RESUMEN
Exploring the interactions between the Ca2+ binding protein calmodulin (CaM) and its target proteins remains a challenging task. Members of the Munc13 protein family play an essential role in short-term synaptic plasticity, modulated via the interaction with CaM at the presynaptic compartment. In this study, we focus on the bMunc13-2 isoform expressed in the brain, as strong changes in synaptic transmission were observed upon its mutagenesis or deletion. The CaMâbMunc13-2 interaction was previously characterized at the molecular level using short bMunc13-2-derived peptides only, revealing a classical 1â5â10 CaM binding motif. Using larger protein constructs, we have now identified for the first time a novel and unique CaM binding site in bMunc13-2 that contains an N-terminal extension of a classical 1â5â10 CaM binding motif. We characterize this motif using a range of biochemical and biophysical methods and highlight its importance for the CaMâbMunc13-2 interaction.
Asunto(s)
Calmodulina/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Espectrometría de Masas , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calmodulina/química , Bovinos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Ratas , PorcinosRESUMEN
The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Espectrometría de Masas/métodos , Albúmina Sérica Bovina/análisis , Albúmina Sérica Bovina/química , Laboratorios , Espectrometría de Masas/instrumentación , Reproducibilidad de los ResultadosRESUMEN
Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal "click reaction" and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2. Graphical abstract á .
Asunto(s)
Compuestos Alílicos/farmacología , Reactivos de Enlaces Cruzados/química , Proteínas/química , Compuestos de Sulfhidrilo/química , Urea/análogos & derivados , Urea/farmacología , Cisteína/química , Glutatión/química , Proteínas Activadoras de la Guanilato-Ciclasa/química , Concentración de Iones de Hidrógeno , Proteínas del Tejido Nervioso/química , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Chemical cross-linking in combination with mass spectrometric analysis of the created cross-linked products is an emerging technology aimed at deriving valuable structural information from proteins and protein complexes. The goal of our protocol is to obtain distance constraints for structure determination of proteins and to investigate protein-protein interactions. We present an integrated workflow for cross-linking/mass spectrometry (MS) based on protein cross-linking with MS-cleavable reagents, followed by enzymatic digestion, enrichment of cross-linked peptides by strong cation-exchange chromatography (SCX), and LC/MS/MS analysis. To exploit the full potential of MS-cleavable cross-linkers, we developed an updated version of the freely available MeroX software for automated data analysis. The commercially available, MS-cleavable cross-linkers (DSBU and CDI) used herein possess different lengths and react with amine as well as hydroxy groups. Owing to the formation of two characteristic 26-u doublets in their MS/MS spectra, many fewer false positives are found than when using classic, non-cleavable cross-linkers. The protocol, exemplified herein for BSA and the whole Escherichia coli ribosome, is robust and widely applicable, and it allows facile identification of cross-links for deriving spatial constraints from purified proteins and protein complexes. The cross-linking/MS procedure takes 2-3 days to complete.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Proteínas/química , Programas Informáticos , Espectrometría de Masas en Tándem/métodos , Animales , Bovinos , Reactivos de Enlaces Cruzados/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Conformación Proteica , Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Flujo de TrabajoRESUMEN
A major challenge in cross-linking/mass spectrometry (MS) is targeting carboxyl functions in proteins under physiological conditions that do not disturb the protein's conformation. Cross-linking of glutamic acid and aspartic acid residues in proteins will greatly expand the scope of structural mass spectrometry. We discovered that carboxyl-reactive cross-linkers have already been employed for many years in cross-linking/MS studies, yet in a completely different context. Diazirine-based cross-linkers, such as photomethionine and succinimidyldiazirine cross-linkers, are currently considered to react nonspecifically upon UV-A photoactivation with all 20 proteinogenic amino acids through a reactive carbene that inserts mainly into C-H bonds. We discovered that the cross-linking capability of diazirines based on X-H (X = C, N, O) insertion is in fact only the tip of the iceberg. Diazirines isomerize to linear diazo compounds that can react with carboxylic acids to yield esters. On top of that, the resulting cross-linked products are MS-cleavable allowing an automated analysis of cross-links via customized software tools. Therefore, diazirines open an entirely new route for photo-cross-linking of carboxylic acids. Previous cross-linking studies using diazirines have to be revisited in the light of these findings.
RESUMEN
During the last two decades, cross-linking combined with mass spectrometry (MS) has evolved as a valuable tool to gain structural insights into proteins and protein assemblies. Structural information is obtained by introducing covalent connections between amino acids that are in spatial proximity in proteins and protein complexes. The distance constraints imposed by the cross-linking reagent provide information on the three-dimensional arrangement of the covalently connected amino acid residues and serve as basis for de-novo or homology modeling approaches. As cross-linking/MS allows investigating protein 3D-structures and protein-protein interactions not only in-vitro, but also in-vivo, it is especially appealing for studying protein systems in their native environment. In this chapter, we describe the principles of cross-linking/MS and illustrate its value for investigating protein 3D-structures and for unraveling protein interaction networks.
Asunto(s)
Espectrometría de Masas , Complejos Multiproteicos/química , Mapas de Interacción de Proteínas , Proteínas/química , Aminoácidos/químicaRESUMEN
INTRODUCTION: Calmodulin (CaM) is a highly conserved Ca2+-binding protein that is exceptionally abundant in the brain. In the presynaptic compartment of neurons, CaM transduces changes in Ca2+ concentration into the regulation of synaptic transmission dynamics. Areas covered: We review selected literature including published CaM interactor screens and outline established and candidate presynaptic CaM targets. We present a workflow of biochemical and structural proteomic methods that were used to identify and characterize the interactions between CaM and Munc13 proteins. Finally, we outline the potential of ion mobility-mass spectrometry (IM-MS) for conformational screening and of protein-protein cross-linking for the structural characterization of CaM complexes. Expert commentary: Cross-linking/MS and native MS can be applied with considerable throughput to protein mixtures under near-physiological conditions, and thus effectively complement high-resolution structural biology techniques. Experimental distance constraints are applicable best when obtained by combining different cross-linking strategies, i.e. by using cross-linkers with different spacer length and reactivity, and by using the incorporation of unnatural photo-reactive amino acids. Insights from structural proteomics can be used to generate CaM-insensitive mutants of CaM targets for functional studies in vitro or ideally in vivo.
Asunto(s)
Calmodulina/genética , Proteómica , Transmisión Sináptica/genética , Secuencia de Aminoácidos/genética , Calcio/metabolismo , Humanos , Espectrometría de Masas , Neuronas/metabolismo , Terminales Presinápticos/metabolismo , Unión ProteicaRESUMEN
Photo-induced cross-linking is a highly promising technique to investigate protein conformations and protein-protein interactions in their natural cellular environment. One strategy relies on the non-directed incorporation of diazirine-containing photo-activatable amino acids into proteins and a subsequent cross-link formation induced by UV-A irradiation. The advantage of this photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can also be targeted, which is advantageous for investigating membrane proteins. Here, we present a simplified protocol that relies on the use of mineral salts medium without any special requirements for the incorporation of photo-methionines into proteins in Escherichia coli cells. The possibility to perform these experiments in E. coli is especially valuable as it is the major system for recombinant protein production. The method is exemplified for the Ca(2+) regulating protein calmodulin containing nine methionines, which were found to be replaced by their photo-activatable analogues. Our protocol allows the facile and stochastic incorporation of photo-methionines as the basis for conducting photo-cross-linking experiments in E. coli in an efficient manner.