Showing papers on "Structural biology published in 2007"
TL;DR: The dream is to 'watch' proteins in action in real time at atomic resolution, which requires addition of a fourth dimension, time, to structural biology so that the positions in space and time of all atoms in a protein can be described in detail.
Abstract: Because proteins are central to cellular function, researchers have sought to uncover the secrets of how these complex macromolecules execute such a fascinating variety of functions. Although static structures are known for many proteins, the functions of proteins are governed ultimately by their dynamic character (or 'personality'). The dream is to 'watch' proteins in action in real time at atomic resolution. This requires addition of a fourth dimension, time, to structural biology so that the positions in space and time of all atoms in a protein can be described in detail.
2,109 citations
TL;DR: Structural comparison of ModB2C2A with Sav1866 suggests a common alternating access and release mechanism, with binding of ATP promoting an outward-facing conformation and dissociation of the hydrolysis products promoting an inward- facing conformation.
Abstract: ATP-binding cassette (ABC) transporter proteins carry diverse substrates across cell membranes. Whereas clinically relevant ABC exporters are implicated in various diseases or cause multidrug resistance of cancer cells, bacterial ABC importers are essential for the uptake of nutrients, including rare elements such as molybdenum. A detailed understanding of their mechanisms requires direct visualization at high resolution and in distinct conformations. Our recent structure of the multidrug ABC exporter Sav1866 has revealed an outward-facing conformation of the transmembrane domains coupled to a closed conformation of the nucleotide-binding domains, reflecting the ATP-bound state. Here we present the 3.1 A crystal structure of a putative molybdate transporter (ModB2C2) from Archaeoglobus fulgidus in complex with its binding protein (ModA). Twelve transmembrane helices of the ModB subunits provide an inward-facing conformation, with a closed gate near the external membrane boundary. The ATP-hydrolysing ModC subunits reveal a nucleotide-free, open conformation, whereas the attached binding protein aligns the substrate-binding cleft with the entrance to the presumed translocation pathway. Structural comparison of ModB2C2A with Sav1866 suggests a common alternating access and release mechanism, with binding of ATP promoting an outward-facing conformation and dissociation of the hydrolysis products promoting an inward-facing conformation.
464 citations
TL;DR: It is shown that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane.
Abstract: The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane.
371 citations
TL;DR: The crystal structure of a periplasmic fragment of YaeT reveals the POTra domain fold and suggests a model for how POTRA domains can bind different peptide sequences, as required for a machine that handles numerous β-barrel protein precursors.
Abstract: Integral β-barrel proteins are found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. The machine that assembles these proteins contains an integral membrane protein, called YaeT in Escherichia coli , which has one or more polypeptide transport–associated (POTRA) domains. The crystal structure of a periplasmic fragment of YaeT reveals the POTRA domain fold and suggests a model for how POTRA domains can bind different peptide sequences, as required for a machine that handles numerous β-barrel protein precursors. Analysis of POTRA domain deletions shows which are essential and provides a view of the spatial organization of this assembly machine.
354 citations
TL;DR: A protocol in which dimethyl sulfate modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA–protein complexes (RNPs) is described.
Abstract: We describe a protocol in which dimethyl sulfate (DMS) modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA-protein complexes (RNPs). The protocol is optimized for RNAs of small to moderate size (< or = 500 nt). The RNA or RNP is first exposed to DMS under conditions that promote formation of the folded structure or complex, as well as 'control' conditions that do not allow folding or complex formation. The positions and extents of modification are then determined by primer extension, polyacrylamide gel electrophoresis and quantitative analysis. From changes in the extent of modification upon folding or protein binding (appearance of a 'footprint'), it is possible to detect local changes in the secondary and tertiary structure of RNA, as well as the formation of RNA-protein contacts. This protocol takes 1.5-3 d to complete, depending on the type of analysis used.
255 citations
TL;DR: The approach, which can be used to obtain orientational restraints at other sites in proteins as well, promises to significantly extend the available information necessary for providing a site-specific characterization of structural properties of transient, low populated states that have to this point remained recalcitrant to detailed analysis.
Abstract: The focus of structural biology is on studies of the highly populated, ground states of biological molecules; states that are only sparsely and transiently populated are more difficult to probe because they are invisible to most structural methods. Yet, such states can play critical roles in biochemical processes such as ligand binding, enzyme catalysis, and protein folding. A description of these states in terms of structure and dynamics is, therefore, of great importance. Here, we present a method, based on relaxation dispersion NMR spectroscopy of weakly aligned molecules in a magnetic field, that can provide such a description by direct measurement of backbone amide bond vector orientations in transient, low populated states that are not observable directly. Such information, obtained through the measurement of residual dipolar couplings, has until now been restricted to proteins that produce observable spectra. The methodology is applied and validated in a study of the binding of a target peptide to an SH3 domain from the yeast protein Abp1p and subsequently used in an application to protein folding of a mutational variant of the Fyn SH3 domain where 1H-15N dipolar couplings of the invisible unfolded state of the domain are obtained. The approach, which can be used to obtain orientational restraints at other sites in proteins as well, promises to significantly extend the available information necessary for providing a site-specific characterization of structural properties of transient, low populated states that have to this point remained recalcitrant to detailed analysis.
169 citations
TL;DR: The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus vectors, and functional activity was determined by binding assays in yeast and mammalian cells.
Abstract: Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.
137 citations
TL;DR: Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis and enable high-throughput screening of compounds that stabilize discrete protein folds.
Abstract: Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.
125 citations
TL;DR: An MS-based proteomic protocol for facile, high-throughput and unbiased discovery of SNO-Cys residues in proteins from complex biological samples is described and identified may provide insights into novel mechanisms and proteins that mediate NO bioactivities in health and disease.
Abstract: Covalent addition of nitric oxide (NO) to Cys-sulfur in proteins, or S-nitrosylation, plays pervasive roles in the physiological and pathophysiological modulation of mammalian protein functions. Knowledge of the specific protein Cys residues that undergo NO addition in different biological settings is fundamental to understanding NO-mediated signal transduction. Here, we describe in detail an MS-based proteomic protocol for facile, high-throughput and unbiased discovery of SNO-Cys residues in proteins from complex biological samples. The approach, termed SNOSID (SNO-Cys site identification), can be used to identify endogenous and chemically induced S-nitrosylation sites in proteins from tissues or cells. Identified SNO-Cys sites may provide insights into novel mechanisms and proteins that mediate NO bioactivities in health and disease. SNOSID builds on the biotin-switch method for covalent addition of disulfide-linked biotin at S-nitrosylation sites on proteins. Biotinylated proteins are then subjected to trypsinolysis and the resulting biotin-tagged peptides are affinity-captured on streptavidin-agarose. After selective elution with beta-mercaptoethanol, the peptides are sequenced using nanoflow liquid chromatography tandem mass spectrometry (nLC-MS/MS). Validation that identified peptide ions as originating from authentic NO-Cys-containing precursor proteins can be provided by establishing that these peptide ions are absent from control samples where S-NO bonds were subjected to prior photolysis, using a UV transilluminator. The protocol requires approximately 2 days for sample processing, including the incubation time for proteolysis. An additional 1-2 days is needed for sample analysis by nLC-MS/MS and data analysis/interpretation.
93 citations
01 Jan 2007
84 citations
TL;DR: Two X-ray structures of EmrE, the smallest ion-coupled multi-drug transporter, raised questions over the veracity of the structural model and represented a cautionary tale about the difficulty of determining the 3D structures of membrane proteins and the dangers of ignoring biochemical results.
TL;DR: It is shown that the variation of NMR dipolar couplings and heteronuclear relaxation rates in α-synuclein closely follows the variations of the bulkiness of amino acids along the polypeptide chain.
Abstract: Natively unfolded proteins play key roles in normal and pathological biochemical processes. This category of proteins remains, however, beyond the reach of classical structural biology because of their inherent conformational heterogeneity. When confined in weakly aligning media, natively unfolded proteins such as α-synuclein, the major component of abnormal aggregates in the brain of patients with Parkinson's disease, display surprisingly variable NMR dipolar couplings as a function of position along the chain, suggesting the presence of residual secondary or tertiary structure. Here we show that the variation of NMR dipolar couplings and heteronuclear relaxation rates in α-synuclein closely follows the variations of the bulkiness of amino acids along the polypeptide chain. Our results demonstrate that the bulkiness of amino acids defines the local conformations and dynamics of α-synuclein and other natively unfolded proteins. Deviations from this random coil behavior can provide insight into residual se...
TL;DR: Structural analysis in conjunction with single-molecule studies has revealed a wealth of new insights into how enzymes use ATP-driven conformational changes to move on nucleic acids using ATP hydrolysis.
TL;DR: The proposed method, which utilizes the information of structure-unknown data, predicts disordered proteins more accurately than other methods and is less affected by training data sparseness.
Abstract: Predicting intrinsically disordered proteins is important in structural biology because they are thought to carry out various cellular functions even though they have no stable three-dimensional structure. We know the structures of far more ordered proteins than disordered proteins. The structural distribution of proteins in nature can therefore be inferred to differ from that of proteins whose structures have been determined experimentally. We know many more protein sequences than we do protein structures, and many of the known sequences can be expected to be those of disordered proteins. Thus it would be efficient to use the information of structure-unknown proteins in order to avoid training data sparseness. We propose a novel method for predicting which proteins are mostly disordered by using spectral graph transducer and training with a huge amount of structure-unknown sequences as well as structure-known sequences. When the proposed method was evaluated on data that included 82 disordered proteins and 526 ordered proteins, its sensitivity was 0.723 and its specificity was 0.977. It resulted in a Matthews correlation coefficient 0.202 points higher than that obtained using FoldIndex, 0.221 points higher than that obtained using the method based on plotting hydrophobicity against the number of contacts and 0.07 points higher than that obtained using support vector machines (SVMs). To examine robustness against training data sparseness, we investigated the correlation between two results obtained when the method was trained on different datasets and tested on the same dataset. The correlation coefficient for the proposed method is 0.14 higher than that for the method using SVMs. When the proposed SGT-based method was compared with four per-residue predictors (VL3, GlobPlot, DISOPRED2 and IUPred (long)), its sensitivity was 0.834 for disordered proteins, which is 0.052–0.523 higher than that of the per-residue predictors, and its specificity was 0.991 for ordered proteins, which is 0.036–0.153 higher than that of the per-residue predictors. The proposed method was also evaluated on data that included 417 partially disordered proteins. It predicted the frequency of disordered proteins to be 1.95% for the proteins with 5%–10% disordered sequences, 1.46% for the proteins with 10%–20% disordered sequences and 16.57% for proteins with 20%–40% disordered sequences. The proposed method, which utilizes the information of structure-unknown data, predicts disordered proteins more accurately than other methods and is less affected by training data sparseness.
TL;DR: The crystal structure of the human Ebp1 is reported, which has the conserved pita bread fold of methionine aminopeptidases, but without the characteristic enzymatic activity.
TL;DR: The much-anticipated structure of the bacterial membrane protein DsbB, the redox partner of DsbA, comprises a flexible redox loop embedded in an antiparallel four-helix bundle, similar to that of soluble eukaryotic Ero1p and Erv2p proteins, theredox partners of PDI.
TL;DR: The results offer no compelling evidence that Naf-BBL folds downhill, particularly as non-truncated, unmodified peripheral subunit binding domains seem to fold cooperatively.
Abstract: There is controversy as to whether homologues from the peripheral subunit binding domain family of small proteins fold 'downhill' (that is, non-cooperatively, in the absence of free-energy barriers between conformations) and whether they modulate their size for biological function. Sadqi et al. claim that Naf-BBL--a naphthylalanine-labelled, truncated version of this domain--folds in this way, on the grounds that they recorded a wide spread of melting temperatures of individual atoms measured by proton nuclear magnetic resonance (NMR) during their thermal denaturation. But their data are not of adequate quality to distinguish, within experimental error, between downhill folding and folding with a cooperative transition. Accordingly, their results offer no compelling evidence that Naf-BBL folds downhill, particularly as non-truncated, unmodified peripheral subunit binding domains seem to fold cooperatively.
TL;DR: This review is focused on surface proteins of bacteria using Streptococcus pneumoniae, a Gram-positive bacterium, as an example, and mechanisms for hyaluronate lyase, pneumococcal surface protein A, pneumolysin, histidine-triad and fibronectin-binding proteins are discussed.
Abstract: Bacteria present a variety of molecules either on their surface or in a cell-free form. These molecules take part in numerous processes in the interactions with their host, with its tissues and other molecules. These molecules are essential to bacterial pathogenesis either during colonization or the spread/invasion stages, and most are virulence factors. This review is focused on such molecules using Streptococcus pneumoniae, a Gram-positive bacterium, as an example. Selected surface proteins are introduced, their structure described, and, whenever available, their mechanisms of function on an atomic level are explained. Such mechanisms for hyaluronate lyase, pneumococcal surface protein A, pneumolysin, histidine-triad and fibronectin-binding proteins are discussed. Elucidation of molecular mechanisms of virulence factors is essential for the understanding of bacteria and their functional properties. Structural biology appears pivotal for these studies, as structural and mechanistic insights facilitate rational approach to the development of new treatments.
TL;DR: This work presents and validate a strategy to rapidly produce, permutate, and posttranslationally modify large, eukaryotic multiprotein complexes by using DNA recombination in a process that is fully automatable.
TL;DR: Recent work is highlighted that is providing insight into the structure and crystal recognition mechanisms of an exemplary salivary protein model system, but which also provides a general approach to studying protein-crystal interactions in molecular detail.
TL;DR: The role of structural biology in systems biology should be to provide enough understanding so that macromolecules can be translated into dots or even into equations devoid of atoms.
TL;DR: The question as to how far prokaryotic homologues can take us towards a structural understanding of the eukaryotic/human membrane proteome(s) is probed.
Abstract: Membrane proteins are core components of many essential cellular processes, and high-resolution structural data is therefore highly sought after. However, owing to the many bottlenecks associated with membrane protein crystallization, progress has been slow. One major problem is our inability to obtain sufficient quantities of membrane proteins for crystallization trials. Traditionally, membrane proteins have been isolated from natural sources, or for prokaryotic proteins, expressed by recombinant techniques. We are however a long way away from a streamlined overproduction of eukaryotic proteins. With this technical limitation in mind, we have probed the question as to how far prokaryotic homologues can take us towards a structural understanding of the eukaryotic/human membrane proteome(s).
TL;DR: This review focuses on a brief description of site-directed spin labeling-derived techniques in the context of their recent applications.
Abstract: Site-directed spin labeling and electron paramagnetic resonance spectroscopy have recently experienced an outburst of multiple applications in protein science. Numerous interesting strategies have been introduced for determining the structure of proteins and its conformational changes at the level of the backbone fold. Moreover, considerable technical development in the field makes the technique an attractive approach for the study of structure and dynamics of membrane proteins and large biological complexes at physiological conditions. This review focuses on a brief description of site-directed spin labeling-derived techniques in the context of their recent applications.
TL;DR: Recently determined ParB-centromere structures have revealed key insights into segrosome assembly, whereas ParA structures have shed light on the mechanism of plasmid separation, which represent important steps in elucidating the molecular details ofplasmid segregation.
TL;DR: Two programs, AnnoLite and AnnoLyze, which use the structural alignments deposited in the DBAli database to reliably and automatically annotate new protein structures.
Abstract: Background
Advances in structural biology, including structural genomics, have resulted in a rapid increase in the number of experimentally determined protein structures. However, about half of the structures deposited by the structural genomics consortia have little or no information about their biological function. Therefore, there is a need for tools for automatically and comprehensively annotating the function of protein structures. We aim to provide such tools by applying comparative protein structure annotation that relies on detectable relationships between protein structures to transfer functional annotations. Here we introduce two programs, AnnoLite and AnnoLyze, which use the structural alignments deposited in the DBAli database.
TL;DR: The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na,K-ATPase.
TL;DR: Improvements in production of NTS1 open up the possibility of direct structural studies, such as solid-state NMR to interrogate the NT-binding site, EM (electron microscopy), and X-ray crystallography and NMR.
Abstract: NT (neurotensin) is an endogenous tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. One receptor for NT, NTS1, belongs to the GPCR (G-protein-coupled receptor) superfamily, has seven putative transmembrane domains, and is being studied by a range of single-molecule, functional and structural approaches. To enable biophysical characterization, sufficient quantities of the receptor need to be expressed and purified in an active form. To this end, rat NTS1 has been expressed in Escherichia coli in an active ligand-binding form at the cell membrane and purified in sufficient amounts for structural biology studies either with or without fluorescent protein [YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein)] fusions. Ligand binding has been demonstrated in a novel SPR (surface plasmon resonance) approach, as well as by conventional radioligand binding measurements. These improvements in production of NTS1 now open up the possibility of direct structural studies, such as solid-state NMR to interrogate the NT-binding site, EM (electron microscopy), and X-ray crystallography and NMR.
TL;DR: Ferguson et al. and Zhou and Bai criticize the quality of the NMR data and atom-by-atom analysis of global 'downhill' folding, also claiming that the data are compatible with two-state folding.
Abstract: Ferguson .
1 and Zhou and Bai
2 criticize the quality of our nuclear magnetic resonance (NMR) data and atom-by-atom analysis of global 'downhill' folding3, also claiming that the data are compatible with two-state folding.