Alcohol-Induced Protein Folding Transitions in Platelet Factor 4: The O-State

Yingqing Yang, Kevin H. Mayo

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


Platelet factor 4 (PF4) (7800 daltons) is an anti-parallel β-sheet, α/β 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 αH 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 M 2-propanol. Simple linear extrapolation to 0 M 2-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 β-structure decreasing by about 20% and α-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 β-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 β-sheet structure and a more stabilized C-terminal α-helix.

Original languageEnglish (US)
Pages (from-to)8661-8671
Number of pages11
Issue number33
StatePublished - 1993


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