Showing papers in "Protein Engineering in 1997"

Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.

Abstract: We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction schemes and can easily be applied on genome-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal signal-anchor sequences is also possible, though with lower precision. Predictions can be made on a publicly available WWW server.

Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method.

Abstract: Prediction of transmembrane α-helices in prokaryotic membrane proteins : the dense alignment surface method

Hydrogen bonds and salt bridges across protein-protein interfaces.

Abstract: To understand further, and to utilize, the interactions across protein-protein interfaces, we carried out an analysis of the hydrogen bonds and of the salt bridges in a collection of 319 non-redundant protein-protein interfaces derived from high-quality X-ray structures. We found that the geometry of the hydrogen bonds across protein interfaces is generally less optimal and has a wider distribution than typically observed within the chains. This difference originates from the more hydrophilic side chains buried in the binding interface than in the folded monomer interior. Protein folding differs from protein binding. Whereas in folding practically all degrees of freedom are available to the chain to attain its optimal configuration, this is not the case for rigid binding, where the protein molecules are already folded, with only six degrees of translational and rotational freedom available to the chains to achieve their most favorable bound configuration. These constraints enforce many polar/charged residues buried in the interface to form weak hydrogen bonds with protein atoms, rather than strongly hydrogen bonding to the solvent. Since interfacial hydrogen bonds are weaker than the intra-chain ones to compete with the binding of water, more water molecules are involved in bridging hydrogen bond networks across the protein interface than in the protein interior. Interfacial water molecules both mediate non-complementary donor-donor or acceptor-acceptor pairs, and connect non-optimally oriented donor-acceptor pairs. These differences between the interfacial hydrogen bonding patterns and the intra-chain ones further substantiate the notion that protein complexes formed by rigid binding may be far away from the global minimum conformations. Moreover, we summarize the pattern of charge complementarity and of the conservation of hydrogen bond network across binding interfaces. We further illustrate the utility of this study in understanding the specificity of protein-protein associations, and hence in docking prediction and molecular (inhibitor) design.

Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification.

Abstract: The Strep-tag, an artificial peptide ligand of streptavidin, has gained use as an affinity handle for the purification and detection of recombinant fusion proteins. In an attempt to achieve tighter complexation of the peptide, streptavidin was engineered and the amino acid residues 44-47 in the flexible loop from 44 to 53, which is close to the binding site, were subjected to random mutagenesis. A fusion between alkaline phosphatase and the Strep-tag II sequence, an improved version of the Strep-tag, was constructed as a molecular probe for peptide binding. By means of a filter-sandwich assay, two streptavidin mutants with significantly stronger binding activity for the Strep-tag II were thus identified from a library of Escherichia coli colonies. Both in an ELISA with the alkaline phosphatase fusion and in a fluorescence titration experiment with the synthetic Strep-tag II peptide, which carried an anthraniloyl group as chromophore, their affinities were found to be higher by more than one order of magnitude compared with wild-type streptavidin. The nature of the amino acid exchanges and an enhanced electrophoretic mobility of the streptavidin tetramers suggest an altered loop conformation to be part of the optimized binding mechanism. When one of the streptavidin mutants was immobilized on a chromatographic column it exhibited clearly improved performance in the purification of Strep-tag II fusion proteins, and desthiobiotin turned out to be a suitable reagent for mild competitive elution.

Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment.

Abstract: associated with the constant domains of the light (CL) and heavy chain (CH1). As a consequence, all residues of the in expression yield between different antibody sequences show former variable/constant domain interface become solvent that protein sequence-related factors are the major remaining exposed. In an analysis of 30 non-redundant Fab structures challenge. Folding efficiency and stability of the antibody it was found that at the former variable/constant domain fragments, as well as protease lability and toxicity of the interface of the Fv fragment the frequency of exposed expressed antibody fragments to the host cells, often severely hydrophobic residues is much higher than in the rest of limit actual production levels, but the rational understanding the Fv fragment surface. We investigated the importance of these problems is only beginning. of these residues for different properties such as folding Knappik and Pluckthun (1995) showed that the primary in vivo and in vitro, thermodynamic stability, solubility of sequence of a particular antibody emerges as the most decisive the native protein and antigen affinity. The experimental factor determining the yield of functional protein. Based on a model system was the scFv fragment of the anti-fluorescein sequence comparison of different antibodies, back-engineering antibody 4-4-20, of which only 2% is native when expressed pin-pointed the effect to just a few point mutations. While in the periplasm of Escherichia coli. To improve its in vivo these particular mutations are only applicable to a few anti- folding, a mutagenesis study of three newly exposed inter- bodies and thus constitute a tailor-made solution, they do show facial residues in various combinations was carried out. that very minor sequence changes can have a dramatic effect The replacement of one of the residues (V84D in VH) led on the in vitro aggregation properties of these molecules and to a 25-fold increase of the functional periplasmic expres- the in vivo functional expression of antibody fragments in the sion yield of the scFv fragment of the antibody 4-4-20. periplasm of E.coli. Similarly, Ullrich et al. (1995) found that With the purified scFv fragment it was shown that the point mutations in the complementarity-determining regions thermodynamic stability and the antigen binding constant (CDRs) can increase the yields in periplasmic antibody frag- are not influenced by these mutations, but the rate of the ment expression. thermally induced aggregation reaction is decreased. Only The observations of Knappik and Pluckthun (1995) indicate a minor effect on the solubility of the native protein that optimizing those parts of the antibody fragment which are was observed, demonstrating that the mutations prevent not directly involved in antigen recognition can significantly aggregation during folding and not of the native protein. improve folding properties and production yields of recom- Since the construction of all scFv fragments leads to the binant Fv and scFv constructs. The causes of the improved exposure of these residues at the former variable/constant expression behavior lie in the decreased aggregation behavior domain interface, this strategy should be generally applic- of these molecules. The understanding of how specific sequence able for improving the in vivo folding of scFv fragments modifications change these properties is still very limited and and, by analogy, also the in vivo folding of other engineered currently under active investigation. protein domains. Normally, the majority of the side chain residues exposed at

Prediction of the stability of protein mutants based on structural environment-dependent amino acid substitution and propensity tables.

Abstract: An approach to the prediction of mutant stability is described using knowledge of amino acid replacements that are tolerated within the families of homologous proteins of known 3-D structure. Amino acid variations in families of homologous proteins are converted to propensity and substitution tables; these provide quantitative information about the existence of an amino acid in a structural environment and the probability of replacement by any other amino acid. The tables are used to calculate a 'stability difference score', analogous to the difference in free energy between a mutant and the wild type. The method has been developed and tested using the high-resolution structures for T4 lysozyme and 159 site-specific mutants. We show that differences in stability scores are correlated with experimentally observed free energy differences and differences in melting temperature. Blind tests, using only structural information derived from the parent wild-type crystal structures, on a combined set of 83 staphylococcal nuclease and 68 barnase mutants showed a correlation of 0.80 in the predicted stability changes with experimental thermodynamic data. Approximately 86% of the predictions were correctly classified as destabilizing or stabilizing.

Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer

Abstract: Single-chain variable fragments (scFvs) of anti-neuraminidase antibody NC10 were constructed by joining the VH and VL domains with 10-residue (Gly4Ser)2 and five-residue (Gly4Ser) linkers; a zero-residue linker scFv was constructed by joining the C-terminal residue of the VH domain to the N-terminus of the VL domain. The scFv with the 10- and five-residue linkers exclusively formed dimeric antibody fragments (M(r) 52000). These were shown to be bivalent and were able to cross-link two neuraminidase tetramers to form a 'sandwich' type complex; each antigen combining site could also bind an anti-idiotype Fab'. The zero-residue linker scFv (M(r) 70000) was shown to form a trimer with three active antigen combining sites, each binding an anti-idiotype Fab' to yield a complex of M(r) 212000. The orientation of the combining sites in the zero-residue linker scFv, however, was such that it could not cross-link tetramers of neuraminidase. BIAcore biosensor experiments showed that the affinity of each individual antigen combining site in both the 10- and five-residue linker scFv dimers and zero-residue linker scFv trimer was essentially the same when the scFvs were immobilized onto the sensor surface. However, when the scFvs were used as the analyte, the dimeric and trimeric scFvs showed an apparent increase in binding affinity due to the avidity of binding the multivalent scFvs.

Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting

Abstract: The complementary determining regions (CDRs) from the fluorescein-binding antibody 4-4-20, which yields almost no soluble protein in periplasmic expression in Escherichia coli, were transplanted to the framework of the humanized antibody 4D5. The resulting single-chain Fv fragment (scFv) 4D5Flu showed both a dramatic improvement in soluble expression, even at 37 degrees C, and an improved thermodynamic stability. Antigen affinity was maintained upon this engineering by paying attention to crucial framework-CDR contacts. This demonstrates that the use of superior frameworks is a robust strategy to improve the physical properties of scFv fragments. We also report that the grafted version was selected in phage display over several competing variants of the same antibody with identical binding constant but poorer folding or stability properties. The selection required four panning rounds and a temperature of 37 degrees C and we show that the underlying reason for this selection is a higher fraction of phages carrying functional scFv molecules.

Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity.

Abstract: Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30-fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid influences the correct folding of the VH domain, presumably by affecting a folding intermediate, but has no effect on antigen binding.

Beta-hairpins in proteins revisited: lessons for de novo design.

Abstract: Beta-Hairpins with short connecting loops (1-5 residues) have been identified from a data set of 250 non-homologous, high resolution (< or =2.0 A) protein crystal structures. The conformational preferences of the loop segments have been analyzed with the specific aim of identifying frequently occurring motifs. Type I' and II' beta-turns were found to have a high propensity for occurrence in two residue loops. For three and four residue loops, the major conformational motif in the linking segments is alphaR-alphaR-alphaL (type I beta-turn followed by a residue in a left-handed helical conformation) and alphaR- alphaR-alphaR-alphaL (a pi-turn motif), respectively. The present larger data set confirms the high occurrences of these motifs which have been identified in earlier analyses. In addition to type I' and type II' beta-turns, several examples of type I beta-turn nucleated two residue loop hairpins, in spite of having an opposing sense of twist to that of type I' beta-turn, have also been observed. Examination of these frequently occurring motifs (flanked by extended conformation [beta]) in the data set reveals that the motifs beta-alphaR-alphaR- alphaL-beta and beta-type I'-beta have equal propensity and type II' indeed having highest propensity to nucleate beta-hairpins. The larger number of examples in this study allows the estimation of the specific amino acid preferences for loop positions in two, three and four residue loops. Small polar residues Asn, Asp, Ser, Thr, Gly and Pro in general have a high propensity for the loop positions but they reveal specific positional preferences in these frequently occurring motifs. There are no strong compositional preferences in the strand segments. Amino acid pair correlations across strands also do not show any significant pattern, with the exception of Cys-Cys pairs. Several Cys- Cys pairs have been identified at the non-hydrogen bonded positions of beta-hairpins; as many as six are disulfide bonded pairs. An examination of longer loop length hairpins reveals that the distortions of hairpins nucleated by tight turns (two residues) are much less frequently observed. The results presented in this study provide inputs for the de novo design of consensus loop segments in synthetic hairpins.

Isolation of anti-T cell receptor scFv mutants by yeast surface display.

Abstract: Yeast surface display and sorting by flow cytometry have been used to isolate mutants of an scFv that is specific for the Vbeta8 region of the T cell receptor. Selection was based on equilibrium binding by two fluorescently labeled probes, a soluble Vbeta8 domain and an antibody to the c-myc epitope tag present at the carboxy-terminus of the scFv. The mutants that were selected in this screen included a scFv with threefold increased affinity for the Vbeta8 and scFv clones that were bound with reduced affinities by the anti-c-myc antibody. The latter finding indicates that the yeast display system may be used to map conformational epitopes, which cannot be revealed by standard peptide screens. Equilibrium antigen binding constants were estimated within the surface display format, allowing screening of isolated mutants without necessitating subcloning and soluble expression. Only a relatively small library of yeast cells (3 x 10[5]) displaying randomly mutagenized scFv was screened to identify these mutants, indicating that this system will provide a powerful tool for engineering the binding properties of eucaryotic secreted and cell surface proteins.

Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris.

Abstract: Diabodies are dimeric antibody fragments held together by associated heavy and light chain variable domains present on different polypeptide chains. To improve their stability we have introduced cysteine residues into the V-domains to promote the disulphide crosslinking of the dimer. A crosslinked bivalent diabody against carcinoembryonic antigen (CEA) and a crosslinked bispecific diabody against CEA and the T-cell co-receptor CD3 were expressed from Pichia pastoris and Escherichia coli by secretion. From Pichia (but not E.coli) the chains were almost quantitatively crosslinked. Compared with the parent diabodies both crosslinked diabodies were more stable to heat (by >7 degrees C) and the crosslinked bivalent diabody showed improved localization to CEA+ human tumour xenografts in nude mice.

Protein distance constraints predicted by neural networks and probability density functions.

Abstract: We predict interatomic Calpha distances by two independent data driven methods. The first method uses statistically derived probability distributions of the pairwise distance between two amino acids, whilst the latter method consists of a neural network prediction approach equipped with windows taking the context of the two residues into account. These two methods are used to predict whether distances in independent test sets were above or below given thresholds. We investigate which distance thresholds produce the most information-rich constraints and, in turn, the optimal performance of the two methods. The predictions are based on a data set derived using a new threshold which defines when sequence similarity implies structural similarity. We show that distances in proteins are predicted more accurately by neural networks than by probability density functions. We show that the accuracy of the predictions can be further increased by using sequence profiles. A threading method based on the predicted distances is presented. A homepage with software, predictions and data related to this paper is available at http://www.cbs.dtu.dk/services/CPHmodels/.

Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold.

Abstract: A subtilisin excreted by the Antarctic Bacillus TA39 has been purified to homogeneity and characterised. Two independent genes subt1 and subt2 are present but only subt1 is expressed significantly in the culture medium. The enzyme displays the usual characteristics of cold enzymes i.e. a high catalytic efficiency at low and moderate temperatures and an increased thermosensitivity originating from a 3D structure probably more flexible than its mesophilic counterpart. This is corroborated by the analysis of the computerized structure which shows a significant decrease in the number and strength of intramolecular weak bonds such as salt bridges and aromatic interactions. The affinity for calcium is also almost three orders of magnitude lower than that of mesophilic subtilisin and the interactions with the solvent are significantly higher thanks to a large increase in the number of Asp residues in the loops connecting secondary structures. The relation between flexibility and activity was investigated by site-directed mutagenesis tending mainly to increase the rigidity of the molecular edifice through the incorporation of additional salt bridge, disulfide bridge, aromatic interaction and by increasing the affinity of the enzyme for calcium. An important stabilization of the molecular structure was achieved through a modification of a calcium ligand T85D. The thermostability of the mutated product expressed in a mesophilic Bacillus reaches that of mesophilic subtilisin. Most important is the fact that this mutation further enhances the specific activity by a factor close to 2 when compared to the wild type enzyme so that the overall activity of the mutated cold enzyme is about 20 times higher than that of mesophilic subtilisin, illustrating the fact that thermostability is not systematically inversely related to specific activity. This opens new perspectives in the use of cold enzymes in biotechnology.

Effectiveness of correlation analysis in identifying protein residues undergoing correlated evolution

Abstract: Various methods for detecting correlation between sites were evaluated by ascertaining their ability to discriminate positively correlated sites from background correlation at randomly evolved sites. A model for generating pairwise correlations of different degrees is also described. An assortment of physicochemical vectors and similarity and difference matrices were used to discriminate correlated change. There was little difference in effectiveness between the different matrices, but there were significant differences between the matrices and the physicochemical vectors. It is shown that all methods investigated exhibit significant inability to screen out background correlation, particularly in the presence of phylogenetic relatedness between the sequences. Methods using the matrices are unable to distinguish positively correlated from negatively correlated, or compensatory, replacements.

Affinity selection of a camelized V(H) domain antibody inhibitor of hepatitis C virus NS3 protease.

Abstract: The HCV genome encodes, within the NS3 gene, a serine protease whose activity specifically cleaves the viral polyprotein precursor. Proteolytic processing of HCV polyprotein precursor by the viral NS3 proteinase is essential for virion maturation and designing specific inhibitors of this protease as possible anti-viral agents is a desirable and practical objective. With a view to studying both the function of HCV NS3 protease and to designing inhibitors of this enzyme, we directed our interest towards engineering macromolecular inhibitors of the viral protease catalytic activity. We describe here the affinity-selection and biochemical characterization of one inhibitor, cV(H)E2, a 'camelized' variable domain antibody fragment, isolated from a phage displayed synthetic repertoire, which is a potent and selective inhibitor of proteolysis by the NS3 enzyme. In addition to being useful as a biological probe to study the function of HCV protease, this inhibitor can serve as a potential pharmacophore model to design antivirals. Moreover, the results suggest a way of engineering improved human-derived small recognition units tailored for enzyme inhibition.

High-resolution crystal structure of M-protease: phylogeny aided analysis of the high-alkaline adaptation mechanism.

Abstract: M-protease is a subtilisin-family serine protease produced by an alkaliphilic Bacillus sp. strain. Optimal enzymatic activity of the protein occurs at pH 12.3. The crystal structure of M-protease (space group P2(1)2(1)2(1), a = 62.3, b = 75.5, c = 47.2 A) has been refined to a crystallographic R-factor of 17.2% at 1.5 A resolution. The alkaline adaptation mechanism of the enzyme was analyzed. Molecular phylogeny construction was used to determine the amino acid substitutions that occurred during the high-alkaline adaptation process. This analysis revealed a decrease in the number of negatively charged amino acids (aspartic acid and glutamic acid) and lysine residues and an increase in arginine and neutral hydrophilic amino acids (histidine, asparagine and glutamine) residues during the course of adaptation. These substitutions increased the isoelectric point of M-protease. Some of the acquired arginine residues form hydrogen bonds or ion pairs to combine both N- and C-terminal regions of M-protease. The substituted residues are localized to a hemisphere of the globular protein molecule where positional shifts of peptide segments, relative to those of the less alkaliphilic subtilisin Carlsberg, are observed. The biased distribution and interactions caused by the substituted residues seem to be responsible for stabilization of the conformation in a high-alkaline condition.

Spontaneous oligomerization of a staphylococcal alpha-hemolysin conformationally constrained by removal of residues that form the transmembrane beta-barrel.

Abstract: Staphylococcal alpha-hemolysin is a water soluble, monomeric, bacterial exotoxin, which forms heptameric pores in membranes. The rate determining step in assembly is the conversion of a heptameric prepore to the fully assembled pore in which the central glycine-rich domain of each subunit inserts into the membrane to form a 14 strand beta barrel. Barrel formation is accompanied by a conformational change in which each N terminus latches onto an adjacent subunit. In the monomer in solution, the central domain is loosely organized and exposed to solvent. In this study, 25 amino acids of the central domain were removed and replaced with the sequence Asp-Gly, which favors the formation of a type I' beta-turn, to yield a mutant devoid of hemolytic activity. Within minutes after synthesis in the absence of membranes, the mutant polypeptide spontaneously assembled into heptamers, as demonstrated by atomic force microscopy. Limited proteolysis suggested that the N termini of the subunits in the heptamers were in the fully assembled pore conformation rather than the prepore conformation. Based on these findings, the deletion is proposed to constrain the central domain and thereby force the creation of a shortened beta barrel, which in turn induces the additional structural changes that normally accompany pore formation. The truncated pore might make a useful framework for the construction of designed membrane active macromolecules.

Mammalian cell expression of dimeric small immune proteins (SIP)

Abstract: We have designed and expressed bivalent small immune proteins (SIP) based on scFv fragments connected through a short linker of four amino acids to the CH3 domain of the human immunoglobulin gamma 1 H-chain. Three different versions have been designed and expressed in mammalian cells. In one construct a cysteine residue was included in the last amino acid of the flexible 15-amino acid long linker connecting the V(L) and V(H) domains, thus creating a disulphide bond stabilized molecule. A version with a shorter (five amino acids) V(L)/V(H) linker was also produced and shown to be efficiently assembled and secreted. All three SIPs form dimers retaining their antigenic specificity in Western blotting and having a comparable functional affinity (avidity) as determined by ELISA.

Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions

Abstract: The complementarity-determining regions (CDRs) of a human kappa light chain were replaced with CDRs from a murine gamma-1 heavy chain and, by use of molecular modeling, key heavy chain framework residues were identified and thus included to preserve the native conformation of the heavy chain CDRs. Co-expression of this hybrid human kappa chain (V[HB]C[L]) with a human kappa chain counterpart (V[L]C[L], engineered to contain murine light chain CDRs) resulted in the secretion of high levels of a heterodimeric protein (V[HB]C[L]::V[L]C[L]) termed 'kappabody'. This protein also had equivalent affinity for antigen as the Fab' of the parent murine IgG1. High-level secretion was also observed for the hybrid chain as homodimers (V[HB]C[L]::V[HB]C[L]), which is not observed for chimeric chains consisting of a heavy chain variable region and light chain constant region, i.e. V[H]C[L] homodimers or single chains are not secreted. This indicates that regions within the variable domain, required for secretion of light chains, reside outside of the hypervariable regions (CDRs) and that the heavy chain CDRs and supporting residues do not prevent secretion. These results demonstrate the possibility of designing small, single-domain molecules possessing a given binding activity which may be secreted at high levels from mammalian cells.

Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli.

Abstract: When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) is expressed in Escherichia coli, it is autophosphorylated at four sites, Ser10, Ser139, Ser338 and Thr197 (49). Three of these sites, Ser10, Ser338 and Thr197, are also found in the mammalian enzyme. To understand the functional importance of these phosphorylation sites, each was replaced with Ala, Glu or Asp. The expression, solubility and phosphorylation state of each mutant protein was characterized by immunoprecipitation following in vivo labeling with 32Pi. When possible, isoforms were resolved and kinetic properties were measured. The two stable phosphorylation sites in the mammalian enzyme, Ser338 and Thr197, were shown to play different roles. Ser338, which stabilizes a turn near the C-terminus, is important for stability. Both rC(S338A) and rC(S338E) were very labile; however, the kinetic properties of rC(S338E) were similar to the wild-type catalytic subunit (C-subunit). Ser338 most likely helps to anchor the C-terminus to the surface of the small lobe. Thr197 is in the activation loop near the cleft interface. Mutagenesis of T197 caused a significant loss of catalytic activity with increases in Kms for both peptide and MgATP, as well as a small decrease in k(cat) indicating that this phosphate is important for the correct orientation of catalytic residues at the active site. Replacement of Ser139, positioned at the beginning of the E-helix, with Ala had no effect on the kinetic parameters, stability or phosphorylation at the remaining sites. In contrast, mutation of Ser10, located at the beginning of the A-helix, produced mostly insoluble, inactive, unphosphorylated protein, suggesting that this region, though far removed from the active site, is structurally important at least for the expression of soluble phosphoprotein in E.coli. Since the mutation of active site residues as well as deletion mutants generate underphosphorylated proteins, these phosphorylations in E.coli all result from autophosphorylation.

Production of human normal adult and fetal hemoglobins in Escherichia coli.

Abstract: A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.

Exploring the limits of nearest neighbour secondary structure prediction

Abstract: that the morphology of the prediction is correct (Zimmermann, 1994), i.e. a minimum length of four residues for α-helices and two for β-strands. Analysis of the results suggests that the current data set of known structures can be considered as

Hyperthermostable mutants of Bacillus licheniformis alpha-amylase: thermodynamic studies and structural interpretation.

Abstract: This paper provides further understanding of the thermodynamic and structural features determining the stability of Bacillus licheniformis alpha-amylase (BLA) at two crucial positions, His133 and Ala209. Results of protein modelling and saturated site-directed mutagenesis at position 133 and 209 have been reported in a previous paper (Declerck et al., 1995, Prot. Engng, 8, 1029-1037). In the first part of the present work, evidence is presented supporting the hypothesis that the stabilizing mutations reduce the rate of initial unfolding of the enzyme during the reversible step of the inactivation reaction and do not modify the irreversible processes undergone subsequently by the unfolded molecules. In the second part, we have examined the three-dimensional structure of BLA which has been determined recently by X-ray analysis (Machius et al., 1995, J. Mol. Biol., 246, 545-559). This analysis showed that our previous predictions made from molecular modelling were partly correct. At position 209, the effect of the stabilizing substitutions can be explained by a groove-filling effect reinforcing the hydrophobic packing between two helices of the central domain, while preserving a well-ordered water structure at the surface. At position 133, the stabilizing substitutions must compensate the loss of the hydrogen bond network in which the original histidine side-chain is involved; this compensation could be achieved through enhanced hydrophobic side-chain interactions within the beta-sheet where residue 133 is located, which correlates with the propensity of the residue to form and maintain a beta-strand conformation of the main chain at this position.

High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties

Abstract: Bovine beta-lactoglobulin (BLG) variant A has been expressed in the methylotropic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the alpha-mating factor prepro-leader peptide from Saccharomyces cerevisiae. P. pastoris Mut+ transformants were obtained by single cross-over integration of the BLG-containing vector into the AOX1 locus. In a fed-batch fermenter, a cell density of approximately 300 mg/ml was achieved by controlled glycerol feeding for a total of 24 h. After 72 h of methanol induction, the secreted BLG reached levels of > 1 g/l. The secreted protein could be purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the secreted BLG revealed that the Glu-Ala spacer repeats inserted between the mature protein and the alpha-factor prepro-leader were still present. The purified protein was characterized by a number of methods, including CD spectroscopy, guanidine-HCl unfolding, crystallization and two-dimensional 1H-NMR spectroscopy. By all of these measures, the physical characteristics of recombinant BLG were indistinguishable from those of the native purified bovine BLG, making it useful as a model for protein folding and other biophysical studies.

Correlation between side chain mobility and conformation in protein structures.

Abstract: Thermal factors of protein atoms as determined by X-ray crystallographic techniques show a tendency to be larger in side chains with unfavourable local conformations rather than in those displaying conformational energy minima. It follows that side chain atoms are more mobile if they are in a non-rotameric configuration and that the stereochemistry of protein structures cannot be fully assessed or simulated without consideration of thermal factors that monitor flexibility in various regions of the protein. The observations should also prove useful in protein folding and design.

Functional expression of adhesive peptides as fusions to Escherichia coli flagellin.

Abstract: An expression system for studying epitopes of adhesion proteins based on fusion of gene fragments into fliC(H7) of Escherichia coli is described. We constructed the system by an in-frame insertion of DNA fragments encoding one, two or three of the fibronectin-binding D repeats present in the fibronectin-binding protein A (FnBPA) of Staphylococcus aureus, into the fliC(H7) gene region encoding the variable domain of the H7 flagellin. The constructs were expressed by in trans complementation in the E. coli strain JT1 which harbours knock-out mutations for the expression of FliC as well as of the mannoside-binding fimbrial adhesin. The resulting chimeric flagella, which contained 39, 77 or 115 heterologous amino acid residues, efficiently bound soluble and immobilized human plasma and cellular fibronectin, and the binding was most efficient with the flagella containing the three D repeats of FnBPA. The chimeric flagella bound to frozen sections of human kidney and to cultured human cells. Antibodies raised against the chimeric flagella bound to Protein A-deficient S. aureus cells and inhibited the binding of staphylococci to immobilized fibronectin. We also expressed peptides, ranging in size between 48 and 302 amino acids, of the collagen-binding YadA adhesin of Yersinia enterocolitica. A fragment of 302 amino acids representing the middle region of YadA was needed for collagen binding. Chimeric flagellar filaments expressing hundreds of intimately associated adhesive epitopes offer versatile tools to analyze adhesin-receptor interactions and functional epitopes of adhesion proteins.

Recombinant ferritin: modulation of subunit stoichiometry in bacterial expression systems.

Abstract: We describe a strategy for the creation of recombinant ferritin heteropolymers which mimic the natural heterogeneity of this protein. This method entailed the co-expression of cDNA for both ferritin H and ferritin L subunits in a single bacterium using either a bicistronic vector, in which both cDNAs were expressed from the vector, or a dual vector expression strategy, in which each subunit was expressed from a separate compatible plasmid in a single bacterial host. Electron microscopy and sucrose density gradient centrifugation demonstrated that ferritin assembled spontaneously in such bacteria to form catalytically active proteins of the expected size and shape. Isoelectric focusing revealed that protein isolated from any of these bacteria exhibited a restricted heterogeneity in subunit composition. Such multi-subunit recombinant ferritins spontaneously assembled in bacteria may be useful in further studies of ferritin assembly and function. Our results further suggest that varying expression levels is a simple way to alter levels of individual components within a multi-subunit recombinant protein, and that this approach may be of general utility in assessing the contribution of individual components to the function of multi-subunit proteins or protein complexes.

Can protein unfolding simulate protein folding

Abstract: Since the discovery that the native structure of a globularprotein can fold spontaneously (Anfinsenet al., 1961), numer-ous experimental and theoretical efforts have been made toelucidate folding pathways, intermediates, transition states,etc. (for recent reviews, see Bryngelson et al., 1995; Fersht,1995, 1997; Karplus and Sˇali, 1995; Ptitsyn, 1995; Mirankerand Dobson, 1996; Shakhnovich, 1997). Protein folding path-ways are of great interest not only in themselves, but alsobecause understanding them is important for both proteinstructure predictions (one has to know what to search for: themost stable chain fold or the one resulting from a rapid foldingpathway) and for de novo protein design (one has to knowwhat to design: a stable fold only or a pathway to this fold also).Molecular dynamics (MD) simulations aim to provide themost detailed description of the behavior of ‘realistic’ atomicmodels of proteins during their folding or unfolding processes.In practice, however, these simulations are used mostly tostudy the unfolding from the native state (Caflisch and Karplus,1994, 1995; Daggett and Levitt, 1994; Hunenberger et al.,1995; Daggett et al., 1996; Li and Daggett, 1996; Williamset al., 1997).The reason is simple. Current MD simulations, even withthemostpowerfulcomputationaltechniques,canbarelyexploremore than a few nanoseconds for an all-atom model of aprotein and its solvent environment. On the other hand, thefolding or unfolding experiment takes not less than hundredsof microseconds (Huang and Oas, 1995; Schindler et al., 1995;Eaton et al., 1997), and usually milliseconds, seconds orminutes (Segawa and Sugihara, 1984; Radford et al., 1992;Itzhaki et al., 1994; Eaton et al., 1997; Roder and Colo´n,1997). Thus, one has to accelerate the events in simulations.This can be done with high temperature, but a high temperaturecorresponds to unfolding conditions. Therefore, unfolding ismuch more tractable than folding for molecular dynamics.To observe anything interesting on the nanosecond simula-tion time-scale, MD has to operate under extreme unfoldingconditions where the protein virtually explodes: usually, atemperature of 500–600 K is used (Caflisch and Karplus, 1994,1995; Daggett and Levitt, 1994; Hunenberger et al., 1995;Daggett et al., 1996; Li and Daggett, 1996) to surmount theunfolding activation barrier over 1 ns. Such a high temperaturemust accelerate the processes by about six orders of magnitude(Karplus and Sˇali, 1995) compared with room temperaturebut, certainly, it allows an investigation of the proteinunfolding process.So far, to the author’s knowledge, no-one has suggested aneffective way to accelerate the folding process, while unfoldingcan be accelerated in different ways. To avoid an unrealisticallyhigh temperature of 500–600 K, some investigators have usedlow pH (strong electrostatic repulsion) combined with a

Identification of membrane spanning beta strands in bacterial porins.

Abstract: The membrane assembly of outer membrane proteins is more complex than that of transmembrane helical proteins owing to the intervention of many charged and polar residues in the membrane. Accordingly, the predictive accuracy of transmembrane beta strands is considerably lower than that of transmembrane alpha helices. In this paper we develop a set of conformational parameters for membrane spanning beta strands. We formulate an algorithm to predict the transmembrane beta strands in the family of bacterial porins based on the conformational parameters and surrounding hydrophobicities of amino acid residues. A Fortran program has been developed which takes the amino acid sequence as the input file and gives the predicted transmembrane beta strand as output. The present method predicts at an accuracy level of 82% for all the bacterial porins considered.