Chemical characterization of the aromatic proton magnetic resonance spectrum of crambin

Abstract

The hydrophobic protein crambin (MW 4715) has an aromatic content of 1 phenylalanyl residue (site 13) and 2 tyrosyl residues (sites 29 and 44). The aromatic residues were 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 2 species. Dynamic 1H-{1H} Overhauser experiments centered on the aromatic doublets of Tyr29 indicate that the 2 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.degree. K. The combined evidence strongly favors the view that the doubled spectrum results from a compositional heterogeneity, most likely a mixture of 2 homologs, rather than from an equilibrium between interconvertible forms. This is important in view of the fact that its crystallographic structure, solved to 1.5-.ANG. resolution (Hendrickson and Teeter 1981), is based on samples containing the 2 species. The 1H NMR evidence is consistent with a model where the Tyr29 side chain is very mobile while both the Phe13 and Tyr44 rings, although flipping fast on the NMR time scale, are more constrained in their motion. Crambin was reacted with tetranitromethane and also iodinated. Under reagent-limiting conditions, Tyr29 nitrates and Tyr44 iodinates, the latter yielding the diiodo derivative. Excess reagent causes Tyr44 to nitrate and Tyr29 to iodinate. This confirms that the reactivity of phenolic groups is controlled by their environments and suggests that probably the iodination of Tyr44 is facilitated by an electrostatic effect from Asp43. Response to nitration or iodination cannot be taken as a single criterion to determine exposure of phenol groups in proteins. Chemical modification of Tyr44 causes shifts of the Ile33 CH3.delta. resonance, asserting proximity of these 2 residues. Upon acquiring a negative charged provided by the modifying group, crambin gains in its water solubility; the nitrated species assists in solubilizing unmodified, native crambin in pure aqueous media. This device opens the possibility of comparing physical properties of crambin with those of other exclusively water-soluble proteins. In particular, crambin NMR spectra can now be related to those of thionin homologs from Graminae that are highly hydrophilic. Comparison with the .alpha.1-purothionin homolog from wheat leads to tentative identification of the crambin Ile34 .delta.-methyl triplet in the proton spectrum.