| Abstract: | The hydrophobic protein crambin (Mr 4715) has an aromatic content of one phenylalanyl residue (site 13) and two tyrosyl residues (sites 29 and 44). The aromatic residues have been studied by 1H NMR spectroscopy at 300 and 600 MHz for crambin dissolved in deuterated glacial acetic acid and in aqueous organic media. In particular, a 3:1 acetone/water mixture affords a solvent system of low viscosity, which yields very narrow line protein spectra. The aromatic proton spectrum is unusual in that signals are doubled. Spectral simulation of modified and unmodified crambin aromatic spin systems can be accomplished only by assuming that the protein is a mixture of two species. Dynamic 1H-[1H] Overhauser experiments centered on the aromatic doublets of Tyr29 indicate that the two species do not interconvert within 2 s. NMR spectra of crambin samples obtained by repeated crystallization in 17:3 acetone/water indicate that the process enriches the mixture with one component that does not convert to the other, even upon heating of the sample to 351 K. The combined evidence strongly favors the view that the doubled spectrum results from a compositional heterogeneity, most likely a mixture of two homologues, rather than from an equilibrium between interconvertible forms. This is important in view of the fact that its crystallographic structure, solved to 1.5-A resolution [Hendrickson, W.A., & Teeter, M. M. (1981) Nature (London) 290, 107-113], is based on samples containing the two species.(ABSTRACT TRUNCATED AT 250 WORDS) |
