Field Of Structural Biology Research Articles

Chemical cross-linking and native mass spectrometry: A fruitful combination for structural biology.

Mass spectrometry (MS) is becoming increasingly popular in the field of structural biology for analyzing protein three-dimensional-structures and for mapping protein-protein interactions. In this review, the specific contributions of chemical crosslinking and native MS are outlined to reveal the structural features of proteins and protein assemblies. Both strategies are illustrated based on the examples of the tetrameric tumor suppressor protein p53 and multisubunit vinculin-Arp2/3 hybrid complexes. We describe the distinct advantages and limitations of each technique and highlight synergistic effects when both techniques are combined. Integrating both methods is especially useful for characterizing large protein assemblies and for capturing transient interactions. We also point out the future directions we foresee for a combination of in vivo crosslinking and native MS for structural investigation of intact protein assemblies.

128 The “physiological” behaviour of DNA/RNA structural motifs in gas phase

In the last decade, molecular modelling methodologies anchored to mass spectrometry (MS) techniques gained their momentum in the field of structural biology. Native MS, where non-covalent interacti...

RNA surveillance and the exosome.

RNA surveillance and the exosome.

Twenty years of RNA.

In the 20 years since the RNA journal was started there have been many exciting and unexpected findings made in the broad field of RNA biology, biochemistry, genetics and structural biology. Below, I will briefly describe a few of the many seminal discoveries.

Back to the future of RNA structure.

Back to the future of RNA structure.

Non-invasive analysis of proteins in living cells using NMR spectroscopy

NMR spectroscopy enables structural analyses of proteins and has been widely used in the structural biology field in recent decades. NMR spectroscopy can be applied to proteins inside living cells, allowing characterization of their structures and dynamics in intracellular environments. The simplest "in-cell NMR" approach employs bacterial cells; in this approach, live Escherichia coli cells overexpressing a specific protein are subjected to NMR. The cells are grown in an NMR active isotope-enriched medium to ensure that the overexpressed proteins are labeled with the stable isotopes. Thus the obtained NMR spectra, which are derived from labeled proteins, contain atomic-level information about the structure and dynamics of the proteins. Recent progress enables us to work with higher eukaryotic cells such as HeLa and HEK293 cells, for which a number of techniques have been developed to achieve isotope labeling of the specific target protein. In this review, we describe successful use of electroporation for in-cell NMR. In addition, (19)F-NMR to characterize protein-ligand interactions in cells is presented. Because (19)F nuclei rarely exist in natural cells, when (19)F-labeled proteins are delivered into cells and (19)F-NMR signals are observed, one can safely ascertain that these signals originate from the delivered proteins and not other molecules.

'Shooting gallery' for membrane proteins provides new insights into complexities of their function and structural dynamics.

'Shooting gallery' for membrane proteins provides new insights into complexities of their function and structural dynamics.

Identification of Novel Intrinsically Disordered Proteins in Eukaryotes

During the past decade, one of the most significant advances in the field of structural biology has been the paradigm-shifting recognition of proteins which are functional under physiological conditions without having a well-defined, rigid 3D structure. Contrary to Anfinsen's dogma indicating that a single unique native structure of a protein is the prerequisite of its function, intrinsically disordered proteins (IDPs) lack stable tertiary structure and exist as ensembles of time-fluctuating structural conformations, while still possessing the ability to carry out functions across a wide spectrum of cellular processes. Due to advantages arising from structural disorder, such as adaptability in partner binding, specific dynamics and enlarged binding surfaces, IDPs are often implicated in complex functions such as cell signaling and gene expression regulation. Bioinformatic predictions suggest that IDPs comprise a significant percentage of numerous eukaryotic proteomes which necessitates high-throughput studies focusing on experimental and computational characterization of such widely-predicted IDPs. In plants, IDPs play important roles in response to abiotic stress, which directly affects plant growth and yield. We have demonstrated through bioinformatic analyses that the genome of industrially important Beta vulgaris L. plant contains a large portion of IDPs. We confirmed the presence and roles in abiotic stress response for several known but also several previously undocumented IDPs using a combination of experimental methods. In order to gain further functional insights, our studies involve molecular dynamics simulations of the newly identified IDPs for ab initio prediction of pre-formed secondary structural elements in short binding regions termed Pre-Structured Motifs. The existence of functionally important transient secondary structure in some IDPs has previously been shown by others using NMR, advancing recent developments of suitable computational approaches for its prediction and thus enabling a new approach to understanding IDP function.

Design and Development of Grid Enabled, G2PU Accelerated Java Application (Protein Sequence Study) for Grid Performance Analysis

Abstract Recent advancement in the field of structural biology has generated huge volume of data and analyzing such data is vital to know the hidden truths of life but such analysis is compute intensive in nature and requires huge computational power resulting in extensive use of high performance computing (Multi Core Computing, G2PU Computing, CPU-GPU Hybrid computing, Cluster, Grid) models. Grid is one of the widely used HPC model which is commonly used in computational biology and to execute a compute intensive tasks on grid, the applications must also be grid enabled. Performance of grid depends on appropriate selection of load balancing strategy (at server and node level) and maximum utilization of computational resources of nodes (multiple cores of CPU and execution units of GPU) by the grid enabled applications. In this paper an attempt has been made to establish an experimental grid, develop a grid enabled application and to operate the grid with its highest possible performance by the grid enabled applications.

創薬等支援技術基盤プラットフォームで取り組む相関構造解析

Protein crystallography has been leading the structural biology field by solving molecular structures at atomic resolution. However, it has been limited in the integration of structural information of macromolecules with a dynamic nature, which can adopt various conformations in time scales of motion even in large complexes. Therefore, it is critical to use multiple methodologies to understand biological phenomena, and connect the interfaces of molecules at both molecular and atomic resolution in macromolecular assembly. “Correlated structural analysis” is a concept in structural life science to fill the gaps between structural biology techniques. In Japan, the Platform for Drug Discovery, Informatics, and Structural Life Science has been launched with diverse expertise in bioinformatics, sample production, and structure determination. The platform supports not only conventional structural studies via X-ray crystallography, but also correlated structural analysis by integrating protein crystallography, small angle X-ray scattering, NMR, and electron microscopy data.

Hierarchical Mechanics of Connective Tissues: Integrating Insights from Nano to Macroscopic Studies

As the key component of the musculoskeletal system, the extracellular matrix of soft connective tissues such as ligaments and tendons is a biological example of fibre-reinforced composite but with a complex hierarchical architecture. To establish a comprehensive structure-function relationship at the respective levels (i.e., from molecule to tissue) of the hierarchical architecture is challenging and requires a multidisciplinary approach, involving the integration of findings from the fields of molecular biology, biochemistry, structural biology, materials science and biophysics. Accordingly, in recent years, some of these fields, namely structural biology, materials science and biophysics, have made significant progress in the microscale and nanoscale studies of extracellular matrix using new tools, such as microelectromechanical systems, optical tweezers and atomic force microscopy, complemented by new techniques in simultaneous imaging and mechanical testing and computer modelling. The intent of this paper is to review the key findings on the mechanical response of extracellular matrix at the respective levels of the hierarchical architecture. The main focus is on the structure and function--the findings are compared across the different levels to provide insights that support the goal of establishing a comprehensive structure-function relationship of extracellular matrix. For this purpose, the review is divided into two parts. The first part explores the features of key structural units of extracellular matrix, namely tropocollagen molecule (the lowest level), microfibril, collagen fibril, collagen fibre and fascicle. The second part examines the mechanics of the structural units at the respective levels. Finally a framework for extracellular matrix mechanics is proposed to support the goal to establish a comprehensive structure-function relationship. The framework describes the integration of the mechanisms of reinforcement by the structural units at the respective levels of the hierarchical architecture in a consistent manner, both to allow comparison of these mechanisms, and to make prediction of the interconnection of these mechanisms that can also assist in the identification of effective mechanical pathways. From a design perspective, this is a step in the direction towards the development of effective strategies for engineering materials to replace or repair damaged tissues, and for exogenous cross-linking therapy to enhance the mechanical properties of injured tissues.

The history of the PDB as a public resource for enabling science

As the crystal structures of biological macromolecules were being determined, a new field of structural biology was born. Inspired by these new structures, the scientific community worked to establish a home to archive and share the data emerging from these experiments. The Protein Data Bank (PDB) was established in 1971 with seven structures. The PDB provides a repository for scientists who generate the data, and an access point for researchers and students to find the information needed to drive additional studies. Today, the PDB contains and supports online access to ~100,000 biomacromolecules that help researchers understand aspects of biology, including medicine, agriculture, and biological energy. The ways in which the interrelationships among science, technology, and community have driven the evolution of the PDB resource for more than forty years will be discussed. The PDB archive is managed by the Worldwide Protein Data Bank (wwpdb.org), whose members are the RCSB PDB, PDBe, PDBj and BMRB.

Protein Crystal Staining for Second Harmonic Generation Imaging

Recently, second harmonic generation (SHG) microscopy has become a useful tool in the field of structural biology for the detection of protein crystals. SHG, or the frequency doubling of light, is a process specific to crystalline media lacking inversion centers. Through theoretical models and experimental data, it is estimated that ~84% of the known protein crystal structures give detectable SHG signal using current SHG microscopy instrumentation. Extending this coverage could be extremely useful to structural biologists who routinely screen entire 96 well plates, with hundreds of crystallization conditions, in search of diffraction-quality protein crystals. A series of SHG active dyes, including Malachite Green (MG) and trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DMI) were investigated to assess their ability to enhance the nonlinear optical (NLO) response across a broad range of protein crystals with varying degrees of inherent SHG activity. MG and DMI were shown to enhance the SHG activity of tetragonal (P43212) lysozyme crystals, a protein that typically generates little to no SHG signal. SHG enhancements for lysozyme of approximately 16000x and 20x were achieved by intercalation of MG and DMI, respectively. These results are consistent with predictions based on the differences in symmetry and structure for the two dyes. The kinetics of the dye intercalation and uptake were investigated with SHG time-lapse images taken of a lysozyme crystal after the addition of MG dye into the crystallization well. Kinetic results indicate that an increase in SHG activity becomes easily noticeable within minutes of exposure to the dyes. These results show a significant progress towards increasing the coverage of SHG microscopy for protein crystal detection.

MoProViewer: a tool to study proteins from a charge density science perspective

Applying the charge density science methods to the structural biology field remains a considerable challenge. Several approaches were followed in the past years, focusing either on multipolar refinement of the rare available subatomic resolution protein structures or on the application of the transferability principle for the evaluation of electrostatic and energetic properties in protein-ligand complexes. However, the usually large size of macromolecules and the consequently large number of multipolar parameters in the Hansen & Coppens formalism [1] obviously complicates the feasibility of such kind of studies. In this presentation, we introduce the tools implemented in the MoProViewer software, part of the MoPro Suite for charge density refinement, especially designed to ease the analysis of a protein structure from a charge density perspective. These tools focus on helping the user in the computation of properties deriving from a refined or transferred [2] protein charge distribution and to manage a large number of atoms described in the multipole formalism in terms for instance of atomic local axis, symmetry or chemical equivalences constraints definitions. Moreover we will present new methods based on the topological analysis of the electrostatic potential [3]. They are designed to allow an original use of the electrostatic properties of a protein-ligand complex structure and rely on the computation and the representation of electrophilic and nucleophilic influence zones of atoms involved in protein-ligand interactions or in a biochemical process. The computational details of these methods as well as application examples on selected protein-ligand complexes will be given.

Traitement de la maladie de Darier par laser CO2 fractionné : un cas

The SARS-CoV-2 pandemic that struck in 2019 has left the world crippled with hundreds of millions of cases and millions of people dead. During this time, we have seen unprecedented support and collaboration amongst scientists to respond to this deadly disease. Advances in the field of structural biology, in particular cryoEM and cryo-electron tomography, have allowed unprecedented structural analysis of SARS-CoV-2. Here, we review the structural work on the SARS-CoV-2 virus and viral components, as well as its cellular assembly process, highlighting some important structural findings that have made significant impact on the protection from and treatment of emerging viral infections.

The myelin membrane-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase: on a highway to structure and function.

The membrane-anchored myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) was discovered in the early 1960s and has since then troubled scientists with its peculiar catalytic activity and high expression levels in the central nervous system. Despite decades of research, the actual physiological relevance of CNPase has only recently begun to unravel. In addition to a role in myelination, CNPase is also involved in local adenosine production in traumatic brain injury and possibly has a regulatory function in mitochondrial membrane permeabilization. Although research focusing on the CNPase phosphodiesterase activity has been helpful, several open questions concerning the protein function in vivo remain unanswered. This review is focused on past research on CNPase, especially in the fields of structural biology and enzymology, and outlines the current understanding regarding the biochemical and physiological significance of CNPase, providing ideas and directions for future research.

Radiation Damage of Myoglobin Crystals in Weak Stationary Electric and Magnetic Fields

Radiation damage is one of the bottlenecks in the field of structural biology. Cryo-cooling of protein crystals provided a breakthrough in the 1980s and resulted in significant reductions in radiation damage. Other factors positively influencing the progression of damage include the application of radical scavengers and reductions in the experimental beam size. Here we study the impact on radiation damage of applying static magnetic and electric fields during protein diffraction experiments, ultimately probing the Lorenz force effect on primary photoelectrons and secondary Auger electrons, which both contribute to the damage process. The design of a special mounting pin using graphene for applying electric fields on a crystalline sample is described. Analyses of myoglobin protein crystals exposed to the fields of ~40 mT and -300 V show a slower global radiation damage rate and also changes in the progression of specific damage process on the molecular level, in particular at doses extending beyond the Garman limit of 30 MGy.

Characterizing protein crystal contacts and their role in crystallization: rubredoxin as a case study.

The fields of structural biology and soft matter have independently sought out fundamental principles to rationalize protein crystallization. Yet the conceptual differences and the limited overlap between the two disciplines have thus far prevented a comprehensive understanding of the phenomenon to emerge. We conduct a computational study of proteins from the rubredoxin family that bridges the two fields. Using atomistic simulations, we characterize the protein crystal contacts, and accordingly parameterize patchy particle models. Comparing the phase diagrams of these schematic models with experimental results enables us to critically examine the assumptions behind the two approaches. The study also reveals features of protein–protein interactions that can be leveraged to crystallize proteins more generally.

High-Resolution NMR Spectroscopy Reveals Structure of Lipoprotein flpp3

Characterization of membrane proteins continues to be an enormous challenge in the field of structural biology compared to their soluble counterparts. Membrane proteins require the presence of a detergent micelle to protect the hydrophobic-exposed residues that are, under native conditions, inserted into a membrane. This provides challenging hurdles for expression, purification, and structural determination. However, for some lipoproteins and in the case of flpp3 from Francisella tularensis, the hydrophilic domain of the protein can be expressed and purified separately from the transmembrane domain. Flpp3 contains a single transmembrane helix at the N-terminus and therefore this technique allows for structural investigation of the majority of the protein without the necessity of a detergent micelle. In this study we report the results of expression, purification, biophysical characterization and NMR analysis of the soluble domain of flpp3 (flpp3-Sol). Sufficient quantities of pure flpp3-Sol are obtained via recombinant expression in E. coli cells followed by purification via Ni-NTA chromatography and gel filtration chromatography. Monodispersity and global secondary structure is investigated by dynamic light scattering (DLS) and circular dichroism (CD) respectively. NMR studies report local secondary structure via secondary chemical shifts and are used to generate a structure-model using CS-ROSETTA. Structures generated from NMR data are compared to structures in the Protein Data Bank using the Dali server and provide insight into possible protein function. The structure of flpp3-Sol is similar to the C-terminal domain of the lipoprotein bamC from E coli, a protein important for the insertion of beta-barrel proteins into the outer membrane of cells via the BAM complex; and as of yet unidentified in F tularensis.

Structural differences observed in arboviruses of the alphavirus and flavivirus genera.

Arthropod borne viruses have developed a complex life cycle adapted to alternate between insect and vertebrate hosts. These arthropod-borne viruses belong mainly to the families Togaviridae, Flaviviridae, and Bunyaviridae. This group of viruses contains many pathogens that cause febrile, hemorrhagic, and encephalitic disease or arthritic symptoms which can be persistent. It has been appreciated for many years that these viruses were evolutionarily adapted to function in the highly divergent cellular environments of both insect and mammalian phyla. These viruses are hybrid in nature, containing viral-encoded RNA and proteins which are glycosylated by the host and encapsulate viral nucleocapsids in the context of a host-derived membrane. From a structural perspective, these virus particles are macromolecular machines adapted in design to assemble into a packaging and delivery system for the virus genome and, only when associated with the conditions appropriate for a productive infection, to disassemble and deliver the RNA cargo. It was initially assumed that the structures of the virus from both hosts were equivalent. New evidence that alphaviruses and flaviviruses can exist in more than one conformation postenvelopment will be discussed in this review. The data are limited but should refocus the field of structural biology on the metastable nature of these viruses.