The folding of immunoglobulin domains requires the formation of a conserved structural disulfide. Therefore, as a general rule, they cannot be functionally expressed in the reducing environment of the cellular cytoplasm. We have previously reported that stability engineering can lead to the cytoplasmic expression of functional immunoglobulin VL domains. Here we apply rational stability engineering by consensus sequence analysis to VH domains. Isolated VH domains tend to aggregate more easily than VL domains; they do not refold quantitatively and are generally more difficult to handle in vitro. To overcome these problems, we successfully predicted and experimentally verified several stabilizing point mutations in the VH domain of a designed, catalytic Fv fragment. The effect of single mutations was additive, and they could be combined in a prototype domain with significantly improved stability against chemical denaturation and a 20-fold increased half time of irreversible thermal denaturation, at physiological temperature. This stabilized, isolated VH domain could be expressed solubly in the reducing cellular cytoplasm of Escherichia coli, at a yield of approximately 1.2 mg/L of shake flask culture. It remains fully functional, as evidenced by the successful reconstitution of an esterolytic Fv fragment with the VL domain. This success provides further evidence that consensus sequence engineering is a rational, plannable route to the construction of intrabodies.

Orthologs typically retain the same function in the course of evolution. Using β-decarboxylating dehydrogenase family as a model, we demonstrate that orthologs can be confidently identified. The strategy is based on our recent findings that substitutions of only a few amino acid residues in these enzymes are sufficient to exchange substrate and coenzyme specificities. Hence, the few major specificity determinants can serve as reliable markers for determining orthologous or paralogous relationships. The power of this approach has been demonstrated by correcting similarity-based functional misassignment and discovering new genes and related pathways, and should be broadly applicable to other enzyme families.

The stability of proteins is known to be affected significantly in the presence of high concentration of salts and is highly pH dependent. Extensive studies have been carried out on the stability of proteins in the presence of simple electrolytes and evaluated in terms of preferential interactions and increase in the surface tension of the medium. We have carried out an in-depth study of the effects of a series of carboxylic acid salts: ethylene diamine tetra acetate, butane tetra carboxylate, propane tricarballylate, citrate, succinate, tartarate, malonate, and gluconate on the thermal stability of five different proteins that vary in their physico-chemical properties: RNase A, cytochrome c, trypsin inhibitor, myoglobin, and lysozyme. Surface tension measurements of aqueous solutions of the salts indicate an increase in the surface tension of the medium that is very strongly correlated with the increase in the thermal stability of proteins. There is also a linear correlation of the increase in thermal stability with the number of carboxylic groups in the salt. Thermal stability has been found to increase by as much as 22 °C at 1 M concentration of salt. Such a high thermal stability at identical concentrations has not been reported before. The differences in the heat capacities of denaturation, ΔCp for RNase A, deduced from the transition curves obtained in the presence of varying concentrations of GdmCl and that of carboxylic acid salts as a function of pH, indicate that the nature of the solvent medium and its interactions with the two end states of the protein control the thermodynamics of protein denaturation. Among the physico-chemical properties of proteins, there seems to be an interplay of the hydrophobic and electrostatic interactions that lead to an overall stabilizing effect. Increase in surface free energy of the solvent medium upon addition of the carboxylic acid salts appears to be the dominant factor in governing the thermal stability of proteins.

Recent studies have pointed out the important role of local water structures in protein conformational stability. Here, we present an accurate and computationally effective way to estimate the free energy contribution of the simplest water structure motif—the water bridge. Based on the combination of empirical parameters for accessible protein surface area and the explicit consideration of all possible water bridges with the protein, we introduce an improved protein solvation model. We find that accounting for water bridge formation in our model is essential to understand the conformational behavior of polypeptides in water. The model formulation, in fact, does not depend on the polypeptide nature of the solute and is therefore applicable to other flexible biomolecules (i.e., DNAs, RNAs, polysaccharides, etc.).

The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.

Transcription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription. TFIIB interacts with a promoter complex containing the TATA-binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF). TFIIB contains a zinc-binding motif near the N-terminus that is directly involved in the interaction with RNA pol II/TFIIF and plays a crucial role in selecting the transcription initiation site. The solution structure of the N-terminal residues 2–59 of human TFIIB was determined by multidimensional NMR spectroscopy. The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and “rubredoxin” turns, three antiparallel β-strands, and disordered loops. The structure is similar to the reported zinc-ribbon motifs in several transcription-related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (Pf TFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM). The zinc-ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the β-sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc-binding site may increase the stability of the β-sheet.

Electrostatic interactions are often critical for determining the specificity of protein-protein complexes. To study the role of electrostatic interactions for assembly of helical bundles, we previously designed a thermostable, heterotrimeric coiled coil, ABC, in which charged residues were employed to drive preferential association of three distinct, 34-residue helices. To investigate the basis for heterotrimer specificity, we have used multiwavelength anomalous diffraction (MAD) analysis to determine the 1.8 Å resolution crystal structure of ABC. The structure shows that ABC forms a heterotrimeric coiled coil with the intended arrangement of parallel chains. Over half of the ion pairs engineered to restrict helix associations were apparent in the experimental electron density map. As seen in other trimeric coiled coils, ABC displays acute knobs-into-holes packing and a buried anion coordinated by core polar amino acids. These interactions validate the design strategy and illustrate how packing and polar contacts determine structural uniqueness.

Dimeric proteins can arise by the swapping of structural domains between monomers. The prevalence of this occurrence is unknown. Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions. Here, this hypothesis is tested and found to be imprecise. The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S-peptide and S-protein) of the protein. The H12A and H119A variants have 105-fold less ribonucleolytic activity than does the wild-type enzyme. Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65 °C results in a 103-fold increase in ribonucleolytic activity. A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid. At pH 6.5 and 65 °C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer–dimer equilibrium. The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37 °C. This value of Kd is only 20-fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo. The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins. Dimers of “monomeric” proteins could be more prevalent than is usually appreciated.

Computer-based database searching and protein multiple sequence alignment has identified a novel clan of zinc metallopeptidases, which, by phylogenetic analysis, has been shown to contain six subfamilies. The family is characterized by four common transmembrane segments and three conserved sequence motifs. The combination of topology analysis and motif identification has detected three potential Zn2+ coordinating residues. Only two of the sequences of this novel zinc metallopeptidase clan possess any functional annotation, one of which is able to cleave its substrate within a cytosol/transmembrane segment junction. A number of observations suggest that the remaining members of this novel clan may also cleave their substrates within transmembrane segments.

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (UA), compact denatured (IM), and expanded helical denatured (H) states, and indicates that the compact denatured state (IM) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (UA and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, α-lactalbumin, β-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.

Domains belonging to the immunoglobulin-like fold are responsible for a wide variety of molecular recognition processes. Here we describe a new family of domains, the HYR family, which is predicted to belong to this fold, and which appears to be involved in cellular adhesion. HYR domains were identified in several eukaryotic proteins, often associated with Complement Control Protein (CCP) modules or arranged in multiple copies. Our analysis provides a sequence and structural basis for understanding the role of these domains in interaction mechanisms and leads to further characterization of heretofore undescribed repeated domains with similar folds found in several bacterial proteins involved in enzymatic activities (some chitinases) or in cell surface adhesion (streptococcal C-alpha antigen).

Comparisons of protein sequence via cyclic training of Hidden Markov Models (HMMs) in conjunction with alignments of three-dimensional structure, using the Combinatorial Extension (CE) algorithm, reveal two putative EF-hand metal binding domains in acetylcholinesterase. Based on sequence similarity, putative EF-hands are also predicted for the neuroligin family of cell surface proteins. These predictions are supported by experimental evidence. In the acetylcholinesterase crystal structure from Torpedo californica, the first putative EF-hand region binds the Zn2+ found in the heavy metal replacement structure. Further, the interaction of neuroligin 1 with its cognate receptor neurexin depends on Ca2+. Thus, members of the α,β hydrolase fold family of proteins contain potential Ca2+ binding sites, which in some family members may be critical for heterologous cell associations.

Staphylococcal nuclease forms three different partially-folded intermediates at low pH in the presence of low to moderate concentration of anions, differing in the amount of secondary structure, globularity, stability, and compactness. Although these intermediates are monomeric at low protein concentration (≤0.25 mg/mL), increasing concentrations of protein result in the formation of dimers and soluble oligomers, ultimately leading to larger insoluble aggregates. Unexpectedly, increasing protein concentration not only led to association, but also to increased structure of the intermediates. The secondary structure, stability, and globularity of the two less-ordered partially-folded intermediates (A1 and A2) were substantially increased upon association, suggesting that aggregation induces structure. An excellent correlation was found between degree of association and amount of structure measured by different techniques, including circular dichroism, fluorescence, Fourier transform infrared spectroscopy (FTIR), and small-angle X-ray scattering. The associated states were also substantially more stable toward urea denaturation than the monomeric forms. A mechanism is proposed, in which the observed association of monomeric intermediates involves intermolecular interactions which correspond to those found intramolecularly in normal folding to the native state.

A statistical analysis of a representative data set of 169 known protein structures was used to analyze the specificity of residue interactions between spatial neighboring strands in β-sheets. Pairwise potentials were derived from the frequency of residue pairs in nearest contact, second nearest and third nearest contacts across neighboring β-strands compared to the expected frequency of residue pairs in a random model. A pseudo-energy function based on these statistical pairwise potentials recognized native β-sheets among possible alternative pairings. The native pairing was found within the three lowest energies in 73% of the cases in the training data set and in 63% of β-sheets in a test data set of 67 proteins, which were not part of the training set. The energy function was also used to detect tripeptides, which occur frequently in β-sheets of native proteins. The majority of native partners of tripeptides were distributed in a low energy range. Self-correcting distance geometry (SECODG) calculations using distance constraints sets derived from possible low energy pairing of β-strands uniquely identified the native pairing of the β-sheet in pancreatic trypsin inhibitor (BPTI). These results will be useful for predicting the structure of proteins from their amino acid sequence as well as for the design of proteins containing β-sheets.

Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.

The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high β-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, β-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p.

Three peptides covering the sequence regions corresponding to the first two (CspB-1), the first three (CspB-2), and the last two (CspB-3) β-strands of CspB, the major cold shock protein of Bacillus subtilis, have been synthesized and analyzed for their conformations in solution and for their precipitation behavior. The peptides are nearly insoluble in water, but highly soluble in aqueous solutions containing 50% acetonitrile (pH 4.0). Upon shifts of the solvent condition toward lower or higher acetonitrile concentrations, the peptides all form fibrils resembling those observed in amyloid associated diseases. These fibrils have been identified and characterized by electron microscopy, binding of the dye congo red, and X-ray fiber diffraction. Characterization of the peptides in solution by circular dichroism and NMR spectroscopy shows that the formation of these fibrils does not require specific preformed secondary structure in the solution state species. While the majority of the soluble fraction of each peptide is monomeric and unstructured, different types of structures including α-helical, β-sheet, and random coil conformations are observed under conditions that eventually lead to fibril formation. We conclude that the absence of tertiary contacts under solution conditions where binding interactions between peptide units are still favorable is a crucial requirement for amyloid formation. Thus, fragmentation of a sequence, like partial chemical denaturation or mutation, can enhance the capacity of specific protein sequences to form such fibrils.

Bile-salt activated lipase (BAL) is a pancreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL (residues 1–538) with two surface mutations (N186D and A298D), which were introduced in attempting to facilitate crystallization, has been determined at 2.3 Å resolution. The crystal form belongs to space group P212121 with one monomer per asymmetric unit, and the protein shows an α/β hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding (residues 115–125 and 270–285).

Cathepsin K is a lysosomal cysteine protease belonging to the papain superfamily. It has been implicated as a major mediator of osteoclastic bone resorption. Wild-type human procathepsin K has been crystallized in a glycosylated and a deglycosylated form. The latter crystals diffract better, to 3.2 Å resolution, and contain four molecules in the asymmetric unit. The structure was solved by molecular replacement and refined to an R-factor of 0.194. The N-terminal fragment of the proregion forms a globular domain while the C-terminal segment is extended and shows substantial flexibility. The proregion interacts with the enzyme along the substrate binding groove and along the proregion binding loop (residues Ser138–Asn156). It binds to the active site in the opposite direction to that of natural substrates. The overall binding mode of the proregion to cathepsin K is similar to that observed in cathepsin L, caricain, and cathepsin B, but there are local differences that likely contribute to the specificity of these proregions for their cognate enzymes. The main observed difference is in the position of the short helix α3p (67p–75p), which occupies the S′ subsites. As in the other proenzymes, the proregion utilizes the S2 subsite for anchoring by placing a leucine side chain there, according to the specificity of cathepsin K toward its substrate.

Human pancreatic α-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 Å confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the kcat/Km values for D197A and D197N were 106–107 times lower than the wild-type enzyme. The decreased kcat/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile.