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Many protein-protein interactions are mediated by small modular domains, which recognize short peptide motifs in their partner proteins. However, for the great majority of these domains, the identity of their partner proteins remains unknown. In this work, a chemical/bioinformatics approach has been developed to identify phosphotyrosyl (pY) proteins that bind to tensin, a protein involved in the formation of actin cytoskeleton and signal transduction. A pY peptide library was chemically synthesized and screened against the Src homology 2 (SH2) domain of tensin to identify the peptide motifs that bind to the SH2 domain. Next, protein databases were searched for proteins containing the SH2 domain-binding peptide motifs. Finally, the potential tensin-binding proteins were confirmed (or disproved) by in vitro pull-down and coimmunoprecipitation assays. This procedure identified phosphoinositide-dependent kinase-1 and downstream of tyrosine kinase 2 as novel tensin-binding proteins. In addition, a cell-permeable pY peptide was designed as tensin SH2 domain inhibitor, which caused the disruption of actin filaments in NIH 3T3 cells. This method should be generally applicable to other modular domains that recognize small peptide motifs.
ACS Chem Biol 2007 Feb 20
PMID:A chemical approach to the identification of tensin-binding proteins. 1731 77

There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein G alpha i1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic G alpha i binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity ( K i approximately 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the betagamma heterodimer, an endogenous G alpha i1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.
ACS Chem Biol 2007 Sep 21
PMID:Design of cyclic peptides that bind protein surfaces with antibody-like affinity. 1789 40

As a naturally occurring nanocapsule abundantly expressed in nearly all-eukaryotic cells, the barrel-shaped vault particle is perhaps an ideal structure to engineer for targeting to specific cell types. Recombinant vault particles self-assemble from 96 copies of the major vault protein (MVP), have dimensions of 72.5 x 41 nm, and have a hollow interior large enough to encapsulate hundreds of proteins. In this study, three different tags were engineered onto the C-terminus of MVP: an 11 amino acid epitope tag, a 33 amino acid IgG-binding peptide, and the 55 amino acid epidermal growth factor (EGF). These modified vaults were produced using a baculovirus expression system. Our studies demonstrate that recombinant vaults assembled from MVPs containing C-terminal peptide extensions display these tags at the top and bottom of the vault on the outside of the particle and can be used to specifically bind the modified vaults to epithelial cancer cells (A431) via the epidermal growth factor receptor (EGFR), either directly (EGF modified vaults) or as mediated by a monoclonal antibody (anti-EGFR) bound to recombinant vaults containing the IgG-binding peptide. The ability to target vaults to specific cells represents an essential advance toward using recombinant vaults as delivery vehicles.
ACS Nano 2009 Jan 27
PMID:Targeting vault nanoparticles to specific cell surface receptors. 1920 45

The understanding of biomineralization and realization of biology-inspired materials technologies depends on understanding the nature of the chemical and physical interactions between proteins and biominerals or synthetically made inorganic materials. Recently, combinatorial genetic techniques permit the isolation of peptides recognizing specific inorganic materials that are used as molecular building blocks for novel applications. Little is known about the molecular structure of these peptides and the specific recognition mechanisms onto their counterpart inorganic surfaces. Here, we report high-resolution atomic force microscopy (AFM), molecular simulation (MS), and geometrical docking studies that detail the formation of an ordered supramolecular self-assembly of a genetically engineered gold binding peptide, 3rGBP(1) ([MHGKTQATSGTIQS](3)), correlating with the symmetry of the Au{111} surface lattice. Using simulated annealing molecular dynamics (SA/MD) studies based on nuclear magnetic resonance (NMR), we confirmed the intrinsic disorder of 3rGBP(1) and identified putative Au docking sites where surface-exposed side chains align with both the <110> and <211> Miller indices of the Au lattice. Our results provide fundamental insight for an atomistic understanding of peptide/solid interfaces and the intrinsic disorder that is inherent in some of these peptide sequences. Analogous to the well-established atomically controlled thin-film heterostructure formation on semiconductor substrates, the basis of today's microelectronics, the fundamental observations of peptide-solid interactions here may well form the basis of peptide-based hybrid molecular technologies of the future.
ACS Nano 2009 Jun 23
PMID:Molecular recognition and supramolecular self-assembly of a genetically engineered gold binding peptide on Au{111}. 1943 57

A 24 x 30 nm ellipsoid nanoparticle containing 84 subunits or 7 dodecamers of the re-engineered core protein of the bacteriophage phi29 DNA packaging motor was constructed. Homogeneous nanoparticles were obtained with simple one-step purification. Electron microscopy and analytical ultracentrifugation were employed to elucidate the structure, shape, size, and mechanism of assembly. The formation of this structure was mediated and stabilized by N-terminal peptide extensions. Reversal of the 84-subunit ellipsoid nanoparticle to its dodecamer subunit was controlled by the cleavage of the extended N-terminal peptide with a protease. The 84 outward-oriented C-termini were conjugated with a streptavidin binding peptide which can be used for the incorporation of markers. This further extends the application of this nanoparticle to pathogen detection and disease diagnosis by signal enhancement.
ACS Nano 2009 Aug 25
PMID:Adjustable ellipsoid nanoparticles assembled from re-engineered connectors of the bacteriophage phi29 DNA packaging motor. 1963 10

An interesting alternative to top-down nanofabrication is to imitate biology, where nanoscale materials frequently integrate organic molecules for self-assembly and molecular recognition with ordered, inorganic minerals to achieve mechanical, sensory, or other advantageous functions. Using biological systems as inspiration, researchers have sought to mimic the nanoscale composite materials produced in nature. Here, we describe a combination of self-assembly, molecular recognition, and templating, relying on an oligonucleotide covalently conjugated to a high-affinity gold-binding peptide. After integration of the peptide-coupled DNA into a self-assembling superstructure, the templated peptides recognize and bind gold nanoparticles. In addition to providing new ways of building functional multinanoparticle systems, this work provides experimental proof that a single peptide molecule is sufficient for immobilization of a nanoparticle. This molecular construction strategy, combining DNA assembly and peptide recognition, can be thought of as programmable, granular, artificial biomineralization. We also describe the important observation that the addition of 1-2% Tween 20 surfactant to the solution during gold particle binding allows the gold nanoparticles to remain soluble within the magnesium-containing DNA assembly buffer under conditions that usually lead to the aggregation and precipitation of the nanoparticles.
ACS Nano 2011 Mar 22
PMID:Organization of inorganic nanomaterials via programmable DNA self-assembly and peptide molecular recognition. 2131 76

This study demonstrates a biological route to programming well-defined protein-inorganic interfaces with an arrayed geometry via modular peptide tag technology. To illustrate this concept, we designed a model multifunctional fusion protein, which simultaneously displays a maltose-binding protein (MBP), a green fluorescence protein (GFPuv) and an inorganic-binding peptide (AgBP2C). The fused combinatorially selected AgBP2C tag controls and site-directs the multifunctional fusion protein to immobilize on silver nanoparticle arrays that are fabricated on specific domain surfaces of ferroelectric LiNbO(3) via photochemical deposition and in situ synthesis. Our combined peptide-assisted biological and ferroelectric lithography approach offers modular design and versatility in tailoring surface reactivity for fabrication of nanoscale devices in environmentally benign conditions.
ACS Appl Mater Interfaces 2012 Apr
PMID:Multifunctional protein-enabled patterning on arrayed ferroelectric materials. 2245 31

Small interfering ribonucleic acid (siRNA) cancer therapies administered by intravenous injection require a delivery system for transport from the bloodstream into the cytoplasm of diseased cells to perform the function of gene silencing. Here we describe nanosized polymeric micelles that deliver siRNA to solid tumors and elicit a therapeutic effect. Stable multifunctional micelle structures on the order of 45 nm in size formed by spontaneous self-assembly of block copolymers with siRNA. Block copolymers used for micelle formation were designed and synthesized to contain three main features: a siRNA binding segment containing thiols, a hydrophilic nonbinding segment, and a cell-surface binding peptide. Specifically, poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLL) comprising lysine amines modified with 2-iminothiolane (2IT) and the cyclo-Arg-Gly-Asp (cRGD) peptide on the PEG terminus was used. Modification of PEG-b-PLL with 2IT led to improved control of micelle formation and also increased stability in the blood compartment, while installation of the cRGD peptide improved biological activity. Incorporation of siRNA into stable micelle structures containing the cRGD peptide resulted in increased gene silencing ability, improved cell uptake, and broader subcellular distribution in vitro and also improved accumulation in both the tumor mass and tumor-associated blood vessels following intravenous injection into mice. Furthermore, stable and targeted micelles inhibited the growth of subcutaneous HeLa tumor models and demonstrated gene silencing in the tumor mass following treatment with antiangiogenic siRNAs. This new micellar nanomedicine could potentially expand the utility of siRNA-based therapies for cancer treatments that require intravenous injection.
ACS Nano 2012 Jun 26
PMID:Targeted polymeric micelles for siRNA treatment of experimental cancer by intravenous injection. 2257 90

Growth in three-electrode electrochemical cells allows quantitative analysis of mechanisms involved in electron flow from dissimilatory metal reducing bacteria to insoluble electron acceptors. In these systems, gold electrodes are a desirable surface to study the electrophysiology of extracellular respiration, yet previous research has shown that certain Shewanella species are unable to form productive biofilms on gold electrodes. To engineer attachment of Shewanella oneidensis to gold, five repeating units of a synthetic gold-binding peptide (5rGBP) were integrated within an Escherichia coli outer membrane protein, LamB, and displayed on the outer surface of S. oneidensis. Expression of LamB-5rGBP increased cellular attachment of S. oneidensis to unpoised gold surfaces but was also associated with the loss of certain outer membrane proteins required for extracellular respiration. Loss of these outer membrane proteins during expression of LamB-5rGBP decreased the rate at which S. oneidensis was able to reduce insoluble iron, riboflavin, and electrodes. Moreover, poising the gold electrode resulted in repulsion of the engineered cells. This study provides a strategy to specifically immobilize bacteria to electrodes while also outlining challenges involved in merging synthetic biology approaches with native cellular pathways and cell surface charge.
ACS Synth Biol 2013 Feb 15
PMID:Electrochemical analysis of Shewanella oneidensis engineered to bind gold electrodes. 2365 72

Bionanocombinatorics is an emerging field that aims to use combinations of positionally encoded biomolecules and nanostructures to create materials and devices with unique properties or functions. The full potential of this new paradigm could be accessed by exploiting specific noncovalent interactions between diverse palettes of biomolecules and inorganic nanostructures. Advancement of this paradigm requires peptide sequences with desired binding characteristics that can be rationally designed, based upon fundamental, molecular-level understanding of biomolecule-inorganic nanoparticle interactions. Here, we introduce an integrated method for building this understanding using experimental measurements and advanced molecular simulation of the binding of peptide sequences to gold surfaces. From this integrated approach, the importance of entropically driven binding is quantitatively demonstrated, and the first design rules for creating both enthalpically and entropically driven nanomaterial-binding peptide sequences are developed. The approach presented here for gold is now being expanded in our laboratories to a range of inorganic nanomaterials and represents a key step toward establishing a bionanocombinatorics assembly paradigm based on noncovalent peptide-materials recognition.
ACS Nano 2013 Nov 26
PMID:Biomolecular recognition principles for bionanocombinatorics: an integrated approach to elucidate enthalpic and entropic factors. 2412 16


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