary
D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D006418:Heme; C017850:cytochrome b562, E coli; D019791:Guanidine