Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.     

Selecting peptides for use in nanoscale materials using phage-displayed combinatorial peptide libraries

Materials that combine inorganic components and biological molecules provide a new paradigm for synthesizing nanoscale and larger structures with tailored physical properties. These synthesis techniques utilize the molecular recognition properties of many biological molecules to nucleate and control growth of the nanoscale structure. Phage-displayed peptide libraries are a powerful tool to identify peptides that selectively recognize and bind to a variety of inorganic surfaces that are utilized in electronic and photonic devices. These libraries have been used extensively to study the peptide-mediated nucleation and growth of some metallic and semiconducting materials, and the application to designed nanostructures has been demonstrated.     

Individuation of monoclonal anti-HPV16 E7 antibody linear peptide epitope by computational biology

We applied computational biology to identify the linear amino acid sequence recognized by a mouse monoclonal antibody raised against the full length HPV16 E7 oncoprotein. Computer-assisted search for the epitopic peptide used two parameters: the capability of E7 peptides to bind to MHC class II molecules, and the similarity level of the oncoprotein sequence to the mouse proteome. We report that anti-E7 mAb recognized the peptide having both high binding potential to MHC II molecules and low level of molecular mimicry to mouse proteome. Peptide ability to bind to MHC II molecules appears a necessary but not sufficient condition to determine peptide immunodominance, by needing to be supported by a low degree of peptide similarity to the host’s proteome.     

A computational method for selecting short peptide sequences for inorganic material binding

Discovering or designing biofunctionalized materials with improved quality highly depends on the ability to manipulate and control the peptide‐inorganic interaction. Various peptides can be used as assemblers, synthesizers, and linkers in the material syntheses. In another context, specific and selective material‐binding peptides can be used as recognition blocks in mining applications. In this study, we propose a new in silico method to select short 4‐mer peptides with high affinity and selectivity for a given target material. This method is illustrated with the calcite (104) surface as an example, which has been experimentally validated. A calcite binding peptide can play an important role in our understanding of biomineralization. A practical aspect of calcite is a need for it to be selectively depressed in mining sites.     

Electrostatic potentials for surfaces of inorganic and molecular crystals

The electrostatic potential above surfaces can be useful in ascertaining how molecules will interact with different crystal faces; thus, it can facilitate our understanding of catalytic and crystal growth phenomena. In this paper, we describe a method for calculating the electrostatic potential above surfaces of inorganic and molecular crystals that takes advantage of the regular periodicity of the surface. In addition, we examine the effect of defects at the surface and discuss several applications, including both molecular and inorganic materials.     

Structures of designed armadillo repeat proteins binding to peptides fused to globular domains

Designed armadillo repeat proteins (dArmRP) are α‐helical solenoid repeat proteins with an extended peptide binding groove that were engineered to develop a generic modular technology for peptide recognition. In this context, the term “peptide” not only denotes a short unstructured chain of amino acids, but also an unstructured region of a protein, as they occur in termini, loops, or linkers between folded domains. Here we report two crystal structures of dArmRPs, in complex with peptides fused either to the N‐terminus of Green Fluorescent Protein or to the C‐terminus of a phage lambda protein D. These structures demonstrate that dArmRPs bind unfolded peptides in the intended conformation also when they constitute unstructured parts of folded proteins, which greatly expands possible applications of the dArmRP technology. Nonetheless, the structures do not fully reflect the binding behavior in solution, that is, some binding sites remain unoccupied in the crystal and even unexpected peptide residues appear to be bound. We show how these differences can be explained by restrictions of the crystal lattice or the composition of the crystallization solution. This illustrates that crystal structures have to be interpreted with caution when protein–peptide interactions are characterized, and should always be correlated with measurements in solution.     

Molecular basis for recognition of an arthritic peptide and a foreign epitope on distinct MHC molecules by a single TCR

KRN TCR transgenic T cells recognize two self-MHC molecules: a foreign peptide, bovine RNase 42-56, on I-Ak and an autoantigen, glucose-6-phosphate isomerase 282-294, on I-Ag7. Because the latter recognition event initiates a disease closely resembling human rheumatoid arthritis, we investigated the structural basis of this pathogenic TCR's dual specificity. While peptide recognition is altered to a minor degree between the MHC molecules, we show that the receptor's cross-reactivity critically depends upon a TCR contact residue completely conserved in the foreign and self peptides. Further, the altered recognition of peptide derives from discrete differences on the MHC recognition surfaces and not the disparate binding grooves. This work provides a detailed structural comparison of an autoreactive TCR's interactions with naturally occurring peptides on distinct MHC molecules. The capacity to interact with multiple self-MHCs in this manner increases the number of potentially pathogenic self-interactions available to a T cell.     

Role of peptide backbone in T cell recognition

T cells recognize self and nonself peptides presented by molecules of the MHC. Amino acid substitutions in the antigenic peptide showed that T cell specificity is highly degenerate. Recently, determination of the crystal structure of several TCR/MHC-peptide complexes suggested that the peptide backbone may significantly contribute to the interaction with the TCR. To directly investigate the role of the peptide backbone in T cell recognition, we performed a methylene-amino scan on the backbone of an antigenic peptide and measured the capacity of such pseudopeptides to bind their cognate MHC molecule, to sensitize target cells for T cell lysis, and to stimulate IL-2 secretion by two T cell hybridomas. For one of these pseudopeptides, we prepared fluorescent tetramers of MHC molecules and compared the staining of two T cell hybridomas. Our results demonstrate that the peptide backbone has an important contribution to TCR binding and suggest that some interactions between the peptide backbone and the TCR may be partially conserved. We discuss this finding in the perspective of TCR plasticity and T cell function.     

Design and immunological properties of topographic immunogenic determinants of a protein antigen (LDH-C4) as vaccines.

Antibodies elicited by immunization with short peptides containing antigenic determinants have been shown, in general, to bind with greatly reduced affinity to the corresponding region in the native proteins. Thus, contiguous linear peptides have not proven to be effective immunogens in generating high affinity neutralizing or protective antibodies and consequently appear to be poor prospects for vaccines. The molecular basis for such reduced reactivity is clear from the crystal structure determination of antibody Fabs bound to protein antigens, which showed the complementarity between interfaces to be lock-and-key-like and extending over a large area (750 A2) involving discontinuous segments of the polypeptide chain. Thus, small perturbations in the secondary and tertiary structure of the antigen have profound effects on the fit of the antigen and its corresponding antibody. Because short peptides are unlikely to assume any particular conformation in solution, the fit is likely to be poor. New strategies are therefore required to produce conformationally stable peptides that mimic the critical structural features of the protein antigenic site. Here we show that a putative topographic determinant of the testis-specific isozyme of lactate dehydrogenase C4 (LDH-C4), designed and synthesized to adopt a well defined alpha-helical secondary and tertiary structure (four-helix bundle motif) in aqueous solutions, is highly immunogenic in both rabbits and mice, inducing IgG antibodies that bind to native LDH-C4. This engineered conformational 40-residue peptide is considerably more effective in inducing antibodies, as compared with the corresponding linear peptide. The antibody response is obtained without coupling the peptide to a carrier protein, suggesting that the peptide contains a T-cell antigenic determinant. The strategy described here to produce a conformationally stable peptide that mimics the native structure may have general applications in vaccine design.     

Recapitulation and design of protein binding peptide structures and sequences

An important objective of computational protein design is the generation of high affinity peptide inhibitors of protein-peptide interactions, both as a precursor to the development of therapeutics aimed at disrupting disease causing complexes, and as a tool to aid investigators in understanding the role of specific complexes in the cell. We have developed a computational approach to increase the affinity of a protein-peptide complex by designing N or C-terminal extensions which interact with the protein outside the canonical peptide binding pocket. In a first in silico test, we show that by simultaneously optimizing the sequence and structure of three to nine residue peptide extensions starting from short (1-6 residue) peptide stubs in the binding pocket of a peptide binding protein, the approach can recover both the conformations and the sequences of known binding peptides. Comparison with phage display and other experimental data suggests that the peptide extension approach recapitulates naturally occurring peptide binding specificity better than fixed backbone design, and that it should be useful for predicting peptide binding specificities from crystal structures. We then experimentally test the approach by designing extensions for p53 and dystroglycan-based peptides predicted to bind with increased affinity to the Mdm2 oncoprotein and to dystrophin, respectively. The measured increases in affinity are modest, revealing some limitations of the method. Based on these in silico and experimental results, we discuss future applications of the approach to the prediction and design of protein-peptide interactions.     

Molecular recognition of a peptide mimic of the Lewis Y antigen by an anti-Lewis Y antibody

Peptides as mimics of carbohydrates display a distinct advantage in vaccine design because of ease of synthesis and their inherent T cell-dependent nature as immunogens. While peptides that mimic carbohydrates have been described, it is not clear how they do so. To further our insight into structural relationships between peptide-mimics and carbohydrate structures, we have analyzed a potential recognition scheme between the murine monoclonal antibody, B3, directed against the tumor-associated antigen Lewis Y oligosaccharide and a peptide identified from phage display screening with B3. The Lewis Y core antigen is a difucosylated structure consisting of four hexose units. The B3 antibody binds to the peptide sequence APWLYGPA in which the putative sequence APWLY is critical for binding to the antibody. Not having experimental structural information for B3, the crystal structure of another anti-Lewis Y antibody, BR96, solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide, provides a molecular basis for LeY antigen recognition and specificity, and how this binding relates to peptide binding. As a guide to place the APWLY motif in the B3 combining site, a fragment library was searched for analogous compounds that have the potential to bind to B3. Our modeling study shows that the B3-peptide complex shares similar recognition features for the difucosylated type 2 lactoseries' structure. This analysis provides a molecular perspective for peptide mimicry of a carbohydrate epitope. © 1998 John Wiley & Sons, Ltd.     

Addressable self-immobilization of lactate dehydrogenase across multiple length scales

Successful nanobiotechnology implementation largely depends on control over the interfaces between inorganic materials and biological molecules. Controlling the orientations of biomolecules and their spatial arrangements on the surface may transform many technologies including sensors, to energy. Here, we demonstrate the self-organization of L -lactate dehydrogenase (LDH), which exhibits enhanced enzymatic activity and stability on a variety of gold surfaces ranging from nanoparticles to electrodes, by incorporating a gold-binding peptide tag (AuBP2) as the fusion partner for Bacillus stearothermophilus LDH (bsLDH). Binding kinetics and enzymatic assays verified orientation control of the enzyme on the gold surface through the genetically incorporated peptide tag. Finally, redox catalysis efficiency of the immobilized enzyme was detected using cyclic voltammetry analysis in enzyme-based biosensors for lactate detection as well as in biofuel cell energy systems as the anodic counterpart. Our results demonstrate that the LDH enzyme can be self-immobilized onto different gold substrates using the short peptide tag under a biologically friendly environment. Depending on the desired inorganic surface, the proposed peptide-mediated path could be extended to any surface to achieve single-step oriented enzyme immobilization for a wide range of applications.     

Identification of peptide mimotopes for the fluorescein hapten binding of monoclonal antibody B13‐DE1

Using 6mer and 12mer phage peptide libraries three unique phage clones were identified which specifically bind to a monoclonal anti‐FITC antibody, B13‐DE1. The two 6mer and one 12mer peptide insert sequences are clearly related to each other and contain a high proportion of hydrophobic amino acids. The peptides are bound by the antibody combining site of B13‐DE1 probably in a similar manner to FITC and represent therefore true peptidic mimics of the fluorescein hapten. No reactivity of the peptides could be demonstrated with another monoclonal anti‐fluorescein antibody or with polyclonal anti‐fluorescein antibodies. Immunization of mice with the peptides resulted in the production of antibodies cross‐reacting with all peptides but not with fluorescein. The results show that phage peptide libraries can be used to isolate mimotope peptides which can mimic low molecular weight structures seen by a specific antibody and probably other recognition molecules. Copyright © 1999 John Wiley & Sons, Ltd.     

Aptamers as functional nucleic acids:<Emphasis Type="Italic">In vitro</Emphasis> selection and biotechnological applications

Aptamers are functional nucleic acids that can specially bind to proteins, peptides, amino acids, nucleotides, drugs, vitamins and other organic and inorganic compounds. The aptamers are identified from random DNA or RNA libraries by a SELEX (systematic evolution of ligands by exponential amplification) process. As aptamers have the advantage, and potential ability to be released from the limitations of antibodies, they are attractive to a wide range of therapeutic and diagnostic applications. Aptamers, with a high-affinity and specificity, could fulfil molecular the recognition needs of various fields in biotechnology. In this work, we reviewed some aptamer selection techniques, properties, medical applications of their molecules and their biotechnological applications, such as ELONA (enzyme linked oligonucleotide assay), flow cytometry, biosensors, electrophoresis, chromatography and microarrays.     

Use of biomolecular templates for the fabrication of metal nanowires

The nano-scale spatial organization of metallic and other inorganic materials into 1D objects is a key task in nanotechnology. Nano-scale fibers and tubes are very useful templates for such organization because of their inherent 1D organization. Fibrillar biological molecules and biomolecular assemblies are excellent physical supports on which to organize the inorganic material. Furthermore, these biological assemblies can facilitate high-order organization and specific orientation of inorganic structures by their utilization of highly specific biological recognition properties. In this minireview, I will describe the use of biomolecules and biomolecular assemblies, including DNA, proteins, peptides, and even viral particles, which are excellent templates for 1D organization of inorganic materials into wires. This ranges from simple attempts at electroless deposition on inert biological templates to the advanced use of structural motifs and specific protein-DNA interactions for nano-bio-lithography as well as the fabrication of multilayer organic and inorganic composites. The potential technological applications of these hybrid biological-inorganic assemblies will be discussed.     

Crystal structures of N-myristoylated lipopeptide-bound HLA class I complexes indicate reorganization of B-pocket architecture upon ligand binding

Rhesus monkeys have evolved MHC-encoded class I allomorphs such as Mamu-B∗098 that are capable of binding N-myristoylated short lipopeptides rather than conventional long peptides; however, it remains unknown whether such antigen-binding molecules exist in other species, including humans. We herein demonstrate that human leukocyte antigen (HLA)-A∗24:02 and HLA-C∗14:02 proteins, which are known to bind conventional long peptides, also have the potential to bind N-myristoylated short lipopeptides. These HLA class I molecules shared a serine at position 9 (Ser9) with Mamu-B∗098, in contrast to most MHC class I molecules that harbor a larger amino acid residue, such as tyrosine, at this position. High resolution X-ray crystallographic analyses of lipopeptide-bound HLA-A∗24:02 and HLA-C∗14:02 complexes indicated that Ser9 was at the bottom of the B pocket with its small hydroxymethyl side chain directed away from the B-pocket cavity, thereby contributing to the formation of a deep hydrophobic cavity suitable for accommodating the long-chain fatty acid moiety of lipopeptide ligands. Upon peptide binding, however, we found the hydrogen-bond network involving Ser9 was reorganized, and the remodeled B pocket was able to capture the second amino acid residue (P2) of peptide ligands. Apart from the B pocket, virtually no marked alterations were observed for the A and F pockets upon peptide and lipopeptide binding. Thus, we concluded that the structural flexibility of the large B pocket of HLA-A∗2402 and HLA-C∗1402 primarily accounted for their previously unrecognized capacity to bind such chemically distinct ligands as conventional peptides and N-myristoylated lipopeptides.     

Kinetics of peptide binding to the class II MHC protein I-Ek

Class II MHC glycoproteins bind short (7-25 amino acid) peptides in an extended type II polyproline-like conformation and present them for immune recognition. Because empty MHC is unstable, measurement of the rate of the second-order reaction between peptide and MHC is challenging. In this report, we use dissociation of a pre-bound peptide to generate the active, peptide-receptive form of the empty class II MHC molecule I-Ek. This allows us to measure directly the rate of reaction between active, empty I-Ek and a set of peptides that vary in structure. We find that all peptides studied, despite having highly variable dissociation rates, bind with similar association rate constants. Thus, the rate-limiting step in peptide binding is minimally sensitive to peptide side-chain structure. An interesting complication to this simple model is that a single peptide can sometimes bind to I-Ek in two kinetically distinguishable conformations, with the stable peptide-MHC complex isomer forming much more slowly than the less-stable one. This demonstrates that an additional free-energy barrier limits the formation of certain specific MHC-peptide complex conformations.     

Self-assembled monolayers and polymer brushes in biotechnology: current applications and future perspectives

The chemistry and topography of a surface affect biological response and are of fundamental importance, especially when living systems encounter synthetic surfaces. Most biomolecules have immense recognition power (specific binding) and simultaneously have a tendency to physically adsorb onto a solid substrate without specific receptor recognition (nonspecific adsorption). Therefore, to create useful materials for many biotechnology applications, interfaces are required that have both enhanced specific binding and reduced nonspecific binding. Thus, in applications such as sensors, the tailoring of surface chemistry and the use of micro or nanofabrication techniques becomes an important avenue for the production of surfaces with specific binding properties and minimal background interference. Both self-assembled monolayers (SAMs) and polymer brushes have attracted considerable attention as surface-active materials. In this review, we discuss both of these materials with their potential applications in biotechnology. We also summarize lithographic methods for pattern formation using combined top-down and bottom-up approaches and briefly discuss the future of these materials by describing emerging new applications.     

Exquisite specificity and peptide epitope recognition promiscuity, properties shared by antibodies from sharks to humans

This review considers definitions of the specificity of antibodies including the development of recent concepts of recognition polyspecificity and epitope promiscuity. Using sets of homologous and unrelated peptides derived from the sequences of immunoglobulin and T cell receptor chains we offer operational definitions of cross-reactivity by investigating correlations of either identities in amino acid sequence, or in hydrophobicity/hydrophilicity profiles with degree of binding in enzyme-linked immunosorbent assays. Polyreactivity, or polyspecificity, are terms used to denote binding of a monoclonal antibody or purified antibody preparation to large complex molecules that are structurally unrelated, such as thyroglobulin and DNA. As a first approximation, there is a linear correlation between degree of sequence identity or hydrophobicity/hydrophilicity and antigenic cross-binding. However, catastrophic interchanges of amino acids can occur where changing of one amino acid out of 16 in a synthetic peptide essentially eliminates binding to certain antibodies. An operational definition of epitope promiscuity for peptides is the case where two peptides show little or no identity in amino acid sequence but bind strongly to the same antibody as shown by either direct binding or competitive inhibition. Analysis of antibodies of humans and sharks, the two most divergent species in evolution to express antibodies and the combinatorial immune response, indicates that the capacity for both exquisite specificity and epitope recognition promiscuity are essential conserved features of individual vertebrate antibodies.     

Redesign of a protein–peptide interaction: Characterization and applications

The design of protein–peptide interactions has a wide array of practical applications and also reveals insight into the basis for molecular recognition. Here, we present the redesign of a tetratricopeptide repeat (TPR) protein scaffold, along with its corresponding peptide ligand. We show that the binding properties of these protein–peptide pairs can be understood, quantitatively, using straightforward chemical considerations. The recognition pairs we have developed are also practically useful for the specific identification of tagged proteins. We demonstrate the facile replacement of these proteins, which we have termed T‐Mods (TPR‐based recognition module), for antibodies in both detection and purification applications. The new protein–peptide pair has a dissociation constant that is weaker than typical antibody–antigen interactions, yet the recognition pair is highly specific and we have shown that this affinity is sufficient for both Western blotting and affinity purification. Moreover, we demonstrate that this more moderate affinity is actually advantageous for purification applications, because extremely harsh conditions are not required to dissociate the T‐Mod‐peptide interaction. The results we present are important, not only because they represent a successful application of protein design but also because they help define the properties that should be sought in other scaffolds that are being developed as antibody replacements.