Alcohol-induced protein folding transitions in platelet factor 4: the O-state.
Platelet factor 4 (PF4) (7800 daltons) is an anti-parallel beta-sheet, alpha/beta class protein whose tertiary structure is stabilized by the presence of two disulfide bonds. Titration of PF4 with 2-propanol or similar low molecular weight, aliphatic alcohols induces reversible protein folding transitions which are observed to be in slow exchange on the 600-MHz 1H NMR time scale. Line fitting of resolved resonances assigned to ring protons of Y60, H35, H23, and alpha H of K50 in native and alcohol-induced states (O-states) allows derivation of folding equilibrium constants and exchange kinetics. Folding exchange rates vary between 5 and 100 s-1 on going from 9.8 to 3.3 M2-propanol. Simple linear extrapolation to 0 M2-propanol yields an O-state to N-state exchange rate of about 500 s-1, i.e., millisecond time scale. At relatively high 2-propanol concentration (>9.5 M), where the O-state is predominant (>90%), NMR spectra suggest a more "unfolded" structure, while CD data indicate the preservation of considerable secondary structure. Increasing 2-propanol from 3.3 to 9.8 M, however, shifts the CD-derived fractional compositions significantly, with overall beta-structure decreasing by about 20% and alpha-helix composition increasing by about 25%. Alcohol-jump experiments, which identify O-state long-lived NHs in the NMR spectrum of native PF4, indicate folding transition reversibility and conservation of about 15 long-lived NHs in native and O-states. Most of these NHs are assigned to residues in anti-parallel beta-sheet structure. Of these 15 NHs, H/D exchange rates, although variably reduced in the O-state, are generally still long-lived compared with random coil H/D exchange. Overall, the PF4 O-state is a stable intermediate with an apparently more highly fluctuating anti-parallel beta-sheet structure and a more stabilized C-terminal alpha-helix.
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Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
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D000433:1-Propanol / Q000494:pharmacology*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides; D006801:Humans; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D010978:Platelet Factor 4 / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects
D004220:Disulfides; D010978:Platelet Factor 4; D006859:Hydrogen; D000433:1-Propanol