Modern Structural Biology Research Articles

Ancient tRNA Synthetase Meets Modern Structural Biology

Use of heavy atoms to calculate the phases in protein X-ray crystallography has now been extended to include site-specific in vivo incorporation of 3-iodo-tyrosine, enabled by the use of an archaeal aminoacyl-tRNA synthetase (Sakamoto et al., 2009).

Towards eukaryotic structural complexomics

Many eukaryotic proteins exist in large multisubunit assemblies and often show compromised folding or activity when their interaction partners are not present. Protein complexes in eukaryotes can contain ten or more subunits with individual polypeptides ranging in size up to several hundred kilodalton, severely restricting the application of conventional cloning strategies and imposing constraints on the choice of the expression host. Modern structural molecular biology often depends on introducing diversity into the specimens under investigation, including mutation, truncation and placement of purification aids. Current recombinant expression methods often require a disproportionate labor investment prior to multiprotein expression, and subsequent to expression and analysis do not provide for rapid revision of the experiment. We have developed reagents and protocols for rapid and flexible multiprotein complex expressions suitable for structural biology, focusing on multigene baculoviral vectors and their recombination mediated assembly. A top priority in protein science is automation. Our strategy can be readily adapted in a robotics setup, for baculovirus/insect cell expression of protein complexes, but likewise also for mammalian or prokaryotic hosts.

Proteins with weakly funneled energy landscapes challenge the classical structure–function paradigm

Unearthing the origin of incredible acceleration of chemical reaction rates by enzymes has occupied the minds of biochemists and molecular biologists for more than a century (1). The modern paradigm of enzymatic action rests on the ideas presented by Haldane and Pauling, namely, that enzymes selectively stabilize transition states in chemical reactions, thus lowering reaction activation energies (2). The widely held microscopic view of enzymatic catalysis envisions a nearly static enzyme reaction chamber carefully sculpted by evolution to match complementarily the shape and electrostatic surface of the reaction transition state. However, recent NMR experiments have suggested that enzyme active sites are mobile on the microsecond to second time scale, commensurate with the time scales for the corresponding catalytic reactions (3). Single-molecule fluorescent spectroscopy studies also point to nontrivial static and dynamic disorder in enzymatic processes, something that is often masked by ensemble averaging in macroscopic kinetic experiments (4). Even in the light of these findings, the central dogma of modern structural biology has remained largely intact; 3D protein structure determines its function. Indeed, this paradigm has worked wonderfully over many decades to explain numerous biological processes, from enzymatic catalysis to signal transduction. Recent works, however, suggested that from one-sixth to one-third of eukaryotic proteins are either disordered or contain large disordered regions (5, 6), hinting that the traditional interpretation of the structure–function paradigm may be too limiting (6). Disordered proteins regulate many transcriptional and signal transduction processes (7). They even exhibit enzymatic activity, as was recently demonstrated experimentally (8). How this is accomplished in the absence of prearranged 3D structure has not been well understood. In a recent issue of PNAS, Roca et al. (9) have used computer simulations to explain the way a molten globule can accelerate a chemical reaction. Their work, combined with recent advances in understanding the way protein folding and functional landscapes are coupled (6, 10), provide essential groundwork for generalizing the structure–function paradigm by using energy landscape ideas.

The Structure of a Two-Disulfide Intermediate Assists in Elucidating the Oxidative Folding Pathway of a Cyclic Cystine Knot Protein

We have determined the three-dimensional structure of a two-disulfide intermediate (Cys(8)-Cys(20), Cys(14)-Cys(26)) on the oxidative folding pathway of the cyclotide MCoTI-II. Cyclotides have a range of bioactivities and, because of their exceptional stability, have been proposed as potential molecular scaffolds for drug design applications. The three-dimensional structure of the stable two-disulfide intermediate shows for the most part identical secondary and tertiary structure to the native state. The only exception is a flexible loop, which is collapsed onto the protein core in the native state, whereas in the intermediate it is more loosely associated with the remainder of the protein. The results suggest that the native fold of the peptide does not represent the free energy minimum in the absence of the Cys(1)-Cys(18) disulfide bridge and that although there is not a large energy barrier, the peptide must transiently adopt an energetically unfavorable state before the final disulfide can form.

Genetic Engineering Combined with Deep UV Resonance Raman Spectroscopy for Structural Characterization of Amyloid-like Fibrils

Elucidating the structure of the cross-beta core in large amyloid fibrils is a challenging problem in modern structural biology. For the first time, a set of de novo polypeptides was genetically engineered to form amyloid-like fibrils with similar morphology and yet different strand length. Differential ultraviolet Raman spectroscopy allowed for separation of the spectroscopic signatures of the highly ordered beta-sheet strands and turns of the fibril core. The relationship between Raman frequencies and Ramachandran dihedral angles of the polypeptide backbone indicates the nature of the beta-sheet and turn structural elements.

Conformational Specificity of the Lac Repressor Coiled-Coil Tetramerization Domain,

Predictive understanding of how the folded, functional shape of a native protein is encoded in the linear sequence of its amino acid residues remains an unsolved challenge in modern structural biology. Antiparallel four-stranded coiled coils are relatively simple protein structures that embody a heptad sequence repeat and rich diversity for tertiary packing of alpha-helices. To explore specific sequence determinants of the lac repressor coiled-coil tetramerization domain, we have engineered a set of buried nonpolar side chains at the a-, d-, and e-positions into the hydrophobic interior of the dimeric GCN4 leucine zipper. Circular dichroism and equilibrium ultracentrifugation studies show that this core variant (GCN4-pAeLV) forms a stable tetrameric structure with a reversible and highly cooperative thermal unfolding transition. The X-ray crystal structure at 1.9 A reveals that GCN4-pAeLV is an antiparallel four-stranded coiled coil of the lac repressor type in which the a, d, and e side chains associate by means of combined knobs-against-knobs and knobs-into-holes packing with a characteristic interhelical offset of 0.25 heptad. Comparison of the side chain shape and packing in the antiparallel tetramers shows that the burial of alanine residues at the e positions between the neighboring helices of GCN4-pAeLV dictates both the antiparallel orientation and helix offset. This study fills in a gap in our knowledge of the determinants of structural specificity in antiparallel coiled coils and improves our understanding of how specific side chain packing forms the teritiary structure of a functional protein.

AIDS Vaccine Development: Challenges and Opportunities

A quarter of a century after AIDS became known as a frightening new disease, substantial progress has been attained on treatments that convert this once certain death sentence into a manageable chronic disease. While some prevention successes have been attained (e.g., screening of the blood supply in industrialized countries), a safe and effective vaccine—the “holy grail” of public health prevention—remains elusive. In the late 1990s, the first HIV vaccine taken to phase III trial, the VaxGen gp120 vaccine (VaxGen, Brisbane, CA, USA), became the basis for substantial debate and controversy between empiricists (generally public health persons who believed that the urgency of the pandemic required taking some risks, including a potentially low-efficacy vaccine as a first step) and reductionists (generally basic scientists and researchers who felt that the gp120 vaccine was unlikely to work given our state of knowledge and who wanted to wait for a better candidate vaccine). With trial results now available, we know that this vaccine was not efficacious. We also know that a phase III trial, although challenging to organize and conduct among persons at high risk, is doable. What else we do and do not know scientifically is summarized nicely in the 19 chapters of this excellently edited, concise (150 pages), softbound book. The book is organized into 5 parts: Global Overview; What Does a Vaccine Need to Do?; Preclinical Development: Design Challenges; Clinical Trials; and From Testing to Deployment. Each chapter, written by experts in each field, is impressive in its balance of compactness (3–4 double-sided pages, including references), technical content, and user-friendliness (abstract and conclusion for each chapter make quick review easy). The authors and editors are to be commended for bringing each of the key topics relevant to HIV vaccines to the reader in a highly accessible form. Key topics include HIV pathogenesis; the twists and turns of what specific knowledge of simian immunodeficiency virus and nonhuman primates is or is not applicable to HIV and humans; and the highly technical nature of modern immunology, virology, and structural biology. The editors were careful to include chapters on important nonscientific aspects of HIV vaccine development, such as clinical site development, regulatory issues, scale-up, and manufacturing. This book provides an excellent introductory overview for the beginning HIV vaccine researcher or any person who needs a more technical primer on the various aspects of the HIV vaccine challenge. The number of HIV vaccine researchers is now increasing, given the support of several organizations (e.g., Bill and Melinda Gates Foundation) and collaborations (e.g., Global HIV Vaccine Enterprise, the Partnership for AIDS Vaccine Evaluation, and the Center for HIV-AIDS Vaccine Immunology). These organizations, collaborations, and researchers are attempting to better organize the human and technical resources needed to challenge this formidable foe on the scale of the Manhattan Project or the March of Dimes search for a polio vaccine. Let us hope that they will eventually succeed.

Multiple Alignment of Membrane Proteins for Measuring Residual Dipolar Couplings Using Lanthanide Ions Bound to a Small Metal Chelator

NMR structure determination of helical integral membrane proteins is one of the most challenging undertakings in modern structural biology. The solubilizing detergent micelle or phospholipid bicelle adds significantly to the overall size of the system, often requiring perdeuteration to obtain useable spectra. However, perdeuteration prevents structure determination using traditional NOE analysis. Residual dipolar coupling constraints are an attractive complement to NOE distance constraints, but alignment methods are limited to strained polyacrylamide gels due to the incompatibility of the solubilizing detergent or lipid with alignment media such as bicelles or phage particles. We demonstrate the use of lanthanide ions bound to the protein through a small thiol-linked metal chelator as a robust method for partial alignment of membrane proteins. This method provides multiple alignment orientations depending on the ion bound and permits RDC measurement of multiple bond vectors. We demonstrate that using this...

Peach: A Simple Perl-Based System for Distributed Computation and Its Application to Cryo-EM Data Processing

A simple distributed processing system named "Peach" was developed to meet the rising computational demands of modern structural biology (and other) laboratories without additional expense by using existing hardware resources more efficiently. A central server distributes jobs to idle workstations in such a way that each computer is used maximally, but without disturbing intermittent interactive users. As compared to other distributed systems, Peach is simple, easy to install, easy to administer, easy to use, scalable, and robust. While it was designed to queue and distribute large numbers of small tasks to participating computers, it can also be used to send single jobs automatically to the fastest currently available computer and/or survey the activity of an entire laboratory's computers. Tests of robustness and scalability are reported, as are three specific electron cryomicroscopy applications where Peach enabled projects that would not otherwise have been feasible without an expensive, dedicated cluster.

HPr as a Model Protein in Structure, Interaction, Folding and Stability Studies

The small size and lack of disulphide bonds or cofactors in the Histidine-containing phosphocarrier protein (HPr) makes it an attractive system with which to study structure, interaction to its enzymatic partners, and its stability and folding. Here we give an overview on the immense work that has been performed on this protein and we will show that HPr has been widely used as a model protein to study important aspects in modern Structural Biology.

Structure Determination of Membrane Proteins by NMR Spectroscopy

AbstractFor Abstract see ChemInform Abstract in Full Text.

Structure determination of membrane proteins by NMR spectroscopy.

Much of postgenomic biochemistry and all of structural biology are based on the premise that the starting point for both understanding specific biochemical processes, such as affinity, reactivity, or transport, and surveying proteomes is determining the three-dimensional structures of proteins. The two well-established methods for structure determination are highly effective when applied to samples of soluble globular proteins and their complexes: witness the enormous growth of the Protein Data Bank.1 However, the vast majority of biological functions are carried out by proteins associated with supramolecular assemblies, whose samples are problematic for both X-ray crystallography and solution NMR spectroscopy, since they are generally difficult to crystallize and do not reorient rapidly even when soluble. The examples of proteins in supramolecular assemblies whose structures have been determined with atomic resolution are exceptional and highlight the importance of developing new methods of experimental protein structure determination. The essential goals of modern structural biology are to have the capability to select proteins for study based on their biological functions and to perform genuinely unbiased surveys of proteomes unfettered by considerations of the solubility, aggregation state, or other physical properties of the polypeptides. NMR spectroscopy has the potential to accomplish these goals, since it can be applied to molecules in all physical states, including the liquid crystalline environments provided by the lipids associated with membrane proteins. Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology.2 Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome.3,4 Furthermore, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. They also provide daunting technical challenges for all methods of protein structure determination, including NMR spectroscopy.5

Light scattering studies on supersaturated protein solutions.

The difficulties associated with protein crystallization is a major obstacle in modern structural biology. The increasing demand from the protein crystal structure community for quick and non-invasive experimental techniques as well as the rapid progress of modern optics and electronics have led during the past decade to a considerable expansion of the laser light scattering techniques. The latter are now very often employed to elucidate the aggregation kinetics of supersaturated protein solutions and the mechanism(s) underlying the early nucleation stages. The experimental verification of the nucleation processes, the prediction of the effective interaction potentials and the development of appropriate diagnostics schemes are discussed.

400 Kv Electron Cryo-Microscopic Imaging Of Large Icosahedral Viruses Towards Atomic Resolution

Three-dimensional structure studies provide important information about the organization of macromolecules, often revealing biological mechanisms and protein structure-function relationships. 400 KV electron cryo-microscopy is an emerging technology that is proving to be a powerful tool for studying the structures of large macromolecular assemblies that are often not tractable using other techniques. Its large depth of field makes it well-suited for imaging large objects to high resolution. In addition, a high accelerating voltage minimizes chromatic aberration yielding images of higher contrast. Recently a 400 KV electron cryo-microscope has been used to image periodic arrays of tubulin to 3.5 Å and single particles at somewhat lower resolutions (13 Å) providing practical demonstrations of its usefulness in modern structural biology. In this paper we present high resolution image data of two large icosahedral viruses: herpes simplex virus IB nucleocapsid (HSV IB) and rice dwarf virus (RDV). Human herpes virus (HSV) is associated with a spectrum of diseases ranging from cold sores to more severe clinical manifestations such as mental retardation.

Challenges facing structural biology beamlines.

Recent advances in structural biology have generated both opportunities and challenges for synchrotron radiation facilities — issues that must be faced by the staffs of modern structural biology beamlines.

The folding process of hen lysozyme: a perspective from the 'new view'.

How a conformationally disordered polypeptide chain rapidly and efficiently achieves its well-defined native structure is still a major question in modern structural biology. Although much progress has been made towards rationalizing the principles of protein structure and dynamics, the mechanism of the folding process and the determinants of the final fold are not yet known in any detail. One protein for which folding has been studied in great detail by a combination of diverse techniques is hen lysozyme. In this article we review the present state of our knowledge of the folding process of this enzyme and focus in particular on recent experiments to probe some of its specific features. These results are then discussed in the context of the 'new view' of protein folding based on energy surfaces and landscapes. It is shown that a schematic energy surface for lysozyme folding, which is broadly consistent with our experimental data, begins to provide a unified model for protein folding through which experimental and theoretical ideas can be brought together.

Hydrogen exchange: the modern legacy of Linderstrøm-Lang.

This discussion, prepared for the Protein Society's symposium honoring the 100th anniversary of Kaj Linderstrøm-Lang, shows how hydrogen exchange approaches initially conceived and implemented by Lang and his colleagues some 50 years ago are contributing to current progress in structural biology. Examples are chosen from the active protein folding field. Hydrogen exchange methods now make it possible to define the structure of protein folding intermediates in various contexts: as tenuous molten globule forms at equilibrium under destabilizing conditions, in kinetic intermediates that exist for less than one second, and as infinitesimally populated excited state forms under native conditions. More generally, similar methods now find broad application in studies of protein structure, energetics, and interactions. This article considers the rise of these capabilities from their inception at the Carlsberg Labs to their contemporary role as a significant tool of modern structural biology.

Loose end

Loose end

Insights into protein folding using physical techniques: studies of lysozyme and alpha-lactalbumin.

Understanding the process of protein folding, during which a disordered polypeptide chain is converted into a compact well-defined structure, is one of the major challenges of modern structural biology. In this article we discuss how a combination of physical techniques can provide a structural description of the events which occur during the folding of a protein. First, we discuss how the rapid kinetic events which take place during in vitro folding can be monitored and deciphered in structural terms. Then we consider how more detailed structural descriptions of intermediates may be obtained from NMR studies of stable, partly folded states. Finally, we discuss how these experimental strategies may be extended to relate the findings of in vitro studies to the events occurring during folding in vivo. The approaches will be illustrated using results primarily from our own studies of the c-type lysozymes and the homologous alpha-lactalbumins. The conclusions from these studies are also related to those from other systems to highlight their unifying features. On the basis of these results we identify some of the determinants of the events in folding and we speculate on the importance of these in driving folding molecules to their native states.

Computer Simulation of Macromolecules

Abstract Computer simulation techniques are now an essential part of modern structural molecular biology. They are used in many different ways in order to study the conformation, dynamics and interactions of proteins and nucleic acids. In this paper, I shall review several of these applications and then focus on three specific areas, namely the conformation and dynamics of proteins including the use of free energy perturbation methods to study mutant proteins, the conformation and dynamics of DNA and DNA-drug complexes, and the use of computers with parallel architectures. Although simulation of molecules as large and complex as proteins and nucleic acids may be considered a grand challenge in itself, there are even greater challenges for the future.