Structural characterization of monellin in the alcohol-denatured state by NMR: evidence for beta-sheet to alpha-helix conversion.
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.
Fan
P
P
Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854-0939.
Bracken
C
C
Baum
J
J
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003903:Deuterium; D017666:Deuterium Oxide; D000431:Ethanol / Q000494:pharmacology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000187:drug effects; D014508:Urea; D014867:Water
D010940:Plant Proteins; C002541:monellin protein, Dioscoreophyllum cumminsii; D014867:Water; D000431:Ethanol; D014508:Urea; D003903:Deuterium; D017666:Deuterium Oxide