RESUMO
c-Src kinase is a multidomain non-receptor tyrosine kinase that aberrantly phosphorylates several signaling proteins in cancers. Although the structural properties of the regulatory domains (SH3-SH2) and the catalytic kinase domain have been extensively characterized, there is less knowledge about the N-terminal disordered region (SH4UD) and its interactions with the other c-Src domains. Here, we used domain-selective isotopic labeling combined with the small-angle neutron scattering contrast matching technique to study SH4UD interactions with SH3-SH2. Our results show that in the presence of SH4UD, the radius of gyration (Rg) of SH3-SH2 increases, indicating that it has a more extended conformation. Hamiltonian replica exchange molecular dynamics simulations provide a detailed molecular description of the structural changes in SH4UD-SH3-SH2 and show that the regulatory loops of SH3 undergo significant conformational changes in the presence of SH4UD, while SH2 remains largely unchanged. Overall, this study highlights how a disordered region can drive a folded region of a multidomain protein to become flexible, which may be important for allosteric interactions with binding partners. This may help in the design of therapeutic interventions that target the regulatory domains of this important family of kinases.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas pp60(c-src) , Domínio Catalítico , Domínios ProteicosRESUMO
Borrelia burgdorferi transmission to the vertebrate host commences with growth of the spirochete in the tick gut and migration from the gut to the salivary glands. This complex process, involving intimate interactions of the spirochete with the gut epithelium, is pivotal to transmission. We utilized a yeast surface display library of tick gut proteins to perform a global screen for tick gut proteins that might interact with Borrelia membrane proteins. A putative fibronectin type III domain-containing tick gut protein (Ixofin3D) was most frequently identified from this screen and prioritized for further analysis. Immunization against Ixofin3D and RNA interference-mediated reduction in expression of Ixofin3D resulted in decreased spirochete burden in tick salivary glands and in the murine host. Microscopic examination showed decreased aggregation of spirochetes on the gut epithelium concomitant with reduced expression of Ixofin3D. Our observations suggest that the interaction between Borrelia and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a "molecular exit" direction for spirochete egress from the gut.
Assuntos
Proteínas de Artrópodes/metabolismo , Fibronectinas/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Doença de Lyme/transmissão , Carrapatos/metabolismo , Animais , Borrelia burgdorferi , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Sortase-mediated ligation was used to attach the photosystem I (PSI) complex from Synechocystis sp. PCC 6803 in a preferential orientation to enhance photoinduced electron flow to a conductive gold surface. Ideally, this method can result in a uniform monolayer of protein, covalently bound unidirectionally to the electrode surface. The exposed C-termini of the psaE subunits of the PSI trimer were targeted to contain an LPETG-sortase recognition sequence to increase noncompeting electron transfer by uniformly orienting the PSI stromal side proximal to the surface. Surface characterization with atomic force microscopy suggested that monolayer formation and optimal surface coverage occurred when the gold surfaces were incubated with peptide at 100 to 500 µM concentrations. When photochronoamperometry with potassium ferrocyanide and ferricyanide as redox mediators was used, photocurrents in the range of 100 to 200 nA/cm(2) were produced, which is an improvement over other attachment techniques for photosystem monolayers that produce approximately 100 nA/cm(2) or less. This work demonstrated that sortase-mediated ligation aided in the control of PSI orientation on modified gold surfaces with a distribution of 94% stromal side proximal and 6% lumenal side proximal to the surface for current-producing PSI.
Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Elétrons , Ouro/química , Complexo de Proteína do Fotossistema I/química , Synechocystis/química , Fontes de Energia Bioelétrica , Técnicas Eletroquímicas , Eletrodos , Transporte de Elétrons , Ferricianetos/química , Ferrocianetos/química , Microscopia de Força Atômica , Modelos Moleculares , Oxirredução , Processos Fotoquímicos , Propriedades de Superfície , Synechocystis/fisiologia , Raios UltravioletaRESUMO
Class II major histocompatibility complex (MHC-II) proteins govern stimulation of adaptive immunity by presenting antigenic peptides to CD4+ T lymphocytes. Many allelic variants of MHC-II exist with implications in peptide presentation and immunity; thus, high-throughput experimental tools for rapid and quantitative analysis of peptide binding to MHC-II are needed. Here, we present an expression system wherein peptide and MHC-II are codisplayed on the surface of yeast in an intracellular association-dependent manner and assayed by flow cytometry. Accordingly, the relative binding of different peptides and/or MHC-II variants can be assayed by genetically manipulating either partner, enabling the application of directed evolution approaches for high-throughput characterization or engineering. We demonstrate the application of this tool to map the side-chain preference for peptides binding to HLA-DR1 and to evolve novel HLA-DR1 mutants with altered peptide-binding specificity.
Assuntos
Antígeno HLA-DR1/metabolismo , Biblioteca de Peptídeos , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Sítios de Ligação/genética , Evolução Molecular Direcionada , Citometria de Fluxo , Antígeno HLA-DR1/química , Antígeno HLA-DR1/genética , Humanos , Modelos Moleculares , Mutação , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Leveduras/genética , Leveduras/metabolismoRESUMO
Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.
Assuntos
Substituição de Aminoácidos , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Leptina/análogos & derivados , Mutação de Sentido Incorreto , Polietilenoglicóis , Animais , Células HEK293 , Humanos , Leptina/antagonistas & inibidores , Leptina/química , Leptina/genética , Leptina/metabolismo , Leptina/farmacologia , Masculino , Camundongos , Mutagênese , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologia , Ligação Proteica , Receptores para Leptina/genética , Receptores para Leptina/metabolismoRESUMO
Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , Leveduras/genética , Animais , Anticorpos Monoclonais/isolamento & purificação , Afinidade de Anticorpos , Humanos , Recombinação GenéticaRESUMO
Yeast surface display has proven to be a powerful tool for the discovery of antibodies and other novel binding proteins and for engineering the affinity and selectivity of existing proteins for their targets. In the decades since the first demonstrations of the approach, the range of yeast display applications has greatly expanded to include many different protein targets and has grown to encompass methods for rapid protein characterization. Here, we briefly summarize the development of yeast display methodologies and highlight several selected examples of recent applications to timely and challenging protein engineering and characterization problems.
Assuntos
Biblioteca de Peptídeos , Saccharomyces cerevisiae , Anticorpos/metabolismo , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/metabolismoRESUMO
Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that bear them.
Assuntos
Nanopartículas , Engenharia de Proteínas , Anticorpos de Cadeia Única/metabolismo , Avidina/metabolismo , Biotina/metabolismo , Cinética , Ligação Proteica , Anticorpos de Cadeia Única/químicaRESUMO
Globular proteins often contain structurally well-resolved internal water molecules. Previously, we reported results from a molecular dynamics study that suggested that buried water (Wat3) may play a role in modulating the structure of the FK506 binding protein-12 (FKBP12) (Park and Saven, Proteins 2005; 60:450-463). In particular, simulations suggested that disrupting a hydrogen bond to Wat3 by mutating E60 to either A or Q would cause a structural perturbation involving the distant W59 side chain, which rotates to a new conformation in response to the mutation. This effectively remodels the ligand-binding pocket, as the side chain in the new conformation is likely to clash with bound FK506. To test whether the protein structure is in effect modulated by the binding of a buried water in the distance, we determined high-resolution (0.92-1.29 A) structures of wild-type FKBP12 and its two mutants (E60A, E60Q) by X-ray crystallography. The structures of mutant FKBP12 show that the ligand-binding pocket is indeed remodeled as predicted by the substitution at position 60, even though the water molecule does not directly interact with any of the amino acids of the binding pocket. Thus, these structures support the view that buried water molecules constitute an integral, noncovalent component of the protein structure. Additionally, this study provides an example in which predictions from molecular dynamics simulations are experimentally validated with atomic precision, thus showing that the structural features of protein-water interactions can be reliably modeled at a molecular level.
Assuntos
Proteína 1A de Ligação a Tacrolimo/química , Água/química , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
Determining structures of large, complex proteins remains challenging, especially for transmembrane proteins, as the protein size increases. Arabidopsis thaliana cellulose synthesis complex is a large, multimeric complex located in the plant cell membrane that synthesizes cellulose microfibrils in the plant cell wall. Despite the biological and economic importance of cellulose and therefore cellulose synthesis, many aspects of the cellulase synthase complex (CSC) structure and function are still unknown. Here, yeast surface display (YSD) is used to determine the full-length expression of A. thaliana cellulose synthase 3 (AtCesA3) fragments. The level of stably-folded AtCesA3 fragments displayed on the yeast surface are determined using flow cytometric analysis of differential surface expression of epitopes flanking the AtCesA3 fragment. This technique provides a fast and simple method for examining folding and expression of protein domains and fragments of complex proteins.
Assuntos
Arabidopsis/genética , Parede Celular/genética , Glucosiltransferases/genética , Complexos Multiproteicos/genética , Arabidopsis/enzimologia , Membrana Celular/química , Membrana Celular/genética , Parede Celular/enzimologia , Celulose/química , Regulação Enzimológica da Expressão Gênica , Glucosiltransferases/química , Complexos Multiproteicos/química , Saccharomyces cerevisiae/genéticaRESUMO
Yeast surface display has become an increasingly popular tool for protein engineering and library screening applications. Recent advances have greatly expanded the capability of yeast surface display, and are highlighted by cell-based selections, epitope mapping, cDNA library screening, and cell adhesion engineering. In this review, we discuss the state-of-the-art yeast display methodologies and the rapidly expanding set of applications afforded by this technology.
Assuntos
Saccharomyces cerevisiae/genética , Animais , Moléculas de Adesão Celular/genética , DNA Complementar/biossíntese , DNA Complementar/genética , Mapeamento de Epitopos , Humanos , Engenharia de ProteínasRESUMO
The methyl-accepting chemotaxis proteins are a family of receptors in bacteria that mediate chemotaxis to diverse signals. To explore the plasticity of these proteins, we have developed a simple method for selecting cells that swim to target attractants. The procedure is based on establishing a diffusive gradient in semi-soft agar plates and does not require that the attractant be metabolized or degraded. We have applied this method to select for variants of the Escherichia coli aspartate receptor, Tar, that have a new or improved response to different amino acids. We found that Tar can be readily mutated to respond to new chemical signals. However, the overall change in specificity depended on the target compound. A Tar variant that could detect cysteic acid still showed a strong sensitivity to aspartate, indicating that the new receptor had a broadened specificity relative to wild-type Tar. Tar variants that responded to phenylalanine or N-methyl aspartate, or that had an increased sensitivity to glutamate showed a strong decrease in their response to aspartate. In at least some of the cases, the maximal level of sensitivity that was obtained could not be attributed solely to substitutions within the binding pocket. The new tar alleles and the techniques described here provide a new approach for exploring the relationship between ligand binding and signal transduction by chemoreceptors and for engineering new receptors for applications in biotechnology.
Assuntos
Proteínas de Bactérias/metabolismo , Quimiotaxia/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Aminoácido/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Proteínas de Bactérias/genética , Células Quimiorreceptoras , Meios de Cultura/química , Ácido Cisteico/metabolismo , Proteínas de Escherichia coli/genética , Ácido Glutâmico/metabolismo , Proteínas de Membrana/genética , Proteínas Quimiotáticas Aceptoras de Metil , Dados de Sequência Molecular , N-Metilaspartato/metabolismo , Fenilalanina/metabolismo , Receptores de Aminoácido/genética , Receptores de Superfície Celular/genética , Alinhamento de SequênciaRESUMO
Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Herein, we show that cells expressing fowl plague virus HA demonstrate discrete switching behavior with respect to the HA conformational change. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. These observations imply a feedback mechanism involving self-catalyzed refolding of HA and thus suggest a mechanism similar to the autocatalytic refolding and aggregation of prions.
Assuntos
Hemaglutininas Virais/metabolismo , Vírus da Influenza A/metabolismo , Modelos Moleculares , Dobramento de Proteína , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Membrana Celular/metabolismo , Ativação Enzimática , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Conformação ProteicaRESUMO
Activated lymphocyte function-associated antigen-1 (LFA-1, alphaLbeta2 integrin) found on leukocytes facilitates firm adhesion to endothelial cell layers by binding to intercellular adhesion molecule-1 (ICAM-1), which is up-regulated on endothelial cells at sites of inflammation. Recent work has shown that LFA-1 in a pre-activation, low-affinity state may also be involved in the initial tethering and rolling phase of the adhesion cascade. The inserted (I) domain of LFA-1 contains the ligand-binding epitope of the molecule, and a conformational change in this region during activation increases ligand affinity. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. Expression of a locked open, high-affinity I domain mutant supports firm adhesion of yeast, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. We find that rolling behavior for these mutants fails to correlate with ligand binding affinity. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow.
Assuntos
Antígeno CD11a/química , Molécula 1 de Adesão Intercelular/química , Antígeno-1 Associado à Função Linfocitária/química , Mutação , Epitopos/química , Humanos , Cinética , Conformação Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Aderências Teciduais , Regulação para CimaRESUMO
Increasing interest in protein immobilization on surfaces has heightened the need for techniques enabling layer-by-layer protein attachment. Here, we report a technique for controlling enzyme-mediated immobilization of layers of protein on the surface using a genetically encoded protecting group. An enterokinase-cleavable peptide sequence was inserted at the N-terminus of bifunctional fluorescent proteins containing Sortase A substrate recognition tags at both ends to control Sortase A-mediated protein immobilization on the surface layer-by-layer. Efficient, sequential immobilization of a second layer of protein using Sortase A required removal of the N-terminal protecting group, suggesting the method enables multilayer synthesis using cyclic deprotection and coupling steps. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:824-831, 2017.
Assuntos
Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Enzimas Imobilizadas/metabolismo , Enteropeptidase/química , Enteropeptidase/metabolismo , Enzimas Imobilizadas/química , Engenharia de ProteínasRESUMO
Engineering proteins that can fold to unique structures remains a challenge. Protein stability has previously been engineered via the observed correlation between thermal stability and eukaryotic secretion level. To explore the limits of an expression-based approach, variants of the highly thermostable three-helix bundle protein alpha3D were studied using yeast surface display. A library of alpha3D mutants was created to explore the possible correlation of protein stability and fold with expression level. Five efficiently expressed mutants were then purified and further studied biochemically. Despite their differences in stability, most mutants expressed at levels comparable with that of wild-type alpha3D. Two other related sequences (alpha3A and alpha3B) that form collapsed, stable molten globules but lack a uniquely folded structure were similarly expressed at high levels by yeast display. Together these observations suggest that the quality control system in yeast is unable to discriminate between well-folded proteins of high stability and molten globules. The present study, therefore, suggests that an optimization of the surface display efficiency on yeast may yield proteins that are thermally and chemically stable yet are poorly folded.
Assuntos
Temperatura Alta , Engenharia de Proteínas , Proteínas/metabolismo , Leveduras , Sequência de Aminoácidos , Clonagem Molecular , Dados de Sequência Molecular , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , Proteínas/genética , TermodinâmicaRESUMO
In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.
Assuntos
Simulação por Computador , Proteínas de Ligação a DNA , DNA/metabolismo , Mutação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas , Fatores de Transcrição , Sequência de Aminoácidos , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Elk-1 do Domínio ets , Proteínas Elk-4 do Domínio etsRESUMO
The Escherichia coli biotin ligase enzyme BirA has been extensively used in recent years to generate site-specifically biotinylated proteins via a biotin acceptor peptide tag. In the present study, BirA was displayed for the first time on the yeast Saccharomyces cerevisiae using the Aga1p-Aga2p platform and assayed using a peptide-tagged protein as the substrate. The enzyme is fully functional and resembles the soluble form in many of its properties, but the yeast-displayed enzyme demonstrates stability and reusability on the time scale of weeks. Thus, the yeast-displayed BirA system represents a facile and highly economical alternative for producing site-specifically biotinylated proteins.
Assuntos
Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Biotecnologia , Biotina/metabolismo , Catálise , Membrana Celular/enzimologia , Clonagem Molecular , Enzimas Imobilizadas , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We report a site-specific, sortase-mediated ligation to immobilize proteins layer-by-layer on a gold surface. Recombinant fluorescent proteins with a Sortase A recognition tag at the C-terminus were immobilized on peptide-modified gold surfaces. We used two sortases with different substrate specificities (Streptococcus pyogenes Sortase A and Staphylococcus aureus Sortase A) to immobilize layers of GFP and mCherry site-specifically on the gold surface. Surfaces were characterized using fluorescence and atomic force microscopy after immobilizing each layer of protein. Fluorescent micrographs showed that both protein immobilization on the modified gold surface and protein oligomerization are sortase-dependent. AFM images showed that either homogenous protein monolayers or layers of protein oligomers can be generated using appropriately tagged substrate proteins. Using Sortase A variants with orthogonal peptide substrate specificities, site-specific immobilization of appropriately tagged GFP onto a layer of immobilized mCherry was achieved without disruption of the underlying protein layer.