Solvent and temperature effects on crambin, a hydrophobic protein, as investigated by proton magnetic resonance.

Abstract

Crambin, a water-insoluble protein of molecular weight 5000, has been studied by 1H-NMR at 270 MHz and 300 MHz as a function of temperature and solvent composition. In (2H4)acetic acid the resonances sharpen up to 75 °C and broaden upon further heating owing to conformational exchange. In (2H7)dimethylformamide, temperature mainly causes monotonic narrowing as a result of high mobility and disaggregation. Crambin, dissolved in acetic acid at 55 °C, dried and redissolved in dimethylformamide, yields a room-temperature spectrum that is considerably narrower than that of the untreated protein dissolved in the same solvent. Acetic acid pretreatment thus interferes with aggregation in dimethylformamide. Both these spectra are closely similar to that of the protein in acetic acid. Given the complementary acid-base nature of acetic acid and dimethylformamide, we conclude that spectra in these two solvents arise from conformations closely related to the native structure. Crambin undergoes extensive unfolding in (2H6)dimethylsulfoxide at 25°C. In contrast, up to 105°C. In contrast, up to 105°C in dimethylformamide and at least up to 85°C in acetic acid the protein structure is only partially altered. These observations substantiate an unprecedented thermal stability for crambin. Proton decoupling experiments are shown that verify vicinal relationships among tyrosyl aromatic resonances. Similar experiments identify a high-field-shifted methyl triplet as arising from an isoleucyl residue. Selective nuclear Overhauser effects are shown that relate this methyl group to a partially constrained tyrosyl ring. This is consistent with the crystallographic model that shows Ile-33 to be in close proximity to Tyr-44.