RESUMEN
Expression of isotopically labeled peptide standards as artificial concatamers (QconCATs) allows for the multiplex quantification of proteins in unlabeled samples by mass spectrometry. We have developed a generalizable QconCAT design strategy, which we term IQcat, wherein concatenated peptides are binned by pI to facilitate MS-sample enrichment by isoelectric focusing. Our method utilizes a rapid (â¼2 weeks), inexpensive and scalable purification of arg/lys labeled IQcat standards in the Escherichia coli auxotroph AT713. With this pipeline, we assess the fidelity of IQcat-based absolute quantification for ten yeast proteins over a broad concentration range in a single information-rich isoelectric fraction. The technique is further employed for a quantitative study of androgen-dependent protein expression in cultured prostate cancer cells.
Asunto(s)
Focalización Isoeléctrica/métodos , Espectrometría de Masas/métodos , Péptidos/análisis , Proteínas/análisis , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Humanos , Focalización Isoeléctrica/economía , Masculino , Espectrometría de Masas/economía , Datos de Secuencia Molecular , Neoplasias de la Próstata/química , Proteómica/economía , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/análisisRESUMEN
The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high-temperature elution in urea-containing buffers, we show that mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde cross-linked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen-activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.
Asunto(s)
Biotina/aislamiento & purificación , Cromatografía de Afinidad/métodos , Receptores Androgénicos/aislamiento & purificación , Estreptavidina/química , Secuencia de Aminoácidos , Biotina/metabolismo , Biotinilación , Línea Celular Tumoral , Humanos , Espectrometría de Masas/normas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Proteómica , Receptores Androgénicos/biosíntesis , Receptores Androgénicos/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Estándares de Referencia , Saccharomyces cerevisiaeRESUMEN
The methylotrophic yeast Pichia pastoris is a powerful eukaryotic platform for the production of heterologous protein. Recent publication of the P. pastoris genome has facilitated strain development toward biopharmaceutical and environmental science applications and has advanced the organism as a model system for the study of peroxisome biogenesis and methanol metabolism. Here we report the development of a P. pastoris arg-/lys- auxotrophic strain compatible with SILAC (stable isotope labeling by amino acids in cell culture) proteomic studies, which is capable of generating large quantities of isotopically labeled protein for mass spectrometry-based biomarker measurements. We demonstrate the utility of this strain to produce high purity human serum albumin uniformly labeled with isotopically heavy arginine and lysine. In addition, we demonstrate the first quantitative proteomic analysis of methanol metabolism in P. pastoris, reporting new evidence for a malate-aspartate NADH shuttle mechanism in the organism. This strain will be a useful model organism for the study of metabolism and peroxisome generation.
Asunto(s)
Marcaje Isotópico , Pichia/genética , Albúmina Sérica/biosíntesis , Secuencia de Aminoácidos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas de Inactivación de Genes , Vectores Genéticos , Humanos , Metanol/metabolismo , Datos de Secuencia Molecular , Peroxisomas/metabolismo , Pichia/enzimología , Pichia/metabolismo , Proteómica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/normas , Estándares de Referencia , Vesículas Secretoras/metabolismo , Albúmina Sérica/metabolismo , Albúmina Sérica/normas , Espectrometría de Masas en Tándem/normas , Regulación hacia ArribaRESUMEN
The four classes of heterotrimeric G-protein alpha subunits act as molecular routers inside cells, gating signals based on a bound guanosine nucleotide (guanosine 5'-triphosphate versus guanosine 5'-diphosphate). Ligands that specifically target individual subunits provide new tools for monitoring and modulating these networks, but are challenging to design due to the high sequence homology and structural plasticity of the Galpha-binding surface. Here we have created an mRNA display library of peptides based on the short Galpha-modulating peptide R6A-1 and selected variants that target a convergent protein-binding surface of Galphas.guanosine 5'-diphosphate. After selection/evolution, the most Galphas-specific peptide, Galphas(s)-binding peptide (GSP), was used to design a second-generation library, resulting in several new affinity- and selectivity-matured peptides denoted as mGSPs. The two-step evolutionary walk from R6A-1 to mGSP-1 resulted in an 8000-fold inversion in binding specificity, altered seven out of nine residues in the starting peptide core, and incorporated both positive and negative design steps. The resulting mGSP-1 peptide shows remarkable selectivity and affinity, exhibiting little or no binding to nine homologous Galpha subunits or human H-Ras, and even discriminates the Galphas splice variant Galphas(l). Selected peptides make specific contacts with the effector-binding region of Galpha, which may explain an interesting bifunctional activity observed in GSP. Overall, our work demonstrates a design of simple, linear, highly specific peptides that target a protein-binding surface of Galphas and argues that mRNA display-based selection/evolution is a powerful route for targeting protein families with high class specificity and state specificity.
Asunto(s)
Evolución Molecular Dirigida , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Biblioteca de Genes , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Ratas , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/metabolismo , Ingeniería de Proteínas/métodos , Secuencias de Aminoácidos , Afinidad de Anticuerpos , Modelos Moleculares , Biblioteca de Péptidos , Péptidos Cíclicos/química , Unión ProteicaRESUMEN
Intracellular Galpha subunits represent potential therapeutic targets for a number of diseases. Here we describe three classes of new molecules that modulate G protein signaling by direct targeting of Galpha. Using messenger RNA display, we have identified unique peptide sequences that bind Galpha i1 . Functionally, individual peptides were found that either enhance or repress basal levels of G protein-activated inwardly rectifying potassium (GIRK) channel signaling, a downstream effector of G protein activation, indicating that the peptides directly turn G proteins on or off in vivo . A third functional class acts as a signaling attenuator; basal GIRK channel activity is unaffected but responses to repeated G protein activation are reduced. These data demonstrate that G protein-directed ligands can achieve physiological effects similar to those resulting from classical receptor targeting and may serve as leads for developing new classes of therapeutics.
Asunto(s)
Proteínas de Unión al GTP/química , Regulación de la Expresión Génica , Péptidos/química , Secuencia de Aminoácidos , Bioquímica/métodos , Línea Celular , ADN Complementario/metabolismo , Electrofisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/efectos de los fármacos , Humanos , Inmunoprecipitación , Ligandos , Datos de Secuencia Molecular , Péptidos/farmacología , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to Galpha(i1). The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of Galpha(i1) and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with Galpha subunits representing all four G protein classes, acting as a core motif for Galpha interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (Galpha(i/0)-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to Galpha(i1-3) in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the Galpha subunits and excludes association with Gbetagamma. R6A-Galpha(i1) complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg(2+), suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using Galpha(i1)/Galpha(s) chimeras identify two regions of Galpha(i1) (residues 1-35 and 57-88) as determinants for strong R6A-G(ialpha1) interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/química , Péptidos/química , Secuencias de Aminoácidos , Clonación Molecular , ADN Complementario/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Vectores Genéticos , Guanosina Difosfato/química , Guanosina Trifosfato/química , Humanos , Hidrólisis , Inmunoprecipitación , Sustancias Macromoleculares/química , Magnesio/química , Modelos Genéticos , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Factores de Tiempo , Tripsina/farmacologíaRESUMEN
N proteins from bacteriophages lambda, P22, and phi21 modulate transcription elongation by binding nascent "boxB" mRNA hairpins. This RNA recognition is mediated by N-terminal arginine-rich peptide sequences capable of interacting with their cognate boxB RNA targets. Here, we have analyzed the affinity and specificity of the peptide-RNA interactions that modulate this transcriptional switch. To do this, we constructed a series of peptides based on the wild-type lambda, P22, and phi21 N protein binding domains ranging from 11 to 22 residues and analyzed their interactions with the leftward and rightward boxB RNA hairpin targets for all three phage. Binding constant (K(d)) values were determined using RNA hairpins labeled with 2-aminopurine (2AP) and monitoring the fluorescence change as peptide was added. K(d)'s demonstrate that lambda and P22 N peptides bind to their cognate boxB targets with high specificity and show equal affinities for their leftward and rightward hairpins. Surprisingly, phi21 shows very little specificity for its cognate targets. Lambda and P22 N peptides exhibit differential modes of recognition with specificity conferred by their amino- and carboxy-terminal modules, respectively. We have generated a reciprocal matrix of substituted peptides to examine the contributions of individual residues to specificity. Amino acid coupling analysis supports a binding model where the Arg8 residue of lambda peptide acts as a conformational hot spot, anchoring the induced loop fold of its boxB hairpin target.
Asunto(s)
Conformación de Ácido Nucleico , Proteínas de la Nucleocápside/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Secuencia de Aminoácidos , Bacteriófago P22/química , Bacteriófago P22/metabolismo , Bacteriófago lambda/química , Bacteriófago lambda/metabolismo , Calorimetría , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas de la Nucleocápside/química , Fragmentos de Péptidos/síntesis química , Unión Proteica , ARN Viral/química , ARN Viral/metabolismo , Proteínas de Unión al ARN/química , Espectrometría de Fluorescencia , Electricidad Estática , Termodinámica , Proteínas Reguladoras y Accesorias Virales/químicaRESUMEN
In vitro peptide and protein selection using mRNA display enables the discovery and directed evolution of new molecules from combinatorial libraries. These selected molecules can serve as tools to control and understand biological processes, enhance our understanding of molecular interactions and potentially treat disease in therapeutic applications. In mRNA display, mRNA molecules are covalently attached to the peptide or protein they encode. These mRNA-protein fusions enable in vitro selection of peptide and protein libraries of >10(13) different sequences. mRNA display has been used to discover novel peptide and protein ligands for RNA, small molecules and proteins, as well as to define cellular interaction partners of proteins and drugs. In addition, several unique applications are possible with mRNA display, including self-assembling protein chips and library construction with unnatural amino acids and chemically modified peptides.
Asunto(s)
Perfilación de la Expresión Génica , ARN/metabolismo , Adenosina Trifosfato/metabolismo , Epítopos/metabolismo , Ligandos , Modelos Biológicos , Estructura Molecular , Conformación de Ácido Nucleico , Péptidos/metabolismo , Conformación Proteica , ARN/genética , Estreptavidina/metabolismoRESUMEN
Arginine-rich peptide motifs (ARMs) capable of binding unique RNA structures play critical roles in transcription, translation, RNA trafficking, and RNA packaging. Bacteriophage ARMs necessary for transcription antitermination bind to distinct boxB RNA hairpin sequences with a characteristic induced alpha-helical structure. Characterization of ARMs from lambdoid phages reveals that the dissociation constant of the P22 bacteriophage model-antitermination complex (P22(N21)-P22boxB) is 200 +/- 56 pM in free solution at physiologic concentrations of monovalent cation, significantly stronger than previously determined by gel mobility shift and polyacrylamide gel coelectophoresis, and 2 orders of magnitude stronger than the tightest known native ARM-RNA interaction at physiological salt. Here, we use a reciprocal design approach to enhance the binding affinity of two separate alpha-helical ARM-RNA interactions; one derived from the native lambda phage antitermination complex and a second isolated using mRNA display selection experiments targeting boxB RNA.