A major challenge in protein design is to create stable scaffolds into which tailored functions can be introduced. Here we present the design, synthesis and characterization of a 61-residue all-beta protein: the minibody. We used a portion of the heavy chain variable domain of an immunoglobulin as a template, obtaining a molecule with a novel beta-sheet scaffold and two regions corresponding to the hypervariable loops H1 and H2. To exploit the potential for creating functional centres in the minibody, we engineered a metal-binding site into it. This site is formed by one histidine in H1 and two in H2. The protein is folded, compact and able to bind metal, thus representing the first designed beta-protein with a novel fold and a tailored function. By randomizing the sequence of the hypervariable loops, we are using the minibody scaffold to construct a conformationally constrained peptide library displayed on phage.

The hepatitis C virus NS3 protein contains a serine protease domain with a chymotrypsin-like fold, which is a target for development of therapeutics. We report the crystal structures of this domain complexed with NS4A cofactor and with two potent, reversible covalent inhibitors spanning the P1-P4 residues. Both inhibitors bind in an extended backbone conformation, forming an antiparallel ␤-sheet with one enzyme ␤-strand. The P1 residue contributes most to the binding energy, whereas P2-P4 side chains are partially solvent exposed. The structures do not show notable rearrangements of the active site upon inhibitor binding. These results are significant for the development of antivirals. Hepatitis C virus (HCV)1 infection is a major health problem that leads to cirrhosis and hepatocellular carcinoma in a substantial number of infected individuals estimated at 100 -200 million worldwide. Immunotherapy or other effective treatments for HCV infection are not yet available, and administration of interferon in combination with ribavirin has several limitations because of toxicity (1). One of the best characterized targets for HCV therapy is the serine protease of NS3 protein. The NS3 protease domain constitutes the N terminus of the NS3 protein, which, when associated to the NS4A polypeptide, gets activated and therefore is responsible for maturation of the viral polyprotein (2).The structure determination of the HCV NS3 protease complexed with a truncated NS4A cofactor (residues 21-34) revealed a shallow, nonpolar P1 specificity pocket. Because of the unusual substrate specificity of this enzyme it has been inferred that the design of highly selective inhibitors that could bind to the NS3 protease would be unlikely (3, 4). We have found that capped tri-peptide ␣-ketoacids, incorporating difluoro aminobutyric acid in the P1 position, are potent, slow binding inhibitors of this enzyme (5). Their mechanism of inhibition is biphasic. The first kinetic phase involves the rapid formation of a noncovalent collision complex with association rate constants Ͼ0.2 s Ϫ1 , and the second kinetic phase consists of a slow isomerization with rate constants between 5 ϫ 10 Ϫ3 and 7.5 ϫ 10 Ϫ3 s Ϫ1 . This results in the formation of a very tight complex with dissociation rate constants between 1.2 ϫ 10 Ϫ5 and 1.8 ϫ 10Ϫ5 s Ϫ1 and with half-lives of 11-16 h. The overall K i values are between 27 and 67 nM (5).The inhibitors described here span the P1-P4 residues and contain an activated carbonyl in an ␣-ketoacid moiety as the active-site serine trap. To investigate the binding mode of these compounds, an hexagonal crystal form of the NS3 protease domain (J strain) complexed with the truncated NS4A cofactor, amenable to soaking experiments, was obtained. The crystal structures of the noninhibited NS3/4A complex (2.4 Å) and with two inhibitors (Fig. 1A), inhibitor I,were solved. EXPERIMENTAL PROCEDURESProtein Expression and Purification-A DNA fragment encoding the serine protease domain of NS3J (amino acids 1-187) was obtained by polymerase c...

A strategy that combines limited proteolysis experiments and mass spectrometric analysis of the fragments generated has been developed to probe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64-residue protein consisting of a P-sheet scaffold based on the heavy-chain variable-domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable HI and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X-ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single-residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X-ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.

The surface topology of the Minibody, a small de novo-designed P-protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values.The results of this study indicate that, while overall the Minibody model is correct, the P-sheet formed by the N-and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini s, Sollazzo M, Tramontano A, Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5902-813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X-ray structure cannot be obtained due to insufficient solubility of the molecule.

~ ~~A transitory cessation of growth was recorded in Sfrepfomyces coelicolor A3(2) at the end of vegetative mycelium formation on solid medium. In the same phase a striking reduction in protein and nucleic acid synthesis was detected. Growth and macromolecular synthesis resumed, nearly reaching the original values, when morphological differentiation occurred. It is concluded that a physiological stress occurs within the bacterial population just before the onset of the morphological differentiation.