In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by approximately 2 kcal/mol (1 cal identical with 4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the 'fast' form. Finally, we have used hirudin fragment 1-47 as a molecular probe of the conformation of thrombin recognition sites in the fast and 'slow' form. From the effects of amino acid substitutions on the affinity of fragment 1-47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation.

Abstract7-Azatryptophan (AW), a noncoded isostere of tryptophan (W), possesses interesting spectral properties. In particular, the presence of a nitrogen atom at position 7 in the indolyl nucleus of AW results in a red shift of the absorption maximum and fluorescence emission by 10 and 46 nm, respectively, compared to W. In the present work, we report the chemical synthesis and the conformational and functional characterization of an analog (denoted as Y3AW) of the N-terminal domain 1-47 of hirudin, a highly potent thrombin inhibitor, in which Tyr 3 has been replaced by AW. The results obtained were compared with those of the cooresponding Y3W analog. We found that the replacement W → AW reduces affinity for thrombin by 10-fold, likely because of the lower hydrophobicity of AW compared with that of W. Measurements of the resonance energy transfer effect, which was observed between Tyr13 and the amino acid at position 3 upon disulfide-coupled folding, demonstrate that AW behaves as a better energy acceptor than W for studying protein renaturation. The interaction of Y3AW with thrombin was studied by exciting the sample at 320 nm and recording the change in fluorescence of Y3AW on binding to the enzyme. Our results indicate that the fluorescence of AW of hirudin 1-47 in the Y3AW-thrombin complex is strongly quenched, possibly because of the presence of two structural water molecules at the hirudin-thrombin interface that can promote the nonradiative decay of AW in the excited state. The data herein reported demonstrate that the incorporation of AW can be of broad applicability in the study of protein folding and protein-protein interaction.Keywords: hirudin; thrombin; 7-azatryptophan; noncoded amino acids; anticoagulants; folding; spectroscopy In its infancy, the approach of protein engineering was essentially restricted to the possibility of chemically modifying, in a rather unspecific fashion, particular amino acid side chains in proteins (for review, see Freedman 1971). More recently, the advent of recombinant DNA technology allowed the site-specifical alteration of a given polypeptide chain at a glance, thus much expanding the tools available to study the molecular mechanisms of protein folding, stability, and function (Fersht and Winter 1992). From this perspective, the incorporation of noncoded amino acids into Abbreviations: (Standard one-letter or three-letter abbreviations are used for all natural amino acids.) 7AI, 7-azaindole; a.m.u., atomic mass units; AW, (L)-7-azatryptophan; W, (L)-tryptophan; BSA, bovine serum albu-