Combination Of Protein Engineering Research Articles

Architecture Effects on the Binding of Cholera Toxin by Helical Glycopolypeptides.

A variety of binding events in biological systems are mediated by multivalent interactions between oligosaccharides and saccharide receptors present on pathogens and cell surfaces. In particular, given the important role of multivalent interaction between proteins and carbohydrates in the initial step of pathogen recognition, many glycosylated molecules and polymers have been synthesized in order to mimic the carbohydrate ligands and to inhibit the binding of the pathogen to its target. In this work, we extend our evaluation of the impact of the architecture of well-defined glycopolypeptides on the inhibition of binding of the cholera toxin B pentamer (CT B(5)) subunit. Here we report the production of two families of α-helical glycopolypeptides which were synthesized via a combination of protein engineering and chemical methods. The presentation of pendant saccharides on the polypeptide backbones, as well as their valencies, can be well controlled via these methods. Control of the backbone conformation, introduced in this report, is also possible via these strategies. The polypeptides and glycopolypeptides were characterized via SDS-PAGE analysis, (1)H NMR, and MALDI-TOF mass spectrometry. Their conformation and hydrodynamic volume were characterized by circular dichroic (CD) spectroscopy and gel permeation chromatography (GPC), respectively. The binding of CT B(5) by these glycopolypeptides was evaluated via direct enzyme-linked immunosorbent assay (DELA). The effects of spacing and conformation were elucidated by comparison of the binding exhibited by helical glycopolypeptides with that of random-coil glycopolypeptides.

Effects of Saccharide Spacing and Chain Extension on Toxin Inhibition by Glycopolypeptides of Well-Defined Architecture.

Many recognition events important in biology are mediated via multivalent interactions between relevant oligosaccharides and multiple saccharide receptors present on lectins, viruses, toxins, and cell surfaces. Because of the important role played by protein-carbohydrate interactions in these pathogenic recognition events and in other human diseases, considerable effort has been devoted toward the development of multivalent polymeric ligands for carbohydrate-binding proteins. In this work, we report the synthesis of new polypeptide-based glycopolymers produced via a combination of protein engineering and chemical methods. These methodologies permit control over the number and the spacing of saccharides on the scaffold, as well as the conformation of the polymer backbone, and allow a more purposeful design of polymers for manipulation of multivalent binding events. Two families of galactose-bearing glycopolypeptides with random coil conformations, [(AG)(3)PEG](y) (y = 10 and 16) and {[(AG)(2)PSG](2)[(AG)(2)PEG][(AG)(2)PSG](2)}(y) (y = 6), have been synthesized. The carboxylic acid functionality of the glutamic acid residues allowed subsequent modification with amino-saccharides to yield the desired glycopolypeptides; selective placement of the glutamic acid group permitted investigation of the effects of multivalency and saccharide spacing on toxin inhibition. In addition, a family of galactose-functionalized PGA-based glycopolymers of varying molecular weights was also synthesized to compare the effects of backbone flexibility and hydrodynamic volume, relative to the recombinant glycopolypeptides, on toxin inhibition. Glycopolypeptides were characterized via (1)H NMR, MALDI-TOF mass spectrometry, SDS-PAGE analysis, and spectrophotometric assays. They were tested as inhibitors of the binding of the cholera toxin B subunit via direct enzyme-linked assays. The data from these experiments confirm the relevance of appropriate saccharide spacing on controlling the binding event and also indicate the influence of chain extension in improving inhibition.

The Kinetic Basis for Dual Recognition in Colicin Endonuclease–Immunity Protein Complexes

The antibacterial activity of E colicin endonucleases (DNases) is counteracted by the binding of immunity proteins; the affinities of cognate and non-cognate complexes differing by up to ten orders of magnitude. Here, we address the mechanism of complex formation using a combination of protein engineering, pre-steady-state kinetics and isothermal titration calorimetry, in order to understand the underlying basis for specificity. Contrary to previous work, we show that a pre-equilibrium mechanism does not explain the binding kinetics. Instead, the data are best explained by a modified induced-fit mechanism where cognate and non-cognate complexes alike form a non-specific, conformationally dynamic encounter complex, most likely centred on conserved interactions at the interface. The dynamics appear to be an intrinsic property of the encounter complex where the proteins move relative to one another, thereby sampling different conformations rather than being "induced" by binding. This allows optimal alignment of interface specificity sites, without producing energetically costly conformational changes, essential for high-affinity binding. Importantly, specificity is achieved without slowing the rate of association, an important requirement for rapid inhibition of the colicin in the producing bacterial cell. A rigid-body rotation model is also consistent with the observation that specificity contacts in colicin-immunity protein complexes can involve different regions of the interface. Such a kinetic discrimination mechanism explains the ability of DNase-specific immunity proteins to display dual recognition specificity, wherein they are broadly cross-reactive yet are highly specific, achieving femtomolar binding affinities in complexes with their cognate DNases.

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with alpha-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 x 10(4)-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 x 10(4)-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.

The Folding and Stability of Human Alpha Class Glutathione Transferase A1-1 Depend on Distinct Roles of a Conserved N-capping Box and Hydrophobic Staple Motif

An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been generated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicate that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabilizing than the first helical residue S154A mutation (N-cap) suggests that the appropriate conformation of the conserved substructure formed by the alpha 6-helix and preceding loop (GST motif II) is crucial for the overall protein stability. The refolding study of GSTA1-1 variants supports the prediction that this subdomain could represent a nucleation site of refolding. The analysis of L153A, I158A, L153G, and L153A/I158A hydrophobic staple mutants indicate that the removal of this motif destabilizes the GSTA1-1 structure as well as its refolding transition state. The hydrophobic staple interaction favors essential inter-domain contacts and, thereby, in contrast to capping interactions, accelerates the enzyme reactivation. Its strict conservation in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.

Functional Significance of the β-Hairpin in the Insecticidal Neurotoxin ω-Atracotoxin-Hv1a

omega-Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta-hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial "hits" from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta-hairpin is a key site for interaction of the toxin with insect calcium channels.

A genetic system yields self-cleaving inteins for bioseparations.

A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.

Protein arrays: Concepts and subjects

To adapt proteins, the materials in life, for use as materials in science and technology, we focused not only on the biological aspects (functional aspects) but also on the material aspects as matter (structural and physical aspects). Engineering with protein arrays will develop under such consideration and advance toward stable devices made of protein molecules. The protein arrays with 2D crystalline order provide a primary model of macroscopic protein-based devices. The combination of protein engineering, the leading edge of life science, and array engineering, the leading edge of materials science, will provide clues to the controlled integration of protein molecules to a form of functional supramolecules on proper surfaces.

Module-module interactions in the cell binding region of fibronectin: stability, flexibility and specificity

The structure of mosaic proteins depends on the nature and strength of interactions between individual modules. Here we investigated the structural significance of module-module interactions in the RGD-depen dent cell binding region of human fibronectin, comprising the ninth and tenth fibronectin type III. A combination of protein engineering, thermodynamics and nuclear magnetic resonance methods was employed to establish a relationship between intermodular protein - protein interactions and the structural properties of the module pair. A poly(glycine) peptide link connecting the C terminus of the ninth and the N terminus of the tenth module was introduced to probe the range of the interaction. NMR studies (Chemical shifts and 15N relaxation) together with equilibrium and kinetic unfolding experiments were carried out on five different single and double module constructs. The results show that non-specific protein - protein interactions provide the bulk of the thermodynamic stabilization and the motional constraint of the two modules. Specific interactions between the two modules are restricted to the wild-type module pair and decline very rapidly with the insertion of additional linker residues. This low level of specificity is nonetheless sufficient to fine-tune the precise module-module orientation and to provide the full biological activity of the wild-type pair. This suggests that individual modules in mosaic proteins can achieve a high degree of motional constraint and mutual stabilization without the requirement for intricate and specific interactions in the module-module interfaces.

Mapping the structures of transition states and intermediates in folding: delineation of pathways at high resolution

The structures of all the intermediates and transition states, from the unfolded state to the native structure, are being determined at the level of individual residues in the folding pathways of barnase and chymotrypsin inhibitor 2 (CI2), using a combination of protein engineering and nuclear magnetic resonance methods. Barnase appears to refold according to a classical framework model in which elements of secondary structure are flickeringly present in the denatured state, consolidate as the reaction proceeds and, when nearly fully formed, dock in the rate-determining step. Unlike barnase, CI2 folds without a kinetically significant folding intermediate. The transition state for its formation has no fully formed elements of secondary structure, and the transition state is like an expanded form of the native structure. CI2 probably represents the folding of an individual domain in a larger protein, whereas barnase represents the folding of a multi-domain protein. The protein engineering methods are being extended to map the pathway in the presence of molecular chaperones. There are parallels between the folding of barnase when bound to GroEL and in solution.

Molecular recognition: design of a biosensor with genetically engineered azurin as redox mediator

The development of a second generation biosensor that uses azurin as mediator for electron transport at the electrode surface is reported. Azurin is a small (14.6 kDa) bacterial protein that is relatively stable and that can be expressed in high yield in a heterologous system ( E. coli) making it an ideal candidate for protein engineering studies and biosensor development. This protein has a role as a natural electron transfer carrier in the bacterial anaerobic respiratory chain and is able to transfer electrons from cytochrome c 551 to nitrate reductase. The active centre of azurin is a type-1 copper centre. The copper ligand His 117 protrudes through the protein surface, and it has been mutated by site-directed mutagenesis into a glycine (His 117Gly). The absence of the side-chain of the glycine residue creates an aperture on the protein surface that makes the copper centre accessible to external ligands. In this way the azurin His 117Gly mutant can accept external ligands, such as 4-methylimidazole, that can be bound to the electrode surface. Attempts to establish fully reversible redox chemistry with the mutated azurin are underway. In principle, this allows the immobilization of azurin on an electrode surface for the construction of a biosensor. In this way azurin can be used as mediator in transferring electrons between the enzymatic system present in solution and the electrode. The combination of protein engineering with electrode surface modification allows further advances in biosensor technology.

Engineering subtilisin BPN′ for site‐specific proteolysis

A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN', which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wild-type subtilisin BPN' is greatly restricted by substitution of the catalytic histidine-containing of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J.A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.