Binding properties of SH3 peptide ligands identified from phage-displayed random peptide libraries

Summary Combinatorial libraries have yielded high-affinity ligands for SH3 domains of a number of different proteins. We have shown that synthetic peptides containing these SH3 ligand sequences serve as specific probes of SH3 domains. Direct binding of the N-terminal biotinylated peptide ligands was conveniently detected in ELISA, filter-blotting, and dot-blotting experiments with the use of streptavidin-conjugated enzymes. In some cases, detection of peptide-SH3 interactions required that the biotinylated peptides first were preconjugated with streptavidin to form a multivalent complex. Interestingly, these nominally tetravalent SH3 peptide ligands cross-react to varying degrees with different SH3 domains. We have used such complexes to screen cDNA expression libraries and have isolated clones that encode both known and novel SH3-domain-containing proteins. Based on the success of this methodology, we propose a general strategy by which ligands of a modular domain-containing protein can be isolated from random peptide libraries and used to screen cDNA expression libraries systematically for novel modular domain-containing proteins.     

Identification of peptide sequences that bind the Thomsen-Friedenreich cancer-associated glycoantigen from bacteriophage peptide display libraries

Summary The goal of this study was to determine if polypeptides that bind specifically to the carcinoma-associated Thomsen-Friedenreich (T) antigen could be isolated from a random peptide bacteriophage display library. T antigen is a carbohydrate antigen that is exposed and immunoreactive on the surfaces of most primary carcinomas and their metastases, while it is masked on normal cells. Tumor-specific surface carbohydrates are often used as markers of cell differentiation and play a role in cell aggregation, which is an important step in the metastatic process. Therefore, peptides that bind and mask T antigen may yield useful carbohydrate-specific probes and provide insight into carbohydrate-mediated tumor-cell aggregation. A 15-amino acid random peptide bacteriophage display library was screened for polypeptides that exhibited high specificity to two glycoproteins which display T antigen on their surfaces. The results suggest that synthetic peptides identified from the bacteriophage display library have high affinities (K_d 1 M) and specificities for proteins and human tumor cells which present T antigen. Thus, random bacteriophage peptide display libraries may be a rich source of sequences that bind to carbohydrate antigen structures.     

Comparative conformational analysis of peptide libraries

Six computer-based combinatorial libraries,including tetrapeptide sequences (generated with fiveamino acids) and conformations (generated with fivemain chain and three side chain rotamers), wereobtained and sequence-conformation probabilities werecalculated with a molecular and statistical mechanicsprocedure. The structural motifs -helix,-sheet, 3₁₀-helix, reverse turn I and-turn were focused in these calculations. Itis shown that sequence-conformation-probabilitysurfaces provide a broad view of structural changesaccompanying changes in sequence. Numerical indicesare defined to enable comparisons between frequenciesof occurrence of these structural motifs in peptidelibraries and in a database of low sequence identityprotein structures. Fine details ofsequence-conformation-probability surfaces show theeffect of point mutations. Broad comparisons betweendifferent regions of these surfaces indicate how toselect the occurrence of structural motifs in thecombinatorial synthesis of peptide chains.     

Directed evolution studies with combinatorial libraries of T4 lysozyme mutants

Summary Gene duplication with divergence to new functions has been an important mechanism in protein evolution. However, the questions of how many new functions can arise from a particular ancestral gene and how many mutational steps are typically required to generate new functions have been difficult to approach experimentally. We have addressed these questions using T4 lysozyme as a model system by synthesizing two combinatorial libraries of >10⁷ mutant T4 lysozyme genes: one library with an average of 14 missense mutations spread throughout the gene and one library in which 13 active site residues have been simultaneously randomized. These libraries were placed under selection inlacZ orpheA deficient strains ofE. coli to investigate whether they sample sufficient diversity to contain mutants with acquired -galactosidase or prephenate dehydratase activities. Although neither selection yielded T4 lysozyme mutants with these new activities, a novelE. coli locus was cloned that weakly complements these mutants, allowing them to form 1 mm colonies in 4–6 weeks. This growth rate corresponds to a turnover number of approximately 1000 or 25 min^– for thelacZ orpheA complementation systems, respectively, thus defining the limits of evolved enzymatic activity detectable in these selections. Thus, the strong selective pressure uncovered an unexpected solution to the biochemical blocks, a frequently observed phenomenon in selection experiments. The characterization of this locus will allow its elimination from futureE. coli complementation schemes.     

Molecular diversification in spider venoms: A web of combinatorial peptide libraries

Summary Spider venoms are a rich source of novel pharmacologically and agrochemically interesting compounds that have received increased attention from pharmacologists and biochemists in recent years. The application of technologies derived from genomics and proteomics have led to the discovery of the enormous molecular diversity of those venoms, which consist mainly of peptides and proteins. The molecular diversity of spider peptides has been revealed by mass spectrometry and appears to be based on a limited set of structural scaffolds. Genetic analysis has led to a further understanding of the molecular evolution mechanisms presiding over the generation of these combinatorial peptide libraries. Gene duplication and focal hypermutation, which has been described in cone snails, appear to be common mechanisms to venomous mollusks and spiders. Post-translational modifications, fine structural variations and new molecular scaffolds are other potential mechanisms of toxin diversification, leading to the pharmacologically complex cocktails used for predation and defense.     

Construction and screening of M13 phage libraries displaying long random peptides

Summary We have constructed two phage display libraries expressing N-terminal pIII fusions in M13 composed of 37 and 43 random amino acid domains, respectively. The D38 library expresses 37 random amino acids with a central alanine residue, and the DC43 library contains 43 random amino acids with a central cysteine flanked by two glycine residues, giving the displayed peptide the potential to form disulfide loops of various sizes. We demonstrate that the majority of random sequences in both libraries are compatible in pentavalent display with phage viability. The M13 phage display vector itself has been engineered to contain a factor Xa protease cleavage site to provide an alternative to acid elution during affinity selection. An in-frame amber mutation has been inserted between the pIII cloning sites to allow for efficient selection against nonrecombinant phage in the library. These libraries have been panned against mAb 7E11-C5, which recognizes the prostate-specific membrane antigen (PSM). Isolated phage display a consensus sequence that is homologous to a region in the PSM molecule.     

Optimization of the synthesis of peptide combinatorial libraries using a one-pot method

Summary Conditions for the synthesis of synthetic peptide combinatorial libraries (SPCLs) from mixtures of amino acids were explored. In a one-pot synthesis, the effect of the starting concentrations of amino acids on the resulting library composition was studied, and the optimum balance of amino acids was determined. Protein sequencing, MALDI-TOF, and amino acid analysis were used for the evaluation of the libraries, and their relative merits are discussed. The effects of continuous-flow automated synthesis instrumentation in conjunction with polyethylene glycol-polystyrene (PEG-PS) graft supports and various cleavage cocktails on the successful synthesis of SPCLs were examined.Abbreviations AA amino acid - Boc tert-butyloxycarbonyl - tBu tert-butyl - Bzl benzyl - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - ESI-MS electrospray ionization mass spectrometry - Fmoc 9-fluorenylmethyloxycarbonyl - HATU N-[(dimethylamino)-1H-1,2,3triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanamimum hexafluorophosphateN-oxide - HOAt 1-hydroxy-7-azabenzotriazole - HPLC highperformance liquid chromatography - MALDI-TOF matrix-assisted laser desorption ionization/time of flight - MBHA methylbenzhydrylamine - PAL 5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxyphenoxy)valeric acid handle - PEG-PS polyethylene glycol-polystyrene graft supports - Pbf 2,2,4,6,7-pentamethylfuran-5-sulphonyl - PNA peptide nucleic acid - PTH phenylthiohydantion - PS polystyrene - Reagent R TFA-thioanisole-1,2-ethanedithiol-anisole (90:5:3:2) - SPCL synthetic peptide combinatorial library - Trt triphenylmethyl - TFA trifluoroacetic acid Amino acids and peptides are abbreviated and designated following the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem., 247 (1972) 977–983]. Amino acid symbols denote thel-configuration unless indicated otherwise.Parts of this paper were presented at the 4th International Symposium on Solid Phase Synthesis and Combinatorial Chemical Libraries, Edinburgh, Scotland, U.K., September 12–16, 1995.     

Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: Deconvolution strategy

Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X₁–X₂–hinge–X₃–X₄–NH₂ (capped) and X₁–hinge–X₂–X₃ (uncapped), where X₁ through X₄ are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.     

Selection of a histidine-containing inhibitor of gelatinases through deconvolution of combinatorial tetrapeptide libraries

Summary A fully automated peptide synthesizer was used to generate tetrapeptide sublibraries from 24 natural and nonnatural amino acids, from which new inhibitors of gelatinases (matrix metalloproteinases MMP-2 and MMP-9) were selected as potential anticancer drugs. MMP-2 and MMP-9 from mouse Balbc/3T3 fibroblasts conditioned media were assayed in their linear range response by zymography to quantify inhibition at each step of the tetrapeptide library deconvolution. The histidine--amino caproic acid-alanine-histidine (His-Ahx-Ala-His) sequence was found to yield optimal inhibition of both MMP-2 and MMP-9. Inhibition by selected tetrapeptides was also evaluated with two other techniques, a native type IV collagen degradation assay and a fluorogenic enzymatic assay, confirming the tetrapeptide potency. The His-Ahx-Ala-His tetrapeptide also inhibited purified human MMP-2 and MMP-9 and the corresponding enzymes present in conditioned media from human tumour cells. Finally, the length of the spacer between the two terminal histidines was found to be crucial to the inhibitory potential. This approach may thus be considered as a successful strategy to yield specific peptide or pseudopeptide inhibitors, although their potency remains moderate, since it was measured before any chemical optimization was undertaken.Abbreviations AA amino acid - Ahx -amino caproic acid - APMA 4-aminophenylmercuric acetate - DNP 2,4-dinitrophenyl - ECM extracellular matrix - MMP matrix metalloproteinase - Nma N-methylanthranilic acid - Nip para-nitrophenylalanine - TIMP tissue inhibitor of metalloproteinase - Z benzyloxycarbonyl     

Low energy effective interaction of XXZ spin interaction in the two-dimensional quantum dot

We investigate the low-energy behavior of the two-dimensional quantum dot. By using the renormalization group analysis with the random matrix theory, we examine the role of anisotropy of the electron–electron interaction and demonstrate the induced instabilities in the universal Hamiltonian. As a result, it is found that anisotropy in general gives rise to four additional phases (eight phases as total), and in certain regions, the anisotropy becomes amplified at low temperature.     

Comments on the design of chemical libraries for screening

Different representations of molecules, based ondistinct sets of properties can yield differentperspectives of the issues involved in library design.In particular, different chemical representations cangive rise to very different estimates of requiredlibrary sizes. We provide a preliminary mathematicalframework that examines the size of libraries requiredto adequately sample the spaces corresponding to somecommonly used property sets. Introduction ofconformational flexibility is also discussed as ameans of increasing coverage of chemical libraries,while at the same time considering the thermodynamicconsequences of flexibility upon detectable activity.Our theoretical analysis reveals that the propertyspaces currently in use are extremely large andunlikely to provide adequate discrimination amongcompounds.     

Changes in the state of polarization of a random electromagnetic beam propagating through tissue

Based on the recently proposed unified theory of coherence and polarization of random electromagnetic beams, we have derived formulae describing changes in the state of polarization of a random electromagnetic beam propagating through tissue. A so-called electromagnetic Gaussian Schell-model beam is used to illustrate the theory. The results may find possible applications in tissue imaging.     

Some aspects of laser-metal vapour interaction

The vapour beam interaction plays an important role in laser machining process, since the vapour generated on the workpiece surface absorbs some fraction of the incident beam and heats the workpiece surface. Consequently, a study of this interaction mechanism is essential. For this purpose, a computer program was developed to investigate the interaction interaction, it provides a foundation to do further studies. It was found that the vapour locally high pressure gradients. Although the analysis presents a simplified picture of the interaction, it provides a foundation to do further studies. It was found that the vapour temperature reaches 5000 K after 10⁻⁶ s for a laser pulse of 10¹¹ W/m² power intensity. The leading edge of the vapour velocity had a velocity of the order of 4000–7000 m/s.     

Signal loss of double transmissions of ultrasonic waves through random rough surfaces

This paper establishes a model evaluating the signal loss of double transmitted acoustic beams through random rough liquid-solid surfaces based on Fresnel approximation and the phase-screen approximation. The numerical solution is replaced with a simple analytical solution through using the exponential substitution approach to remove the nonlinear integral terms. Therefore, the real-time inspection by using C-scan imaging systems of flaws in materials can be achieved through the proposed model. The research results show that the signal loss of double transmissions from random rough surfaces mainly depends on two factors: the root-mean-square (RMS) of the roughness and the depth of the flaw in materials. The experimentally measured signal loss is in good agreement with the theoretical predictions. The evaluation of the signal loss can be useful for improving the accuracy and reliability of the non-destructive testing (NDT).     

Numerical simulation of random magnetic anisotropy with solid magnetization grains

Random anisotropy model (RAM) was investigated by means of numerical simulation. Magnetization of magnetically interacting grains with randomly oriented uniaxial anisotropy was calculated using the Landau-Lifshitz-Gilbert equation where the magnetization in a particular grain is assumed to align in the same direction (single spin model). Calculations were carried out for 10×10×10 three dimensional cells changing cell sizes from 5 to 25 nm. The relation between coercive forces and grain sizes was obtained to be H_C∼D^5.7 from the simulated magnetization curves. This result fits the primitive theory H_C∼D⁶ and the experimental results.     

Changes in the polarization ellipse of random electromagnetic beams propagating through the turbulent atmosphere

During the last few years, changes in the state of polarization of a class of random electromagnetic beams (so-called electromagnetic Gaussian Schell-model beams), propagating in free space have been investigated. In the present paper, we extend the analysis to propagation of such beams in homogeneous, isotropic, non-absorbing atmospheric turbulence. We find that the effects of turbulence on the state of polarization are most significant when the atmospheric fluctuations are weak or moderate, whereas in a strong regime of atmospheric fluctuations the state of polarization of the beam returns to its original state. Our results might find possible useful applications for sensing, imaging and communication through the atmosphere.     

Changes in the polarization ellipse of random electromagnetic beams propagating through the turbulent atmosphere

During the last few years, changes in the state of polarization of a class of random electromagnetic beams (so-called electromagnetic Gaussian Schell-model beams), propagating in free space have been investigated. In the present paper, we extend the analysis to propagation of such beams in homogeneous, isotropic, non-absorbing atmospheric turbulence. We find that the effects of turbulence on the state of polarization are most significant when the atmospheric fluctuations are weak or moderate, whereas in a strong regime of atmospheric fluctuations the state of polarization of the beam returns to its original state. Our results might find possible useful applications for sensing, imaging and communication through the atmosphere.     

The angle-of-arrival variance of optical waves propagating through turbulent atmosphere in the presence of random jitter

Dynamic wind loads, thermal expansion and other mechanical reasons cause optical platform mounts to sway. The sway distorts the alignment between transmitters and receivers, causing random pointing jitter, the outcome of which is the phase fluctuation of optical waves from the transmitter. Furthermore, atmospheric turbulence causes fluctuations in the phase of those optical waves. By using the extended Huygens-Fresnel principle, both phase fluctuations are considered and an analytic formulation for the angle-of-arrival variance of optical waves propagating through turbulent atmosphere in the presence of motion-induced pointing jitter is derived. The result shows that the angle-of-arrival variance of optical waves under the combined impact of motion-induced pointing jitter and atmospheric turbulence is the sum of the angle-of-arrival variances induced by them independently.     

The dynamics of one-dimensional random quantum XY system with Dzyaloshinskii-Moriya interaction

In this paper,the effects of random variables on the dynamics of the s = 1/2 XY model with the Dzyaloshinskii-Moriya interaction are studied.By means of the recurrence relation method in the high-temperature limit,we calculate the spin autocorrelation functions as well as the corresponding spectral densities for the cases that the exchange couplings between spins or external magnetic fields satisfy the double-Gaussian distribution.It is found that when the standard deviation of random exchange coupling δJ(or the standard deviation of random external field δB) is small,the dynamics of the system undergoes a crossover from a collective-mode behavior to a central-peak one.However,when δJ(or δB) is large,the crossover vanishes,and the system shows a central-peak behavior or the most disordered one.We also analyze the cases in which the exchange couplings or the external fields satisfy the bimodal and the Gaussian distributions.Our results show that for all the cases considered,the dynamics of the above system is similar to that of the one-dimensional random XY model.     

Preparation of entangled simultaneous nonresonant atomic states through atom-field interaction

A scheme is proposed for generating a three-atom maximal entanglement W state. It is based on the simultaneous nonresonant interaction of atoms with a single-mode cavity field. Our scheme is insensitive to the cavity field, so the cavity field in our scheme can be initially in thermal states.