RESUMO
The conjugation of proteins with polymers offers immense biotechnological potential by creating novel macromolecules. This article presents experimental findings on the structural properties of maltose-binding protein (MBP) conjugated with linear biodegradable polyphosphoester polymers with different molecular weights. We studied isotopic effects on both proteins and polymers. Circular dichroism and fluorescence spectroscopy and small-angle neutron scattering reveal that the conjugation process destabilizes the protein, affecting the secondary more than the tertiary structure, even at room temperature, and that the presence of two domains in the MBP may contribute to its observed instability. Notably, unfolding temperatures differ between native MBP and the conjugates. In particular, this study sheds light on the complex interplay of factors such as the deuteration influencing protein stability and conformational changes in the conjugation processes. The perdeuteration influences the hydrogen bond network and hydrophobic interactions in the case of the MBP protein. The perdeuteration of the protein influences the hydrogen bond network and hydrophobic interactions. This is evident in the decreased thermal stability of deuterated MBP protein, in the conjugate, especially with high-molecular-mass polymers.
Assuntos
Deutério , Proteínas Ligantes de Maltose , Estabilidade Proteica , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismo , Deutério/química , Polímeros/química , Ligação de Hidrogênio , Interações Hidrofóbicas e HidrofílicasRESUMO
Cysteine conjugation is widely used to constrain phage displayed peptides for the selection of cyclic peptides against specific targets. In this study, the nontoxic Bi3+ ion was used as a cysteine conjugation reagent to cross-link peptide libraries without compromising phage infectivity. We constructed a randomized 3-cysteine peptide library and cyclized it with Bi3+, followed by a selection against the maltose-binding protein as a model target. Next-generation sequencing of selection samples revealed the enrichment of peptides containing clear consensus sequences. Chemically synthesized linear and Bi3+ cyclized peptides were used for affinity validation. The cyclized peptide showed a hundred-fold better affinity (0.31 ± 0.04 µM) than the linear form (39 ± 6 µM). Overall, our study proved the feasibility of developing Bi3+ constrained bicyclic peptides against a specific target using phage display, which would potentially accelerate the development of new peptide-bismuth bicycles for therapeutic or diagnostic applications.
Assuntos
Biblioteca de Peptídeos , Peptídeos Cíclicos , Peptídeos Cíclicos/química , Cisteína/química , Proteínas Ligantes de Maltose/metabolismo , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Ciclização , Peptídeos/química , Sequência de AminoácidosRESUMO
Polyphosphate exhibits a unique post-translational modification-like function, known as histidine polyphosphate modification (HPM), marked by a robust non-covalent interaction with histidine repeat proteins. Here, we present a protocol for detecting HPM of human proteins via maltose-binding protein-tagged expression in E. coli. We describe steps for detecting HPM by observing electrophoretic mobility shifts on NuPAGE gels followed by western blot. We then detail procedures for analyzing the influence of ionic strength and pH on HPM. For complete details on the use and execution of this protocol, please refer to Neville et al.1.
Assuntos
Escherichia coli , Histidina , Proteínas Ligantes de Maltose , Polifosfatos , Processamento de Proteína Pós-Traducional , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Polifosfatos/metabolismo , Polifosfatos/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Proteínas Ligantes de Maltose/química , Histidina/metabolismo , Histidina/genética , Histidina/química , Western BlottingRESUMO
Fusion proteins (FPs) are frequently utilized as a biotechnological tool in the determination of macromolecular structures using X-ray methods. Here, we explore the use of different protein tags in various FP, to obtain initial phases by using them in a partial molecular replacement (MR) and constructing the remaining FP structure with ARP/wARP. Usually, the tag is removed prior to crystallization, however leaving the tag on may facilitate crystal formation, and structural determination by expanding phases from known to unknown segments of the complex. In this study, the Protein Data Bank was mined for an up-to-date list of FPs with the most used protein tags, Maltose Binding Protein (MBP), Green Fluorescent Protein (GFP), Thioredoxin (TRX), Glutathione transferase (GST) and the Small Ubiquitin-like Modifier Protein (SUMO). Partial MR using the protein tag, followed by automatic model building, was tested on a subset of 116 FP. The efficiency of this method was analyzed and factors that influence the coordinate construction of a substantial portions of the fused protein were identified. Using MBP, GFP, and SUMO as phase generators it was possible to build at least 75 % of the protein of interest in 36 of the 116 cases tested. Our results reveal that tag selection has a significant impact; tags with greater structural stability, such as GFP, increase the success rate. Further statistical analysis identifies that resolution, Wilson B factor, solvent percentage, completeness, multiplicity, protein tag percentage in the FP (considering amino acids), and the linker length play pivotal roles using our approach. In cases where a structural homologous is absent, this method merits inclusion in the toolkit of protein crystallographers.
Assuntos
Proteínas de Fluorescência Verde , Proteínas Ligantes de Maltose , Proteínas Recombinantes de Fusão , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismo , Cristalografia por Raios X/métodos , Glutationa Transferase/genética , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Modelos Moleculares , Bases de Dados de Proteínas , Cristalização/métodos , Conformação ProteicaRESUMO
Cells contain membraneless compartments that assemble due to liquid-liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bioinspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non-phase-separating domain. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and influence droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations coassemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates.
Assuntos
Condensados Biomoleculares/química , Proteínas/química , Tensoativos/química , Adsorção , Simulação por Computador , Glutationa Transferase/química , Proteínas Intrinsicamente Desordenadas/química , Proteínas Ligantes de Maltose/química , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes de Fusão/químicaRESUMO
With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose-binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions.
Assuntos
Cobre/química , Transferência Ressonante de Energia de Fluorescência , Proteínas Ligantes de Maltose/metabolismo , beta-Alanina/análogos & derivados , Sequência de Aminoácidos , Fluorometria , Células HEK293 , Humanos , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Modelos Químicos , Mutação , Conformação Proteica em alfa-Hélice , Relação Estrutura-Atividade , Fatores de Tempo , beta-Alanina/químicaRESUMO
Bacillus subtilis has five type I signal peptidases, one of these, SipW, is an archaeal-like peptidase. SipW is expressed in an operon (tapA-sipW-tasA) and is responsible for removing the signal peptide from two proteins: TapA and TasA. It is unclear from the signal peptide sequence of TasA and TapA, why an archaeal-like signal peptidase is required for their processing. Bioinformatic analysis of TasA and TapA indicates that both contain highly similar signal peptide cleavage sites, both predicted to be cleaved by Escherichia coli signal peptidase I, LepB. We show that expressing full length TasA in E. coli is toxic and leads to cell death. To determine if this phenotype is due to the inability of the E. coli LepB to process the TasA signal peptide, we fused the TasA signal peptide and two amino acids of mature TasA (up to P2') to both maltose binding protein (MBP) and ß-lactamase (Bla). We observed a defect in secretion, indicated by an abundance of unprocessed protein with both TasA-MBP and TasA-Bla fusions. A series of mutations in both TasA-MBP and TasA-Bla were made around the junction of the TasA signal peptide and the fusion protein. Both of these studies indicate that residues around the predicted TasA signal sequence cleavage site, particularly the sequence from P3 to P2', inhibit processing by LepB. The cell death observed when TasA and TasA signal sequence fusion proteins are expressed is likely due to the TasA signal peptide blocking LepB and thereby the general secretion pathway.
Assuntos
Proteínas de Bactérias/genética , Proteínas Ligantes de Maltose/genética , Proteínas de Membrana/genética , Serina Endopeptidases/genética , beta-Lactamases/genética , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/patogenicidade , Proteínas de Bactérias/química , Morte Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas Ligantes de Maltose/química , Proteínas de Membrana/química , Mutação/genética , Oligopeptídeos/química , Oligopeptídeos/genética , Ligação Proteica , Sinais Direcionadores de Proteínas/genética , Serina Endopeptidases/química , beta-Lactamases/químicaRESUMO
Maltose binding protein (MBP) has a long history as an expression tag with the ability to increase the solubility of fused proteins. A critical step for obtaining a sufficient amount of the MBP fusion protein is purification. Commercially available amylose matrix for the affinity purification of MBP fusion proteins has two main issues: (i) low (micromolar) affinity and (ii) the limited number of uses due to the cleavage of polysaccharide matrix by the amylases, present in the crude cell extract. Here, we present a new affinity purification approach based on the protein-protein interaction. We developed the affinity matrix which contains immobilized Designed Ankyrin Repeat Protein off7 (DARPin off7) - previously identified MBP binder with nanomolar affinity. The functionality of the DARPin affinity matrix was tested on the purification of MBP-tagged green fluorescent protein and flavodoxin. The affinity purification of the MBP fusion proteins, based on the MBP-DARPin off7 interaction, enables the purification of the fusion proteins in a simple two-steps procedure. The DARPin affinity matrix - easy to construct, resistant to amylase, insensitive to maltose contamination, and reusable for multiple purification cycles - provides an alternative approach to commercially available affinity matrices for purification of proteins containing the MBP tag.
Assuntos
Cromatografia de Afinidade , Escherichia coli , Proteínas Ligantes de Maltose , Engenharia de Proteínas , Proteínas Recombinantes de Fusão , Proteínas de Repetição de Anquirina Projetadas/biossíntese , Proteínas de Repetição de Anquirina Projetadas/química , Proteínas de Repetição de Anquirina Projetadas/genética , Proteínas de Repetição de Anquirina Projetadas/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ligantes de Maltose/biossíntese , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Atypical S1 and S11 split inteins have been used for N-terminal or C-terminal protein labeling. Here we reported a novel site-specific internal protein labeling method based on two atypical split inteins, Ter DnaE3 S11 and Rma DnaB S1. Protein-peptide trans-splicing activity was first demonstrated in vitro between a short peptide (Flag tag, FLAG) and two recombinant proteins (Maltose binding protein, MBP, and Thioredoxin, Trx) by trans-splicing between MBP-TE3S11N (MBP-N fragment of Ter DnaE3 S11), TE3S11C-FLAG-RBS1N (C fragment of Ter DnaE3 S11-FLAG-N fragment of Rma DnaB S1), and RBS1C-Trx (C fragment of Rma DnaB S1-Trx). To minimize the middle synthetic peptide (TE3S11C-linker-RBS1N), we reduced the number of native extein amino acids, which may play a role in protein trans-splicing. The results showed at least 3 (CKG) native extein amino acids were required for detectable trans-splicing activity. This method was further demonstrated to be effective in facilitating the incorporation of fluorescent probe (FITC) to the internal site of recombinant protein, generating the FITC-labeled protein. Besides the fluorescent group, these two split inteins can also be useful for adding any desirable chemical groups into a protein of interest, which may include biotin, modified and unnatural amino acids, or drug molecules.
Assuntos
Fluoresceína-5-Isotiocianato/química , Inteínas , Proteínas Ligantes de Maltose/química , Oligopeptídeos/química , Engenharia de Proteínas , Processamento de Proteína , Tiorredoxinas/química , Trans-Splicing , Proteínas Ligantes de Maltose/genética , Oligopeptídeos/genética , Tiorredoxinas/genéticaRESUMO
The junction adhesion molecule (JAM) family of proteins play central roles in the tight junction (TJ) structure and function. In contrast to claudins (CLDN) and occludin (OCLN), the other membrane proteins of the TJ, whose structure is that of a 4α-helix bundle, JAMs are members of the immunoglobulin superfamily. The JAM family is composed of four members: A, B, C and 4. The crystal structure of the extracellular domain of JAM-A continues to be used as a template to model the secondary and tertiary structure of the other members of the family. In this article, we have expressed the extracellular domains of JAMs fused with maltose-binding protein (MBP). This strategy enabled the work presented here, since JAM-B, JAM-C and JAM4 are more difficult targets due to their more hydrophobic nature. Our results indicate that each member of the JAM family has a unique tertiary structure in spite of having similar secondary structures. Surface plasmon resonance (SPR) revealed that heterotypic interactions among JAM family members can be greatly favored compared to homotypic interactions. We employ the well characterized epithelial cadherin (E-CAD) as a means to evaluate the adhesive properties of JAMs. We present strong evidence that suggests that homotypic or heterotypic interactions among JAMs are stronger than that of E-CADs.
Assuntos
Caderinas/química , Claudinas/química , Proteínas Ligantes de Maltose/química , Ocludina/química , Antígenos CD/química , Cromatografia , Dicroísmo Circular , Biologia Computacional , Simulação por Computador , Escherichia coli/metabolismo , Humanos , Moléculas de Adesão Juncional/metabolismo , Cinética , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Ressonância de Plasmônio de Superfície , Junções Íntimas/metabolismoRESUMO
Maltose binding protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The monoclonal antibody B48 binds MBP tightly and has no cross-reactivity to other proteins in an Escherichia coli lysate. This high level of specificity suggested that MBP contains an epitope that could prove useful as a purification and visualization tag for proteins expressed in E. coli. To discover the MBP epitope, a co-crystal structure was determined for MBP bound to its antibody and four amino acids of MBP were identified as critical for the binding interaction. Fusions of various fragments of MBP to the glutathione S-transferase protein were engineered in order to identify the smallest fragment still recognized by the α-MBP antibody. Stabilization of the epitope via mutational engineering resulted in a minimized 14 amino-acid tag.
Assuntos
Epitopos/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas Ligantes de Maltose/química , Cristalografia por Raios X , Epitopos/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Ligantes de Maltose/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Infection by Enterotoxigenic Escherichia coli is a common cause of diarrhea in animals. The development of vaccines against enterotoxins can effectively control the infection. We have previously constructed a recombinant antigen SLS fused by STa, LTB and STb enterotoxin and it showed a high immunogenicity in mice. Herein, we evaluated the expression of SLS in three different E. coli cells with corresponding plasmids. SLS proteins expressed in E. coli BL21 (DE3) and Rosetta-gami B (DE3) were aggregated as inclusion bodies, and the proteins solubility were not obviously promoted in low temperature combined with adjustment of inducer concentration. In contrast, SLS protein with maltose-binding protein (MBP) yielded from TB1 (DE3) cells were partially soluble. After increasing the IPTG concentration in the medium up to 2 mM and incubating at 37 â for 4 h, the soluble protein yield reached the highest level (4.533 mg/0.2 L culture), which was significantly higher than the expression of SLS protein in Rosetta-gami B (DE3) (P < 0.05). Therefore, the TB1-pMAL expression system can be used for mass extraction and purification of SLS antigen prior to measuring its immunogenicity in pregnant mammals.
Assuntos
Enterotoxinas , Escherichia coli , Plasmídeos/genética , Proteínas Recombinantes de Fusão , Escherichia coli/genética , Escherichia coli/metabolismo , Corpos de Inclusão/química , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/isolamento & purificação , Proteínas Ligantes de Maltose/metabolismo , SolubilidadeRESUMO
Chemotaxis allows bacteria to detect specific compounds and move accordingly. This pathway involves signal detection by chemoreceptors (MCPs). Attributing a chemoreceptor to a ligand is difficult because there is a lot of redundancy in the MCPs that recognize a single ligand. We propose a methodology to define which chemoreceptors bind a given ligand. First, an MCP is overproduced to increase sensitivity to the ligand(s) it recognizes, thus promoting accumulation of cells around an agarose plug containing a low attractant concentration. Second, the ligand-binding domain (LBD) of the chemoreceptor is fused to maltose-binding protein (MBP), which facilitates purification and provides a control for a thermal shift assay (TSA). An increase in the melting temperature of the LBD in the presence of the ligand indicates that the chemoreceptor directly binds it. We showed that overexpression of two Shewanella oneidensis chemoreceptors (SO_0987 and SO_1056) promoted swimming toward an agarose plug containing a low concentration of chromate. The LBD of each of the two chemoreceptors was fused to MBP. A TSA revealed that only the LBD from SO_1056 had its melting temperature increased by chromate. In conclusion, we describe an efficient approach to define chemoreceptor-ligand pairs before undertaking more-sophisticated biochemical and structural studies.
Assuntos
Proteínas de Bactérias/química , Shewanella/química , Proteínas de Bactérias/genética , Ligantes , Proteínas Ligantes de Maltose/química , Temperatura de TransiçãoRESUMO
Coupling of side chain dynamics over long distances is an important component of allostery. Methionine side chains show the largest intrinsic flexibility among methyl-containing residues but the actual degree of conformational averaging depends on the proximity and mobility of neighboring residues. The 13 C NMR chemical shifts of the methyl groups of methionine residues located at long distances in the same protein show a similar scaling with respect to the values predicted from the static X-ray structure by quantum methods. This results in a good linear correlation between calculated and observed chemical shifts. The slope is protein dependent and ranges from zero for the highly flexible calmodulin to 0.7 for the much more rigid calcineurin catalytic domain. The linear correlation is indicative of a similar level of side-chain conformational averaging over long distances, and the slope of the correlation line can be interpreted as an order parameter of the global side-chain flexibility.
Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Metionina/química , Calcineurina/química , Domínio Catalítico , Teoria da Densidade Funcional , Proteínas Ligantes de Maltose/químicaRESUMO
Ribosome biogenesis in eukaryotes requires the participation of several transactivation factors that are involved in the modification, assembly, transport and quality control of the ribosomal subunits. One of these factors is the Large subunit GTPase 1 (Lsg1), a protein that acts as the release factor for the export adaptor named Nonsense-mediated mRNA decay 3 protein (Nmd3) and facilitates the incorporation of the last structural protein uL16 into the 60S subunit. Here, we characterised the recombinant yeast Lsg1 and studied its catalysis and binding properties for guanine nucleotides. We described the interaction of Lsg1 with guanine nucleotides alone and in the presence of the complex Nmd3â¢60S using fluorescence spectroscopy. Lsg1 has a greater affinity for GTP than for GDP suggesting that in the cell cytoplasm it exists mainly bound to the former. In the presence of 60S subunits loaded with Nmd3, the affinity of Lsg1 for both nucleotides increases but to a larger extent towards GTP. From this observation together with the excess of GTP present in the cytoplasm of exponentially growing cells over that of GDP, we can infer that the pre-ribosomal particle composed by Nmd3â¢60S acts as a GTP Stabilising Factor for Lsg1. Additionally, Lsg1 undergoes different conformational changes depending on its binding partner or the guanine nucleotides it interacts with. Steady-state kinetic analysis of free Lsg1 indicated slow GTP hydrolysis with values of kcat 1â¯min-1 and Km of 34⯵M.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina Difosfato/química , Guanosina Trifosfato/química , Cinética , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/enzimologia , Subunidades Ribossômicas Maiores de Eucariotos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , TermodinâmicaRESUMO
TREM2 has been identified by genomic analysis as a potential and novel target for the treatment of Alzheimer's disease. To enable structure-based screening of potential small molecule therapeutics, we sought to develop a robust crystallization platform for the TREM2 Ig-like domain. A systematic set of constructs containing the structural chaperone, maltose binding protein (MBP), fused to the Ig domain of TREM2, were evaluated in parallel expression and purification, followed by crystallization studies. Using protein crystallization and high-resolution diffraction as a readout, a MBP-TREM2 Ig fusion construct was identified that generates reproducible protein crystals diffracting at 2.0 Å, which makes it suitable for soaking of potential ligands. Importantly, analysis of crystal packing interfaces indicates that most of the surface of the TREM2 Ig domain is available for small molecule binding. A proof of concept co-crystallization study with a small library of fragments validated potential utility of this system for the discovery of new TREM2 therapeutics.
Assuntos
Cristalização/métodos , Glicoproteínas de Membrana , Chaperonas Moleculares , Receptores Imunológicos , Proteínas Recombinantes de Fusão , Humanos , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Cyclic α-maltosyl-(1â6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic α-nigerosyl-(1â6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM.
Assuntos
Arthrobacter/metabolismo , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/metabolismo , Calorimetria , Cristalografia por Raios X , Ciclodextrinas/metabolismo , Dissacarídeos/metabolismo , Redes e Vias Metabólicas , Modelos Moleculares , Conformação Proteica , Domínios ProteicosRESUMO
Methyl-NMR enables atomic-resolution studies of structure and dynamics of large proteins in solution. However, resonance assignment remains challenging. The problem is to combine existing structural informational with sparse distance restraints and search for the most compatible assignment among the permutations. Prior classification of peaks as either from isoleucine, leucine, or valine reduces the search space by many orders of magnitude. However, this is hindered by overlapped leucine and valine frequencies. In contrast, the nearest-neighbor nuclei, coupled to the methyl carbons, resonate in distinct frequency bands. Here, we develop a framework to imprint additional information about passively coupled resonances onto the observed peaks. This depends on simultaneously orchestrating closely spaced bands of resonances along different magnetization trajectories, using principles from control theory. For methyl-NMR, the method is implemented as a modification to the standard fingerprint spectrum (the 2D-HMQC). The amino acid type is immediately apparent in the fingerprint spectrum. There is no additional relaxation loss or an increase in experimental time. The method is validated on biologically relevant proteins. The idea of generating new spectral information using passive, adjacent resonances is applicable to other contexts in NMR spectroscopy.
Assuntos
Espectroscopia de Ressonância Magnética , Aminoácidos/química , Simulação por Computador , Humanos , Proteínas Ligantes de Maltose/química , Metilação , Reprodutibilidade dos Testes , Streptococcus pyogenes/metabolismoRESUMO
The peptide hormone amylin receptor is a complex of the calcitonin receptor (CTR) and an accessory protein called receptor activity-modifying proteins (RAMPs). The soluble extracellular domain (ECD) of CTR is an important binding site of peptide hormone calcitonin. RAMPs also have an ECD and the association of CTR ECD with RAMP ECD enhances the affinity of peptide hormone amylin. However, the mechanism of how RAMP ECD association enhances amylin affinity remains elusive. Here, we report evidence supporting direct molecular interaction between an antagonistic amylin analog AC413 and RAMP2 ECD. We measured FITC-labeled peptide affinity for purified receptor ECD using fluorescence polarization (FP). We first found that RAMP2 ECD addition to maltose-binding protein (MBP)-tagged CTR ECD and an engineered MBP-tagged RAMP2 ECD-CTR ECD fusion protein (MBP-RAMP2-CTR ECD fusion) enhanced AC413 affinity. This suggests that these recombinant ECD systems represent functional amylin receptors. Interestingly, AC413 C-terminal residue Tyr25 (Y25) to Pro mutation eliminated its selective affinity for the MBP-RAMP2-CTR ECD fusion suggesting the critical role of the AC413 C-terminal residue in amylin receptor selectivity. Our structural model of the RAMP2 ECD:CTR ECD complex predicted molecular interaction of AC413 C-terminal residue Y25 with RAMP2 Glu101 (E101). Our FP peptide-binding assay showed that the RAMP2 E101A mutation of MBP-RAMP2-CTR ECD fusion decreased AC413 affinity by 7-fold, while the affinity of AC413 with the Y25P mutation was minimally changed. Consistently, AC413 binding affinity for the MBP-free RAMP2-CTR ECD fusion protein was also markedly decreased by the RAMP2 E101A mutation, while the affinity of AC413 with the Y25P mutation was moderately decreased. Together, our results support the molecular interaction between the AC413 C-terminal residue Y25 and RAMP2 E101 expanding our understanding of how the accessory protein RAMP2 enhances affinity of peptide hormone amylin for its receptor.
Assuntos
Polarização de Fluorescência , Peptídeos/química , Proteína 2 Modificadora da Atividade de Receptores/química , Animais , Humanos , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Modelos Moleculares , Peptídeos/genética , Ratos , Proteína 2 Modificadora da Atividade de Receptores/genética , Salmão , Alinhamento de SequênciaRESUMO
High-speed AFM revealed the conformational change of fused in sarcoma (FUS) from a compact to an extended structure upon binding of non-coding RNA, which is supposed to allow FUS to bind to CBP/p300 for transcriptional interference. Thus, a mechanistic insight into transcription regulation by FUS and non-coding RNA is provided.