Showing papers on "Structural biology published in 2005"

Chemistry in living systems.

Abstract: Dissecting complex cellular processes requires the ability to track biomolecules as they function within their native habitat. Although genetically encoded tags such as GFP are widely used to monitor discrete proteins, they can cause significant perturbations to a protein's structure and have no direct extension to other classes of biomolecules such as glycans, lipids, nucleic acids and secondary metabolites. In recent years, an alternative tool for tagging biomolecules has emerged from the chemical biology community—the bioorthogonal chemical reporter. In a prototypical experiment, a unique chemical motif, often as small as a single functional group, is incorporated into the target biomolecule using the cell's own biosynthetic machinery. The chemical reporter is then covalently modified in a highly selective fashion with an exogenously delivered probe. This review highlights the development of bioorthogonal chemical reporters and reactions and their application in living systems.

Molecular dynamics and protein function

Abstract: A fundamental appreciation for how biological macromolecules work requires knowledge of structure and dynamics. Molecular dynamics simulations provide powerful tools for the exploration of the conformational energy landscape accessible to these molecules, and the rapid increase in computational power coupled with improvements in methodology makes this an exciting time for the application of simulation to structural biology. In this Perspective we survey two areas, protein folding and enzymatic catalysis, in which simulations have contributed to a general understanding of mechanism. We also describe results for the F1 ATPase molecular motor and the Src family of signaling proteins as examples of applications of simulations to specific biological systems.

The structural biology of type ii fatty acid biosynthesis

Abstract: ▪ Abstract The type II fatty acid synthetic pathway is the principal route for the production of membrane phospholipid acyl chains in bacteria and plants. The reaction sequence is carried out by a series of individual soluble proteins that are each encoded by a discrete gene, and the pathway intermediates are shuttled between the enzymes as thioesters of an acyl carrier protein. The Escherichia coli system is the paradigm for the study of this system, and high-resolution X-ray and/or NMR structures of representative members of every enzyme in the type II pathway are now available. The structural biology of these proteins reveals the specific three-dimensional features of the enzymes that explain substrate recognition, chain length specificity, and the catalytic mechanisms that define their roles in producing the multitude of products generated by the type II system. These structures are also a valuable resource to guide antibacterial drug discovery.

Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase

Abstract: Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic alpha-helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.

Structural insights into a yeast prion illuminate nucleation and strain diversity

Abstract: Self-perpetuating changes in the conformations of amyloidogenic proteins play vital roles in normal biology and disease. Despite intense research, the architecture and conformational conversion of amyloids remain poorly understood. Amyloid conformers of Sup35 are the molecular embodiment of the yeast prion known as [PSI], which produces heritable changes in phenotype through self-perpetuating changes in protein folding. Here we determine the nature of Sup35's cooperatively folded amyloid core, and use this information to investigate central questions in prion biology. Specific segments of the amyloid core form intermolecular contacts in a 'Head-to-Head', 'Tail-to-Tail' fashion, but the 'Central Core' is sequestered through intramolecular contacts. The Head acquires productive interactions first, and these nucleate assembly. Variations in the length of the amyloid core and the nature of intermolecular interfaces form the structural basis of distinct prion 'strains', which produce variant phenotypes in vivo. These findings resolve several problems in yeast prion biology and have broad implications for other amyloids.

Solving the membrane protein folding problem

Abstract: One of the great challenges for molecular biologists is to learn how a protein sequence defines its three-dimensional structure. For many years, the problem was even more difficult for membrane proteins because so little was known about what they looked like. The situation has improved markedly in recent years, and we now know over 90 unique structures. Our enhanced view of the structure universe, combined with an increasingly quantitative understanding of fold determination, engenders optimism that a solution to the folding problem for membrane proteins can be achieved.

The importance of sequence diversity in the aggregation and evolution of proteins

Abstract: The tendency for proteins to aggregate is a problem that any living system must overcome. Conditions such as Alzheimer's disease and late-onset diabetes show what can happen if aggregation is not controlled. Two systems that suppress aggregation, molecular chaperones and quality control proteins, have been widely studied, but little is known about how selective pressure on amino acid sequences might contribute. A study of the giant molecule titin now shows that the sequences of multidomain proteins may have have evolved to reduce the probability of misfolding and aggregation. Titin is well suited to this study as its component immunoglobulin domains unfold and refold as an intrinsic part of muscle action. The results show that although equivalent immunoglobulin domains from different organisms frequently have around 95% sequence identity, sequence identity between adjacent domains in a given protein is only about 25%. Calculations suggest that neighbouring domain sequence identity over 30–40% could cause misfolding and unwanted inter-domain aggregation. Incorrect folding of proteins, leading to aggregation and amyloid formation, is associated with a group of highly debilitating medical conditions1,2 including Alzheimer's disease and late-onset diabetes. The issue of how unwanted protein association is normally avoided in a living system is particularly significant in the context of the evolution of multidomain proteins, which account for over 70% of all eukaryotic proteins3, where the effective local protein concentration in the vicinity of each domain is very high. Here we describe the aggregation kinetics of multidomain protein constructs of immunoglobulin domains and the ability of different homologous domains to aggregate together. We show that aggregation of these proteins is a specific process and that the efficiency of coaggregation between different domains decreases markedly with decreasing sequence identity. Thus, whereas immunoglobulin domains with more than about 70% identity are highly prone to coaggregation, those with less than 30–40% sequence identity do not detectably interact. A bioinformatics analysis of consecutive homologous domains in large multidomain proteins shows that such domains almost exclusively have sequence identities of less than 40%, in other words below the level at which coaggregation is likely to be efficient. We propose that such low sequence identities could have a crucial and general role in safeguarding proteins against misfolding and aggregation.

A network of orthogonal ribosome·mRNA pairs

Abstract: Synthetic biology promises the ability to program cells with new functions. Simple oscillators, switches, logic functions, cell-cell communication and pattern-forming circuits have been created by the connection of a small set of natural transcription factors and their binding sites in different ways to produce different networks of molecular interactions. However, the controlled synthesis of more complex synthetic networks and functions will require an expanded set of functional molecules with known molecular specificities. Here, we tailored the molecular specificity of duplicated Escherichia coli ribosome x mRNA pairs with respect to the wild-type ribosome and mRNAs to produce multiple orthogonal ribosome x orthogonal mRNA pairs that can process information in parallel with, but independent of, their wild-type progenitors. In these pairs, the ribosome exclusively translates the orthogonal mRNA, and the orthogonal mRNA is not a substrate for cellular ribosomes. We predicted and measured the network of interactions between orthogonal ribosomes and orthogonal mRNAs, and showed that they can be used to post-transcriptionally program the cell with Boolean logic.

Structure and different conformational states of native AMPA receptor complexes

Abstract: Ionotropic glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. Their modulation is believed to affect learning and memory, and their dysfunction has been implicated in the pathogenesis of neurological and psychiatric diseases. Despite a wealth of functional data, little is known about the intact, three-dimensional structure of these ligand-gated ion channels. Here, we present the structure of native AMPA receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; AMPA-Rs) purified from rat brain, as determined by single-particle electron microscopy. Unlike the homotetrameric recombinant GluR2 (ref. 3), the native heterotetrameric AMPA-R adopted various conformations, which reflect primarily a variable separation of the two dimeric extracellular amino-terminal domains. Members of the stargazin/TARP family of transmembrane proteins co-purified with AMPA-Rs and contributed to the density representing the transmembrane region of the complex. Glutamate and cyclothiazide markedly altered the conformational equilibrium of the channel complex, suggesting that desensitization is related to separation of the N-terminal domains. These data provide a glimpse of the conformational changes of an important ligand-gated ion channel of the brain.

Solution structure of a protein denatured state and folding intermediate.

Abstract: The most controversial area in protein folding concerns its earliest stages. Questions such as whether there are genuine folding intermediates, and whether the events at the earliest stages are just rearrangements of the denatured state or progress from populated transition states, remain unresolved. The problem is that there is a lack of experimental high-resolution structural information about early folding intermediates and denatured states under conditions that favour folding because competent states spontaneously fold rapidly. Here we have solved directly the solution structure of a true denatured state by nuclear magnetic resonance under conditions that would normally favour folding, and directly studied its equilibrium and kinetic behaviour. We engineered a mutant of Drosophila melanogaster Engrailed homeodomain that folds and unfolds reversibly just by changing ionic strength. At high ionic strength, the mutant L16A is an ultra-fast folding native protein, just like the wild-type protein; however, at physiological ionic strength it is denatured. The denatured state is a well-ordered folding intermediate, poised to fold by docking helices and breaking some non-native interactions. It unfolds relatively progressively with increasingly denaturing conditions, and so superficially resembles a denatured state with properties that vary with conditions. Such ill-defined unfolding is a common feature of early folding intermediate states and accounts for why there are so many controversies about intermediates versus compact denatured states in protein folding.

Heptameric (L12)6/L10 rather than canonical pentameric complexes are found by tandem MS of intact ribosomes from thermophilic bacteria.

Abstract: Ribosomes are universal translators of the genetic code into protein and represent macromolecular structures that are asymmetric, often heterogeneous, and contain dynamic regions. These properties pose considerable challenges for modern-day structural biology. Despite these obstacles, high-resolution x-ray structures of the 30S and 50S subunits have revealed the RNA architecture and its interactions with proteins for ribosomes from Thermus thermophilus, Deinococcus radiodurans, and Haloarcula marismortui. Some regions, however, remain inaccessible to these high-resolution approaches because of their high conformational dynamics and potential heterogeneity, specifically the so-called L7/L12 stalk complex. This region plays a vital role in protein synthesis by interacting with GTPase factors in translation. Here, we apply tandem MS, an approach widely applied to peptide sequencing for proteomic applications but not previously applied to MDa complexes. Isolation and activation of ions assigned to intact 30S and 50S subunits releases proteins S6 and L12, respectively. Importantly, this process reveals, exclusively while attached to ribosomes, a phosphorylation of L12, the protein located in multiple copies at the tip of the stalk complex. Moreover, through tandem MS we discovered a stoichiometry for the stalk protuberance on Thermus thermophilus and other thermophiles and contrast this assembly with the analogous one on ribosomes from mesophiles. Together with evidence for a potential interaction with the degradosome, these results show that important findings on ribosome structure, interactions, and modifications can be discovered by tandem MS, even on well studied ribosomes from Thermus thermophilus.

Mass spectrometry in biophysics : conformation and dynamics of biomolecules

Abstract: Preface. 1 General Overview of Basic Concepts in Molecular Biophysics. 1.1. Covalent Structure of Biopolymers. 1.2. Noncovalent Interactions and Higher-order Structure. 1.3. The Protein Folding Problem. 1.4. Protein Energy Landscapes and the Folding Problem. 1.5. Protein Dynamics and Function. References. 2 Overview of "Traditional" Experimental Arsenal to Study Biomolecular Structure and Dynamics. 2.1. X-Ray Crystallography. 2.2. Solution Scattering Techniques. 2.3. NMR Spectroscopy. 2.4. Other Spectroscopic Techniques. 2.5. Other Biophysical Methods to Study Macromolecular Interactions and Dynamics. References. 3 Overview of Biological Mass Spectrometry. 3.1. Basic Principles of Mass Spectrometry. 3.2. Methods of Producing Biomolecular Ions. 3.3. Mass Analysis. 3.4. Tandem Mass Spectrometry. 3.5. Brief Overview of Common Mass Analyzers. References. 4 Mass Spectrometry-Based Approaches to Study Biomolecular Higher-Order Structure. 4.1. Biomolecular Topography: Contact and Proximity Maps via Chemical Cross-Linking. 4.2. Mapping Solvent-Exposed Regions: Footprinting Methods. 4.3. Emerging Low-Resolution Methods: Zero-Interference Approaches. References. 5 Mass Spectrometry-based Approaches to Study Biomolecular Dynamics: Equilibrium Intermediates. 5.1. Monitoring Equilibrium Intermediates: Protein Ion Charge State Distributions (ESI MS). 5.2. Chemical Labeling and Trapping Equilibrium States in Unfolding Experiments. 5.3. Structure and Dynamics of Intermediate Equilibrium States: Use of Hydrogen Exchange. 5.4. Measurements of Local Patterns of Hydrogen Exchange. References. 6 Kinetic Studies by Mass Spectrometry. 6.1. Kinetics of Protein Folding. 6.2. Kinetics by Mass Spectrometry. 6.3. Kinetics of Enzyme Catalysis. References. 7 Protein Interaction: A Closer Look at the "Structure-Dynamics-Function" Triad. 7.1. Protein-Ligand Interactions: Characterization of Noncovalent Complexes Using Direct ESI MS Measurements. 7.2. Indirect Characterization of Noncovalent Interactions: Measurements Under Native Conditions. 7.3. Indirect Characterization of Noncovalent Interactions: Exploiting Protein Dynamics Under Denaturing Conditions. 7.4. Understanding Protein Action: Mechanistic Insights from the Analysis of Structure and Dynamics Under Native Conditions. 7.5. Understanding Protein Action: Mechanistic Insights from the Analysis of Structure and Dynamics Under Denaturing Conditions. References. 8 Synergism Between Biophysical Techniques. 8.1. Hen Egg White Lysozyme. 8.2. Molecular Chaperones. References. 9 Other Biopolymers and Synthetic Polymers of Biological Interest. 9.1. DNA. 9.2. RNA. 9.3. Oligosaccharides. 9.4. "Passive" Polymers of Biotic and Abiotic Origin. References. 10 Biomolecular Ions in a Solvent-Free Environment. 10.1. General Considerations: Role of Solvent in Maintaining Biomolecular Structure and Modulating its Dynamics. 10.2. Experimental Methods to Study Biomolecular Structure in Vacuo. 10.3. Protein and Peptide Ion Behavior in a Solvent-Free Environment. 10.4. Protein Hydration in the Gas Phase: Bridging "Micro" and "Macro". References. 11 Mass Spectrometry on the March: Where Next? From Molecular Biophysics to Structural Biology, Perspectives and Challenges. 11.1. Assembly and Function of Large Macromolecular Complexes: From Oligomers to Subcellular Structures to ... Organisms? 11.2. Structure and Dynamics of Membrane Proteins. 11.3. Macromolecular Trafficking and Cellular Signaling. 11.4. In Vivo versus in Vitro Behavior of Biopolymers. References. Appendix: Physics of Electrospray. Index.

New EMBO Member's Review The many faces of protease-protein inhibitor interaction

Abstract: Proteases and their natural protein inhibitors are among the most intensively studied protein–protein complexes. There are about 30 structurally distinct inhibitor families that are able to block serine, cysteine, metalloand aspartyl proteases. The mechanisms of inhibition can be related to the catalytic mechanism of protease action or include a mechanism-unrelated steric blockage of the active site or its neighborhood. The structural elements that are responsible for the inhibition most often include the Nor the C-terminus or exposed loop(s) either separately or in combination of several such elements. During complex formation, no major conformational changes are usually observed, but sometimes structural transitions of the inhibitor and enzyme occur. In many cases, convergent evolution, with respect to the inhibitors’ parts that are responsible for the inhibition, can be inferred from comparisons of their structures or sequences, strongly suggesting that there are only limited ways to inhibit proteases by proteins. The EMBO Journal (2005) 24, 1303–1310. doi:10.1038/ sj.emboj.7600611; Published online 3 March 2005 Subject Categories: structural biology; proteins

Imaging molecular interactions in living cells.

Abstract: Hormones integrate the activities of their target cells through receptor-modulated cascades of protein interactions that ultimately lead to changes in cellular function. Understanding how the cell assembles these signaling protein complexes is critically important to unraveling disease processes, and to the design of therapeutic strategies. Recent advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins, now allow the assembly of these signaling protein complexes to be tracked within the organized microenvironment of the living cell. Here, we review some of the recent developments in the application of imaging techniques to measure the dynamic behavior, colocalization, and spatial relationships between proteins in living cells. Where possible, we discuss the application of these different approaches in the context of hormone regulation of nuclear receptor localization, mobility, and interactions in different subcellular compartments. We discuss measurements that define the spatial relationships and dynamics between proteins in living cells including fluorescence colocalization, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy. These live-cell imaging tools provide an important complement to biochemical and structural biology studies, extending the analysis of protein-protein interactions, protein conformational changes, and the behavior of signaling molecules to their natural environment within the intact cell.

Prion protein remodelling confers an immediate phenotypic switch.

Abstract: The prospects of limiting the spread of transmissible spongiform encephalopathies such as Creutzfeldt–Jakob disease depend in part on identifying the most infectious forms of the prions that carry the diseases. A study of modified scrapie prions shows that clusters of 14 to 28 prion proteins are the most infectious and that clusters of less than six molecules have virtually no infectivity. That could have implications for the treatment of diseases such as Alzheimer's and Parkinson's, characterized by deposition of prion-related amyloid fibrils. It's possible that efforts to alleviate symptoms by destabilizing these large protein aggregates might make things worse by producing smaller, more infective particles. Two other papers in this issue tackle fundamental aspects of the biology of prions and amyloid fibrils. The conversion of the yeast protein Sup35 to its prion form does not need to happen during the synthesis of Sup35 — mature and fully functional molecules can readily join a prion seed. This remodelling of the mature protein is accompanied by the immediate loss of its activity. And a study of a ‘designed’ amyloid fibril made from ribonuclease A reveals that amyloid containing native-like molecules can retain enzyme activity. This involves a domain swap with the neighbouring protein, and supports the ‘zipper-spine model’ for β-amyloid structures. In a variety of systems, proteins have been linked to processes historically limited to nucleic acids, such as infectivity and inheritance1,2. These atypical proteins, termed prions3, lack sequence homology but are collectively defined by their capacity to adopt multiple physical and therefore functional states in vivo. Newly synthesized prion protein generally adopts the form already present in the cell, and this in vivo folding bias directs the near faithful transmission of the corresponding phenotypic state1,2. Switches between the prion and non-prion phenotypes can occur in vivo2; however, the fate of existing protein during these transitions and its effects on the emergence of new traits remain major unanswered questions. Here, we determine the changes in protein-state that induce phenotypic switching for the yeast prion Sup35/[PSI+]. We show that the prion form does not need to be specified by an alternate misfolding pathway initiated during Sup35 synthesis but instead can be accessed by mature protein. This remodelling of protein from one stable form to another is accompanied by the loss of Sup35 activity, evoking a rapid change in cellular phenotype within a single cell cycle.

Decharging of Globular Proteins and Protein Complexes in Electrospray

Abstract: Electrospray ionization mass spectrometry (ESI-MS) is a valuable tool in structural biology for investigating globular proteins and their biomolecular interactions. During the electrospray ionization process, proteins become desolvated and multiply charged, which may influence their structure. Reducing the net charge obtained during the electrospray process may be relevant for studying globular proteins. In this report we demonstrate the effect of a series of inorganic and organic gas-phase bases on the number of charges that proteins and protein complexes attain. Solution additives with very strong gas-phase basicities (GB) were identified among the so-called "proton sponges". The gas-phase proton affinities (PA) of the compounds that were added to the aqueous protein solutions ranged from 700 to 1050 kJ mol(-1). Circular dichroism studies showed that in these solutions the proteins retain their globular structures. The size of the proteins investigated ranged from the 14.3 kDa lysozyme up to the 800 kDa tetradecameric chaperone complex GroEL. Decharging of the proteins in the electrospray process by up to 60 % could be achieved by adding the most basic compounds rather than the more commonly used ammonium acetate additive. This decharging process probably results from proton competition events between the multiply protonated protein ions and the basic additives just prior to the final desolvation. We hypothesize that such globular protein species, which attain relatively few charges during the ionization event, obtain a gas-phase structure that more closely resembles their solution-phase structure. Thus, these basic additives can be useful in the study of the biologically relevant properties of globular proteins by using mass spectrometry.

Structural biology: proteins flex to function.

Abstract: Static pictures of protein structures are so prevalent that it is easy to forget they are dynamic molecular machines. Characterizing their intrinsic motions may be necessary to understand how they work.

Structural biology: prying into prions.

Abstract: Various aberrant protein forms are the subject of intense research. It is not easy to probe their structures, but studies that have done so provide telling information about their biological properties.

Efficient strategy for the rapid backbone assignment of membrane proteins.

Abstract: Investigations of membrane proteins pose one of the biggest current challenges in structural biology. Recent advances in protein production techniques based on cell-free transcription/translation methods have, however, opened new opportunities in this area. Here, we report an efficient protocol for the backbone assignment of membrane proteins as the first step of NMR-based structure determination.

Novel cyanine-AMP conjugates for efficient 5′ RNA fluorescent labeling by one-step transcription and replacement of [γ-32P]ATP in RNA structural investigation

Abstract: Two novel fluorescent cyanine-AMP conjugates, F550/570 and F650/670, have been synthesized to serve as transcription initiators under the T7 phi2.5 promoter. Efficient fluorophore labeling of 5' RNA is achieved in a single transcription step by including F550/570 and F650/670 in the transcription solution. The current work makes fluorescently labeled RNA readily available for broad applications in biochemistry, molecular biology, structural biology and biomedicine. In particular, site-specifically fluorophore-labeled large RNAs prepared by the current method may be used to investigate RNA structure, folding and mechanism by various fluorescence techniques. In addition, F550/570 and F650/670 may replace [gamma-32P]ATP to prepare 5' labeled RNA for RNA structural and functional investigation, thereby eliminating the need for the unstable and radio-hazardous [gamma-32P]ATP.

Structure of Apaf-1 in the auto-inhibited form: a critical role for ADP.

Abstract: The pathway of apoptosis is conserved in the three model species: mammals, Drosophila, and C. elegans. The apoptotic protease-activating factor 1, an essential protein conserved in all three species, is responsible for the activation of the initiator caspase-9 in mammalian cells. The structure of the auto-inhibited form of Apaf-1 reveals a critical role for ADP, which serves as an organizing center for four adjoining domains. The ADP-binding pocket contains features that are important for designing other nucleotide analogs. ATP binding is a prerequisite for the formation of the apoptosome. Despite strong sequence conservation between Apaf-1 and its orthologues in Drosophila and C. elegans, it is unclear whether they employ similar mechanisms for their own activation and for activating caspases. Much of the underlying mechanisms remain to be investigated by structural biology and biochemistry.