Detection and structure determination of an equilibrium unfolding intermediate of Rd-apocytochrome b562: native fold with non-native hydrophobic interactions.
The absence of detectable kinetic and equilibrium folding intermediates by optical probes is commonly taken to indicate that protein folding is a two-state process. However, for some small proteins with apparent two-state behavior, unfolding intermediates have been identified in native-state hydrogen exchange or kinetic unfolding experiments monitored by nuclear magnetic resonance. Rd-apocytochrome b(562), a four-helix bundle, is one such protein. Here, we found another unfolding intermediate for Rd-apocytochrome b(562). It is based on a cooperative transition of (15)N chemical shifts of amide protons as a function of urea concentrations before the global unfolding. We have solved the high-resolution structure of the protein at 2.8 M urea, which is after this cooperative transition but before the global unfolding. All four helices remained intact, but a number of hydrophobic core residues repacked. This intermediate provides a possible structural interpretation for the kinetic unfolding intermediates observed using nuclear magnetic resonance methods for several proteins and has important implications for theoretical studies of protein folding.
Feng
Hanqiao
H
Laboratory of Biochemistry, National Cancer Institute, NIH, Building 37, Room 6114E, Bethesda, MD 20892, USA.
Vu
Ngoc-Diep
ND
Bai
Yawen
Y
D003198:Computer Simulation; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D014508:Urea / Q000378:metabolism
D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D014508:Urea; C017850:cytochrome b562, E coli