Hirudin is the most potent and specific inhibitor of thrombin, a key enzyme in the coagulation process existing in equilibrium between its procoagulant (fast) and anticoagulant (slow) form. In a previous study, we described the solid-phase synthesis of a Trp3 analogue of fragment 1-47 of hirudin HM2, which displayed approximately 5-fold higher thrombin inhibitory potency relative to that of the natural product [De Filippis, V., et al. (1995) Biochemistry 34, 9552-9564]. By combining automated and manual peptide synthesis, here we have produced in high yields seven analogues of fragment 1-47 containing natural and non-natural amino acids. In particular, we have replaced Val1 with tert-butylglycine (tBug), Ser2 with Arg, and Tyr3 with Phe, cyclohexylalanine (Cha), Trp, alpha-naphthylalanine (alphaNal), and beta-naphthylalanine (betaNal). The crude reduced peptides are able to fold almost quantitatively into the disulfide-cross-linked species, whose unique alignment (Cys6-Cys14, Cys16-Cys28, and Cys22-Cys37) has been shown to be identical to that of the natural fragment. The results of conformational characterization provide evidence that synthetic peptides retain the structural features of the natural species, whereas thrombin inhibition data indicate that the synthetic analogues are all more potent inhibitors of thrombin. In particular, Val --> tBug exchange leads to a 3-fold increase in binding, interpreted as arising from a favorable reduction of the entropy of binding, due to the presence of the more symmetric side chain of tBug relative to that of Val. The S2R analogue binds 24- and 125-fold more tightly than the natural fragment to the fast or slow form of thrombin. These results are explained by considering that Arg2 may favorably couple to Glu192, a key residue involved in the slow to fast transition, thus stabilizing the slow form. Replacement of Tyr3 with more hydrophobic residues having different side chain orientations and electronic structures improves binding by 2-40-fold, suggesting that nonpolar interactions and shape-dependent packing effects strongly influence binding at this position. Overall, these results provide new insights for elucidating the mechanism of hirudin-thrombin recognition at the molecular level and highlight new strategies for designing more potent and selective inhibitors of thrombin.

The introduction into peptide chains of alpha-aminoisobutyric acid (Aib) has proven to stabilize the helical structure in short peptides by restricting the available range of polypeptide backbone conformations. In order to evaluate the potential stabilizing effect of Aib at the protein level, we have studied the conformational and stability properties of Aib-containing analogs of the carboxy-terminal subdomain 255-316 of thermolysin. Previous NMR studies have shown that this disulfide-free 62-residue fragment forms a dimer in solution and that the global 3D structure of each monomer (3 alpha-helices encompassing residues 260-274, 281-295, and 301-311) is largely coincident with that of the corresponding region in the X-ray structure of intact thermolysin. The Aib analogs of fragment 255-316 were prepared by a semisynthetic approach in which the natural fragment 255-316 was coupled to synthetic analogs of peptide 303-316 using V8-protease in 50% (v/v) aqueous glycerol [De Filippis, V., and Fontana, A. (1990) Int. J. Pept. Protein Res. 35, 219-227]. The Ala residue in position 304, 309, or 312 of fragment 255-316 was replaced by Aib, leading to the singly substituted fragments Ala304Aib, Ala309Aib, and Ala312Aib. Moreover, fragment Ala304Aib/Ala309Aib with a double Ala-->Aib exchange in positions 304 and 309 was produced. Far- and near-UV circular dichroism measurements demonstrated that both secondary and tertiary structures of the natural fragment 255-316 are fully retained upon Ala-->Aib substitution(s). Thermal unfolding measurements, carried out by recording the ellipticity at 222 nm upon heating, showed that the melting temperatures (Tm) of analogs Ala304Aib and Ala309Aib were 2.2 and 5.4 degrees C higher than that of the Ala-containing natural species (Tm = 63.5 degrees C), respectively, whereas the Tm of the Ala312Aib analog was lowered by -0.6 degree C. The enhanced stability of the Ala304Aib analog can be quantitatively explained on the basis of a reduced backbone entropy of unfolding due to the restriction of the conformational space allowed to Aib in respect to Ala, while the larger stabilization observed for the Ala309Aib analog can be accounted for by both entropic and hydrophobic effects. In fact, whereas Ala304 is a surface residue, Ala309 is shielded from the solvent, and thus the enhanced stability of fragment Ala309Aib is also due to the burial of an additional -CH3 group with respect to the natural fragment. The slightly destabilizing effect of the Ala-->Aib exchange in position 312 appears to derive from unfavorable strain energy effects, since phi and psi values for Ala312 are out of the allowed angles for Aib. Of interest, the simultaneous incorporation of Aib at positions 304 and 309 leads to a significant and additive increase of +8 degrees C in Tm. The results of this study indicate that the rational incorporation of Aib into a polypeptide chain can be a general procedure to significantly stabilize proteins.

Interleukin-6 (IL-6), a four-helix bundle protein, is a multifunctional cytokine which plays an important role in the regulation of the immune system, hematopoiesis, and inflammatory response, as well as in the pathogenesis of multiple myeloma. We have previously shown that a single-disulfide variant of human IL-6, lacking 22 N-terminal amino acids and the disulfide bond connecting Cys-45 and Cys-51 in the 185-residue chain of the wild-type protein, fully retains the conformational, stability, and functional properties of the full-length human IL-6 [Breton et al. (1995) Eur. J. Biochem. 227, 573-581]. In this study, we have investigated the conformational and stability properties of mutant IL-6 at acidic pH (A-state). Using far- and near-ultraviolet (UV) circular dichroism (CD), fluorescence emission, and second-derivative absorption spectroscopy, we have established that mutant IL-6 at pH 2.0 fully retains the helical secondary structure of the native protein at pH 7.5, while the tertiary interactions are much weaker. At variance from the native species, mutant IL-6 in the A-state binds 1-anilinonaphthalene-8-sulfonic acid (ANS), a property considered most typical of a protein in the molten globule state. The pH-induced conformational change from the native to the A-state, monitored either by near-UV CD or by ANS-binding measurements, shows a transition midpoint at pH approximately 4.5, thus indicating that the partial unfolding of the protein is mediated by the titration of glutamic and/or aspartic acid residues. At pH 2.0, the thermal denaturation of mutant IL-6 occurs as a broad process of low cooperativity with a transition at 50-60 degrees C, whereas at pH 7.5 the thermal unfolding is cooperative and characterized by a transition midpoint at 65 degrees C. Of interest, the unfolding of the A-state is not complete even up to approximately 85 degrees C. The urea-mediated unfolding profile of mutant IL-6, measured by far-UV CD, is essentially identical at both pH 7.5 and 2.0, with a midpoint of the cooperative unfolding transition at 5.5 +/- 0.1 M denaturant. Both thermal and urea denaturations of the A-state are complex and cannot fit to a two-state model for unfolding. The unusual stability of mutant IL-6 in acid is also reflected by the resistance to proteolysis at pH 3.6-4.0 by Staphylococcus aureus V8 protease or cathepsin D, an acid protease released by machrophages upon inflammatory stimulation. It is suggested that the molten globule state of IL-6 at acidic pH can play a role in the biological activity of this cytokine, which can exert its activity also at mildly acidic pH, as in inflammation sites.

The mouse Major Urinary Proteins (MUPs) contain a conserved β-barrel structure with a characteristic central hydrophobic pocket that binds a variety of volatile compounds. After release of urine, these molecules are slowly emitted in the environment where they play an important role in chemical communication. MUPs are highly polymorphic and conformationally stable. They may be of interest in the construction of biosensor arrays capable of detection of a broad range of analytes. In this work, 14 critical amino acids in the binding pocket involved in ligand interactions were identified in MUP20 using in silico techniques and 7 MUP20 mutants were synthesised and characterised to produce a set of proteins with diverse ligand binding profiles to structurally different ligands. A single amino acid substitution in the binding pocket can dramatically change the MUPs binding affinity and ligand specificity. These results have great potential for the design of new biosensor and gas-sensor recognition elements.

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.