Biophysical Techniques in Structural Biology.

Over the past six decades, steadily increasing progress in the application of the principles and techniques of the physical sciences to the study of biological systems has led to remarkable insights into the molecular basis of life. Of particular significance has been the way in which the determination of the structures and dynamical properties of proteins and nucleic acids has so often led directly to a profound understanding of the nature and mechanism of their functional roles. The increasing number and power of experimental and theoretical techniques that can be applied successfully to living systems is now ushering in a new era of structural biology that is leading to fundamentally new information about the maintenance of health, the origins of disease, and the development of effective strategies for therapeutic intervention. This article provides a brief overview of some of the most powerful biophysical methods in use today, along with references that provide more detailed information about recent applications of each of them. In addition, this article acts as an introduction to four authoritative reviews in this volume. The first shows the ways that a multiplicity of biophysical methods can be combined with computational techniques to define the architectures of complex biological systems, such as those involving weak interactions within ensembles of molecular components. The second illustrates one aspect of this general approach by describing how recent advances in mass spectrometry, particularly in combination with other techniques, can generate fundamentally new insights into the properties of membrane proteins and their functional interactions with lipid molecules. The third reviewdemonstrates the increasing power of rapidly evolving diffraction techniques, employing the very short bursts of X-rays of extremely high intensity that are now accessible as a result of the construction of free-electron lasers, in particular to carry out time-resolved studies of biochemical reactions. The fourth describes in detail the application of such approaches to probe the mechanism of the light-induced changes associated with bacteriorhodopsin's ability to convert light energy into chemical energy.

ProNAB: database for binding affinities of protein-nucleic acid complexes and their mutants.

Protein-nucleic acid interactions are involved in various biological processes such as gene expression, replication, transcription, translation and packaging. The binding affinities of protein-DNA and protein-RNA complexes are important for elucidating the mechanism of protein-nucleic acid recognition. Although experimental data on binding affinity are reported abundantly in the literature, no well-curated database is currently available for protein-nucleic acid binding affinity. We have developed a database, ProNAB, which contains more than 20 000 experimental data for the binding affinities of protein-DNA and protein-RNA complexes. Each entry provides comprehensive information on sequence and structural features of a protein, nucleic acid and its complex, experimental conditions, thermodynamic parameters such as dissociation constant (Kd), binding free energy (ΔG) and change in binding free energy upon mutation (ΔΔG), and literature information. ProNAB is cross-linked with GenBank, UniProt, PDB, ProThermDB, PROSITE, DisProt and Pubmed. It provides a user-friendly web interface with options for search, display, sorting, visualization, download and upload the data. ProNAB is freely available at https://web.iitm.ac.in/bioinfo2/pronab/ and it has potential applications such as understanding the factors influencing the affinity, development of prediction tools, binding affinity change upon mutation and design complexes with the desired affinity.

Supramolecular Architectures of Nucleic Acid/Peptide Hybrids.

Supramolecular architectures that are built artificially from biomolecules, such as nucleic acids or peptides, with structural hierarchical orders ranging from the molecular to nano-scales have attracted increased attention in molecular science research fields. The engineering of nanostructures with such biomolecule-based supramolecular architectures could offer an opportunity for the development of biocompatible supramolecular (nano)materials. In this review, we highlighted a variety of supramolecular architectures that were assembled from both nucleic acids and peptides through the non-covalent interactions between them or the covalently conjugated molecular hybrids between them.

Tetrahedral Framework Nucleic Acids Promote Corneal Epithelial Wound Healing in Vitro and in Vivo.

Poor post-traumatic wound healing can affect the normal function of damaged tissues and organs. For example, poor healing of corneal epithelial injuries may lead to permanent visual impairment. It is of great importance to find a therapeutic way to promote wound closure. Tetrahedral framework nucleic acids (tFNAs) are new promising nanomaterials, which can affect the biological behavior of cells. In the experiment, corneal wound healing is used as an example to explore the effect of tFNAs on wound healing. Results show that the proliferation and migration of human corneal epithelial cells are enhanced by exposure to tFNAs in vitro, possibly relevant to the activation of P38 and ERK1/2 signaling pathway. An animal model of corneal alkali burn is established to further identify the facilitation effect of tFNAs on corneal wound healing in vivo. Clinical evaluations and histological analyses show that tFNAs can improve the corneal transparency and accelerate the re-epithelialization of wounds. Both in vitro and in vivo experiments show that tFNAs can play a positive role in corneal epithelial wound healing.

Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics with the potential to detect and treat diseases. Despite tremendous advancements in NANP development, their immunotoxicity, one of the major impediments in clinical translation of traditional therapeutic nucleic acids (TNAs), has never been fully characterized. Here, we describe the first systematically studied immunological recognition of 25 representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells, which agrees with predictions based on the experiences with TNAs. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on NANP shapes, connectivities, and compositions. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.

Homology threading to generate RNA polymerase structures.

Homology threading is a powerful technology for generating structural models based on homologous structures. Here we use threading to generate four complex RNA polymerase models. The models appear to be as useful as x-ray crystal structures or cryo-electron microscopy structures to support research projects.

How to Analyze and Present SAS Data for Publication.

SAS is a powerful technique to investigate oligomeric state and domain organization of macromolecules, e.g. proteins and nucleic acids, under physiological, functional and even time resolved conditions. However, reconstructing three dimensional structures from SAS data is inherently ambiguous, as no information about orientation and phase is available. In addition experimental artifacts such as radiation damage, concentration effects and incorrect background subtraction can hinder the interpretation of even lead to wrong results. In this chapter, explanations on how to analyze data and how to assess and minimize the influence of experimental artifacts on the data. Furthermore, guidelines on how to present the resulting data and models to demonstrate the data supports the conclusion being made and that it is not biased by artifacts, will be given.

Designing and Performing Biological Solution Small-Angle Neutron Scattering Contrast Variation Experiments on Multi-component Assemblies.

Solution small-angle neutron scattering (SANS) combined with contrast variation provides information about the size and shape of individual components of a multi-component biological assembly, as well as the spatial arrangements between the components. The large difference in the neutron scattering properties between hydrogen and deuterium is key to the method. Isotopic substitution of deuterium for some or all of the hydrogen in either the molecule or the solvent can greatly alter the scattering properties of the biological assembly, often with little or no change to its biochemical properties. Thus, SANS with contrast variation provides unique information not easily obtained using other experimental techniques.If used correctly, SANS with contrast variation is a powerful tool for determining the solution structure of multi-component biological assemblies. This chapter discusses the principles of SANS theory that are important for contrast variation, essential considerations for experiment design and execution, and the proper approach to data analysis and structure modeling. As sample quality is extremely important for a successful contrast variation experiment, sample issues that can affect the outcome of the experiment are discussed as well as procedures used to verify the sample quality. The described methodology is focused on two-component biological complexes. However, examples of its use for multi-component assemblies are also discussed.

SAS-Based Structural Modelling and Model Validation.

Small angle scattering of X-rays (SAXS) and neutrons (SANS) is a structural technique to study disordered systems with chaotic orientations of scattering inhomogeneities at low resolution. An important example of such systems are solutions of biological macromolecules. Rapid development in the methodology for solution scattering data interpretation and model building during the last two decades brought the analysis far beyond the determination of just few overall structural parameters (which was the only possibility in the past) and ensured SAS a firm position in the methods palette of the modern life sciences. The advances in the methodology include ab initio approaches for shape and domain structure restoration from scattering curves without a priori structural knowledge, classification and validation of the models, evaluation of potential ambiguity associated with the reconstruction. In rigid body and hybrid modelling approaches, solution scattering is synergistically used with other structural techniques utilizing the complementary information such as atomic models of the components, intramolecular contacts, subunits orientations etc. for the reconstruction of complex systems. The usual requirement of the sample monodispersity has been loosed recently and the technique can now address such systems as weakly bound oligomers and transient complexes. These state-of-the-art methods are described together with the examples of their applications and the possible ways of post-processing of the models.

A kinetic zipper model with intrachain interactions applied to nucleic acid hairpin folding kinetics.

Single-stranded DNA and RNA hairpin structures with 4-10 nucleotides (nt) in the loop and 5-8 basepairs (bp) in the stem fold on 10-100 µs timescale. In contrast, theoretical estimate of first contact time of two ends of an ideal semiflexible polymer of similar lengths (with persistence length ~2-nt) is 10-100 ns. We propose that this three-orders-of-magnitude difference between these two timescales is a result of roughness in the folding free energy surface arising from intrachain interactions. We present a statistical mechanical model that explicitly includes all misfolded microstates with nonnative Watson-Crick (WC) and non-WC contacts. Rates of interconversion between different microstates are described in terms of two adjustable parameters: the strength of the non-WC interactions (ΔG(nWC)) and the rate at which a basepair is formed adjacent to an existing basepair (k(bp)(+)). The model accurately reproduces the temperature and loop-length dependence of the measured relaxation rates in temperature-jump studies of a 7-bp stem, single-stranded DNA hairpin with 4-20-nt-long poly(dT) loops, with ΔG(nWC) ≈ -2.4 kcal/mol and k(bp)(+) ≥ (1 ns)(-1), in 100 mM NaCl. Thus, our model provides a microscopic interpretation of the slow hairpin folding times as well as an estimate of the strength of intrachain interactions.

On modeling biomolecular-surface nonbonded interactions: application to nucleobase adsorption on single-wall carbon nanotube surfaces.

In this work we explored the selectivity of single nucleobases towards adsorption on chiral single-wall carbon nanotubes (SWCNTs) by density functional theory calculations. Specifically, the adsorption of molecular models of guanine (G), adenine (A), thymine (T), and cytosine (C), as well as of AT and GC Watson-Crick (WC) base pairs on chiral SWCNT C(6, 5), C(9, 1) and C(8, 3) model structures, was analyzed in detail. The importance of correcting the exchange-correlation functional for London dispersion was clearly demonstrated, yet limitations in modeling such interactions by considering the SWCNT as a molecular model may mask subtle effects in a molecular-macroscopic material system. The trend in the calculated adsorption energies of the nucleobases on same diameter C(6, 5) and C(9, 1) SWCNT surfaces, i.e., G > A > T > C, was consistent with related computations and experimental work on graphitic surfaces, however contradicting experimental data on the adsorption of single-strand short homo-oligonucleotides on SWCNTs that demonstrated a trend of G > C > A > T (Albertorio et al 2009 Nanotechnology 20 395101). A possible role of electrostatic interactions in this case was partially captured by applying the effective fragment potential method, emphasizing that the interplay of the various contributions in modeling nonbonded interactions is complicated by theoretical limitations. Finally, because the calculated adsorption energies for Watson-Crick base pairs have shown little effect upon adsorption of the base pair farther from the surface, the results on SWCNT sorting by salmon genomic DNA could be indicative of partial unfolding of the double helix upon adsorption on the SWCNT surface.

Lipoplex nanostructures reveal a general self-organization of nucleic acids.

BACKGROUND: Lipid and plasmid DNA complexes (Lx) were designed for gene transfer and were studied comprehensibly to elucidate their formation and ultrastructure. METHODS: We compared supramolecular self-assembly into stable Lx containing nucleic acids of various types and lengths using Cryo-Electron Microscopy, Small Angle X-ray Scattering and dynamic light scattering. RESULTS: Analysis of these complexes showed that they reproducibly formed monodisperse and spherical multilamellar particles. The same concentric and lamellar structure with different packing regimes was produced by circular double-stranded DNA, linear double-stranded DNA, single-stranded DNA, oligodeoxynucleotides or RNA. Strikingly, thousands of oligonucleotide molecules seem to align with one another and to behave as longer nucleic acid molecules in forming structurally similar particles. Neither excess cationic lipids nor excess DNA of different forms changed significantly the mean diameter and the size distribution of Lx particles. This suggests a role for Lx formation of steric size in addition to the conventional thermodynamic mechanism. The Lx ultrastructure is highly ordered and crystalline and is in a lamellar and/or hexagonal phase. Increasing NA size led to an increased proportion of Lx in a hexagonal structure phase as in the case of T4 phage virus DNA. These observations were made using two Lx made from different lipids exhibiting negative and positive charged surface. We also demonstrated structural similarities between the supramolecular auto-organization of Lx and that found in some viruses. In particular, both synthetic and viral particles have an ultrastructure that exhibits a phase transition between lamellar and hexagonal phases. GENERAL SIGNIFICANCE: Taken together, our data point towards the possible existence of a ubiquitous organization of genetic materials, at least with cationic lipids, that has implications for both therapy and the origins of life.

Solidifying framework nucleic acids with silica.

Soft matter can serve as a template to guide the growth of inorganic components with well-controlled structural features. However, the limited design space of conventional organic and biomolecular templates restricts the complexity and accuracy of templated growth. In past decades, the blossoming of structural DNA nanotechnology has provided us with a large reservoir of delicate-framework nucleic acids with design precision down to a single base. Here, we describe a DNA origami silicification (DOS) approach for generating complex silica composite nanomaterials. By utilizing modified silica sol-gel chemistry, pre-hydrolyzed silica precursor clusters can be uniformly coated onto the surface of DNA frameworks; thus, user-defined DNA-silica hybrid materials with ~3-nm precision can be achieved. More importantly, this method is applicable to various 1D, 2D and 3D DNA frameworks that range from 10 to >1,000 nm. Compared to pure DNA scaffolds, a tenfold increase in the Young's modulus (E modulus) of these composites was observed, owing to their soft inner core and solid silica shell. We further demonstrate the use of solidified DNA frameworks to create 3D metal plasmonic devices. This protocol provides a platform for synthesizing inorganic materials with unprecedented complexity and tailored structural properties. The whole protocol takes ~10 d to complete.

SwS: a solvation web service for nucleic acids.

UNLABELLED: SwS, based on a statistical analysis of crystallographic structures deposited in the NDB, is designed to provide an exhaustive overview of the solvation of nucleic acid structural elements through the generation of 3D solvent density maps. A first version (v1.0) of this web service focuses on the interaction of DNA, RNA and hybrid base pairs linked by two or three hydrogen bonds with water, cations and/or anions. Data provided by SwS are updated on a weekly basis and can be used by: (i) those involved in molecular dynamics simulation studies for validation purposes; (ii) crystallographers for help in the interpretation of solvent density maps; and all those involved in (iii) drug design and, more generally, in (iv) nucleic acid structural studies. SwS provides also statistical data related to the frequency of occurrence of different types of base pairs in crystallographic structures and the conformation of the involved nucleotides. This web service has been designed to allow a maximum of flexibility in terms of queries and has also been developed with didactic considerations in mind. AVAILABILITY: http://www-ibmc.u-strasbg.fr/arn/sws.html

Preparing Frozen-Hydrated Protein-Nucleic Acid Assemblies for High-Resolution Cryo-EM Imaging.

High-resolution image acquisition and structure determination by cryo-electron microscopy is becoming increasingly streamlined. Preparing electron-microscopy grids of suitable quality remains, however, a critical bottleneck. Strategies to achieve particle monodispersity, optimal sample concentration and suitable ice thickness can vary from specimen to specimen. In this book chapter we describe our protocols for negative-stain grid and cryo-grid preparation, which we apply to studying protein-nucleic acid complexes.

Direct visualization of a protein nuclear architecture.

Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus.

Imaging and structural studies of DNA-protein complexes and membrane ion channels.

In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABAA channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 Å, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

Electron Cryomicroscopy of Viruses at Near-Atomic Resolutions.

Recently, dozens of virus structures have been solved to resolutions between 2.5 and 5.0 Å by means of electron cryomicroscopy. With these structures we are now firmly within the "atomic age" of electron cryomicroscopy, as these studies can reveal atomic details of protein and nucleic acid topology and interactions between specific residues. This improvement in resolution has been the result of direct electron detectors and image processing advances. Although enforcing symmetry facilitates reaching near-atomic resolution with fewer particle images, it unfortunately obscures some biologically interesting components of a virus. New approaches on relaxing symmetry and exploring structure dynamics and heterogeneity of viral assemblies have revealed important insights into genome packaging, virion assembly, cell entry, and other stages of the viral life cycle. In the future, novel methods will be required to reveal yet-unknown structural conformations of viruses, relevant to their biological activities. Ultimately, these results hold the promise of answering many unresolved questions linking structural diversity of viruses to their biological functions.

Many Ways to Derivatize Macromolecules and Their Crystals for Phasing.

Due to the availability of many macromolecular models in the Protein Data Bank, the majority of crystal structures are currently solved by molecular replacement. However, truly novel structures can only be solved by one of the versions of the special-atom method. The special atoms such as sulfur, phosphorus or metals could be naturally present in the macromolecules, or could be intentionally introduced in a derivatization process. The isomorphous and/or anomalous scattering of X-rays by these special atoms is then utilized for phasing. There are many ways to obtain potentially useful derivatives, ranging from the introduction of special atoms to proteins or nucleic acids by genetic engineering or by chemical synthesis, to soaking native crystals in solutions of appropriate compounds with heavy and/or anomalously scattering atoms. No approach guarantees the ultimate success and derivatization remains largely a trial-and-error process. In practice, however, there is a very good chance that one of a wide variety of the available procedures will lead to successful structure solution.

Study on the interaction between nucleic acid and Eu3+-oxolinic acid and the determination of nucleic acid using the resonance light scattering technique.

At pH 9.75, the resonance light scattering (RLS) intensity of OA-Eu3+ system is greatly enhanced by nucleic acid. Based on this phenomenon, a new quantitative method for nucleic acid in aqueous solution has been developed. Under the optimum condition, the enhanced RLS is proportional to the concentration of nucleic acid in the range of 1.0x10(-9) to 1.0x10(-6)g/ml for herring sperm DNA, 8.0x10(-10) to 1.0x10(-6) g/ml for calf thymus DNA and 1.0x10(-9) to 1.0x10(-6) g/ml for yeast RNA, and their detection limits are 0.020, 0.011 and 0.010 ng/ml, respectively. Synthetic samples and actual samples were satisfactorily determined. In addition, the interaction mechanism between nucleic acid and OA-Eu3+ is also investigated.