A conformational study of thioredoxin and its tryptic fragments.

The absorption, fluorescence, and circular dichroism properties of Escherichia coli thioredoxin, and of its tryptic fragments thioredoxin-T-(1-73) and thioredoxin-T-(74-108), in water and in trifluoroethanol, have been investigated as a function of pH and temperature in order to gain information about their conformational behavior. Both reduced and oxidized thioredoxin have a remarkable conformational stability as judged from CD spectra at various pH values and temperatures. The percentage of secondary structure in solution was calculated using the procedures suggested in the literature, but no satisfactory agreement with the x-ray data could be obtained. The reasons for this discrepancy are discussed. The fluorescence spectrum shows an intense tyrosine contribution, whose intensity is strongly pH-dependent in the acidic region. An interaction with a carboxylate group (tentatively Tyr-49 with Asp-104) is suggested. The pH dependence of the fluorescence intensity of tryptophan in reduced thioredoxin is also surprisingly marked, and a group with pK = 6.4, having a high quenching efficiency in the deprotonated form, is deemed responsible for this behavior. This is ascribed to one of the two reactive cysteine residues, whose negative charge is probably stabilized by Lys-36. Various spectroscopic/conformational studies have been carried out on the two fragments (1-73) and (74-108). The salient result is that the (1-73) fragment, which assumes a largely aperiodic structure at neutral and alkaline pH, is able to refold in a globular and stable structure when the pH is below approximately 3.0. This process is attended by marked changes in the fluorescence and CD spectra. The refolding mechanism and its implications are discussed.