Trifluoroethanol stabilizes the pH 4 folding intermediate of sperm whale apomyoglobin.
2,2,2-Trifluoroethanol (TFE) is known to stabilize peptide helices by strengthening hydrogen bonds. On the other hand, TFE destabilizes native proteins, as we confirm here, presumably by weakening the hydrophobic interaction. The stability of the pH 4 folding intermediate of apomyoglobin is known to depend both on the strength of the individual A, G, and H helices and on hydrophobic interactions between helices. We ask which effect of TFE dominates in this case: strengthening helices or weakening hydrophobic interactions between helices? Protein stability is measured by denaturant-induced unfolding curves, and two-state unfolding is tested by monitoring both far-UV CD and tryptophan fluorescence emission. Low concentrations of TFE strongly stabilize the pH 4 folding intermediate. Moreover, low concentrations of TFE compensate for helix-destabilizing mutations in the A and G helices. Consequently, enhancing helix propensity, rather than weakening the hydrophobic interaction, is the dominant effect of TFE on the folding intermediate. This result agrees with earlier mutational evidence that helix propensities are very important in determining the stability of the pH 4 intermediate. Although TFE destabilizes native holomyoglobin, as well as native lysozyme and ribonuclease A, nevertheless, TFE stabilizes native apomyoglobin.
Luo
Y
Y
Department of Biochemistry, Stanford University School of Medicine, CA 94305-5307, USA. yluo@cmgm.stanford.edu
Baldwin
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RL
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry / Q000187:drug effects; D009211:Myoglobin / Q000737:chemistry / Q000187:drug effects*; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000187:drug effects; D014270:Trifluoroethanol / Q000494:pharmacology*; D014907:Whales
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D014270:Trifluoroethanol; D012259:Ribonuclease, Pancreatic; D009113:Muramidase