Structural characterization of monellin in the alcohol-denatured state by NMR: Evidence for .beta.-sheet to .alpha.-helix conversion

Abstract

Two-dimensional 1H NMR spectroscopy and hydrogen exchange methods have been used to characterize the alcohol-denatured state of monellin. Monellin is a sweet tasting protein composed of two chains. In the native state, the A-chain consists entirely of beta-structure, and the B-chain contains both alpha- and beta-structure. Upon addition of either 50% ethanol or 50% trifluoroethanol (TFE), the native structure of monellin is disrupted resulting in an alcohol-denatured state with properties different from those of the random coil state. In the alcohol-denatured state, the far-UV circular dichroism (CD) spectrum displays a higher helical content relative to the native state and the intensity of the near-UV CD signal is completely lost. One-dimensional NMR studies show that there are approximately 14 amide protons protected from exchange with solvent in the alcohol-denatured state and that large portions of the protein exchange at a rate that is comparable to the exchange rate of the protein in urea. Utilizing hydrogen exchange trapping techniques, the slowly exchanging residues are identified at pH 2.0 in 50% ethanol and 50% TFE (A10-A15, A18, A19, A21, A24, and A39) and are found to be clustered on one region of the A-chain. Preliminary 2D NMR assignments show that in the alcohol-denatured state the A-chain of monellin undergoes structural reorganization, with one strand of the native state beta-sheet on the A-chain (residues A17-A30) becoming an alpha-helix in the alcohol-denatured state. The secondary structure of the A-chain in the alcohol-denatured state is different from the native state structure, although the slowly exchanging residues are similar.

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

“…It is noteworthy that NMR studies of a synthetic sequence corresponding to helix C of HEWL in aqueous TFE establish a high helical structure of this peptide in solution (Bolin et al, 1996). Recently, aqueous organic solvents have been used in generating partially folded states of some proteins (Harding et al, 1991;Fan et al ., 1993;Alexandrescu et al, 1994;Bychkova et al, 1996;Kamatari et al, 1996). A partially denatured state of hen lysozyme has been obtained in a 50% T-water mixture (Buck et al, 1993).…”

Conformational diversity of acid‐denatured cytochrome c studied by a matrix analysis of far‐UV CD spectra

“…To support this view experimentally, a comprehensive description on the structure of protein in a full range of the conformational space is necessary. The D, MG, and H conformers and their variants of various proteins have been characterized extensively by many experimental methods (Roder et al, 1988;Hughson et al, 1990;Dill & Shortle, 1991;Jeng & Englander, 1991;Sosnick & Trewhella, 1992;Fan et al, 1993;Alexandrescu et al, 1994;Konno et al, 1995). However, limited sensitivity and resolution of any experimental methods make this problem still largely unsolved.…”

Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation

“…Tamburro et al (1968) studied the effects of trifluoroethanol (TFE) on the conformations of the ribonuclease S-peptide; TFE was found to stabilize the small peptide in the same (a-helical) confor-mation that it adopts in the native protein. Since then, alcohols have been used widely to examine the conformational (particularly helical) propensities of peptides (Nelson & Kallenbach, 1986;Lehrman et al, 1990;Segawa et al, 1991;Sonnichsen et al, 1992) and to induce conformational changes in intact proteins (Stone et al, 1985;Wilkinson & Mayer, 1986;Dufour & HaertlC, 1990;Jackson & Mantsch, 1992;Buck et al, 1993;Fan et al, 1993). The primary physical mechanisms by which alcohols effect these changes are still unresolved.…”