Showing papers in "Quarterly Reviews of Biophysics in 2000"

Single-particle electron cryo-microscopy: towards atomic resolution.

Abstract: 4. Single particles and angular reconstitution 323 4.1 Preliminary filtering and centring of data 323 4.2 Alignments using correlation functions 324 4.3 Choice of first reference images 324 4.4 Multi-reference alignment of data 325 4.5 MSA eigenvector/eigenvalue data compression 328 4.6 MSA classification 330 4.7 Euler angle determination (‘ angular reconstitution ’) 332 4.8 Sinograms and sinogram correlation functions 332 4.9 Exploiting symmetry 335 4.10 Three-dimensional reconstruction 337 4.11 Euler angles using anchor sets 339 4.12 Iterative refinements 339

NMR structures of biomolecules using field oriented media and residual dipolar couplings.

Abstract: 2. Theoretical treatment of dipolar interactions 376 2.1 Anisotropic interactions as probes of macromolecular structure and dynamics 376 2.1.1 The dipolar interaction 376 2.1.2 Averaging in the solution state 377 2.2 Ordering of a rigid body 377 2.2.1 The Saupe order tensor 378 2.2.2 Orientational probability distribution function 380 2.2.3 The generalized degree of order 380 2.3 Molecular structure and internal dynamics 381

Structures of helical junctions in nucleic acids.

Abstract: Our knowledge of the architectural principles of nucleic acid junctions has seen significant recent advances. The conformation of DNA junctions is now well understood, and this provides a new basis for the analysis of important structural elements in RNA. The most significant new data have come from X-ray crystallography of four-way DNA junctions; incidentally showing the great importance of serendipity in science, since none of the three groups had deliberately set out to crystallise a junction. Fortunately the results confirm, and of course extend, the earlier conformational studies of DNA junctions in almost every detail. This is important, because it means that these methods can be applied with greater confidence to new systems, especially in RNA. Methods like FRET, chemical probing and even the humble polyacrylamide gel can be rapid and very powerful, allowing the examination of a large number of sequence variants relatively quickly. Molecular modelling in conjunction with experiments is also a very important component of the general approach. Ultimately crystallography provides the gold standard for structural analysis, but the other, simple approaches have considerable value along the way. At the beginning of this review I suggested two simple folding principles for branched nucleic acids, and it is instructive to review these in the light of recent data. In brief, these were the tendency for pairwise coaxial stacking of helical arms, and the importance of metal ion interactions in the induction of folding. We see that both are important in a wide range of systems, both in DNA and RNA. The premier example is the four-way DNA junction, which undergoes metal ion-induced folding into the stacked X-structure that is based on coaxial stacking of arms. As in many systems, there are two alternative ways to achieve this depending on the choice of stacking partners. Recent data reveal that both forms often exist in a dynamic equilibrium, and that the relative stability of the two conformers depends upon base sequence extending a significant distance from the junction. The three-way junction has provided a good test of the folding principles. Perfect three-way (3H) DNA junctions seem to defy these principles in that they appear reluctant to undergo coaxial stacking of arms, and exhibit little change in conformation with addition of metal ions. Modelling suggests that such a junction is stereochemically constrained in an extended conformation. However, upon inclusion of a few additional base pairs at the centre (to create a 3HS2 junction for example) the additional stereochemical flexibility allows two arms to undergo coaxial stacking. Such a junction exhibits all the properties consistent with the general folding principles, with ion-induced folding into a form based on pairwise coaxial stacking of arms in one of two different conformers. The three-way junction is therefore very much the exception that proves the rule. It is instructive to compare the folding of corresponding species in DNA and RNA, where we find both similarities and differences. The RNA four-way junction can adopt a structure that is globally similar to the stacked X-structure (Duckett et al. 1995a), and the crystal structure of the DNAzyme shows that the stacked X-conformation can include one helical pair in the A-conformation (Nowakowski et al. 1999). However, modelling suggests that the juxtaposition of strands and grooves will be less satisfactory in RNA, and the higher magnesium ion concentration required to fold the RNA junction indicates a lower stability of the antiparallel form. Perhaps the biggest difference between the properties of the DNA and RNA four-way junctions is the lack of an unstacked structure at low salt concentrations for the RNA species. This clearly reflects a major difference in the electrostatic interactions in the RNA junction. In general the folding of branched DNA provides some good indications on the likely folding of the corresponding RNA species, but caution is required in making the extrapolation because the two polymers are significantly different. A number of studies point to the flexibility and malleability of branched nucleic acids, and this turns out to have particular significance in their interactions with proteins. Proteins such as the DNA junction-resolving enzymes exhibit considerable selectivity for the structure of their substrates, which is still not understood at a molecular level. Despite this, it appears to be universally true that these proteins distort the global, and in some cases at least the local, structure of the junctions. The somewhat perplexing result is that the proteins appear to distort the very property that they recognise. In general it seems that four-way DNA junctions are opened to one extent or another by interaction with proteins. (ABSTRACT TRUNCATED)

RNA secondary structure: physical and computational aspects.

Abstract: 1. Background to RNA structure 2001.1 Types of RNA 2001.1.1 Transfer RNA (tRNA) 2001.1.2 Messenger RNA (mRNA) 2011.1.3 Ribosomal RNA (rRNA) 2011.1.4 Other ribonucleoprotein particles 2021.1.5 Viruses and viroids 2021.1.6 Ribozymes 2021.2 Elements of RNA secondary structure 2031.3 Secondary structure versus tertiary structure 2052. Theoretical and computational methods for RNA secondary structure determination 2082.1 Dynamic programming algorithms 2082.2 Kinetic folding algorithms 2102.3 Genetic algorithms 2122.4 Comparative methods 2133. RNA thermodynamics and folding mechanisms 2163.1 The reliability of minimum free energy structure prediction 2163.2 The relevance of RNA folding kinetics 2183.3 Examples of RNA folding kinetics simulations 2213.4 RNA as a disordered system 2274. Aspects of RNA evolution 2334.1 The relevance of RNA for studies of molecular evolution 2334.1.1 Molecular phylogenetics 2344.1.2 tRNAs and the genetic code 2344.1.3 Viruses and quasispecies 2354.1.4 Fitness landscapes 2354.2 The interaction between thermodynamics and sequence evolution 2364.3 Theory of compensatory substitutions in RNA helices 2384.4 Rates of compensatory substitutions obtained from sequence analysis 2405. Conclusions 2466. Acknowledgements 2467. References 246This article takes an inter-disciplinary approach to the study of RNA secondary structure, linking together aspects of structural biology, thermodynamics and statistical physics, bioinformatics, and molecular evolution. Since the intended audience for this review is diverse, this section gives a brief elementary level discussion of the chemistry and structure of RNA, and a rapid overview of the many types of RNA molecule known. It is intended primarily for those not already familiar with molecular biology and biochemistry.Ribonucleic acid consists of a linear polymer with a backbone of ribose sugar rings linked by phosphate groups. Each sugar has one of the four ‘bases’ adenine, cytosine, guanine and uracil (A, C, G, and U) linked to it as a side group. The structure and function of an RNA molecule is specific to the sequence of bases. The phosphate groups link the 5′ carbon of one ribose to the 3′ carbon of the next. This imposes a directionality on the backbone. The two ends are referred to as 5′ and 3′ ends, since one end has an unlinked 5′ carbon and one has an unlinked 3′ carbon. The chemical differences between RNA and DNA (deoxyribonucleic acid) are fairly small: one of the OH groups in ribose is replaced by an H in deoxyribose, and DNA contains thymine (T) bases instead of U. However, RNA structure is very different from DNA structure. In the familiar double helical structure of DNA the two strands are perfectly complementary in sequence. RNA usually occurs as single strands, and base pairs are formed intra-molecularly, leading to a complex arrangement of short helices which is the basis of the secondary structure. Some RNA molecules have well-defined tertiary structures. In this sense, RNA structures are more akin to globular protein structures than to DNA.The role of proteins as biochemical catalysts and the role of DNA in storage of genetic information have long been recognised. RNA has sometimes been considered as merely an intermediary between DNA and proteins. However, an increasing number of functions of RNA are now becoming apparent, and RNA is coming to be seen as an important and versatile molecule in its own right.

NMR spectroscopy: a multifaceted approach to macromolecular structure.

Abstract: 1. Introduction 292. Landmarks in NMR of macromolecules 322.1 Protein structures and methods development 322.1.1 Sequential assignment method 322.1.2 Triple-resonance experiments 342.1.3 Structures of large proteins 362.2 Protein–nucleic acid complexes 372.3 RNA structures 382.4 Membrane-bound systems 393. NMR spectroscopy today 403.1 State-of-the-art structure determination 413.2 New methods 443.2.1 Residual dipolar couplings 443.2.2 Direct detection of hydrogen bonds 443.2.3 Spin labeling 453.2.4 Segmental labeling 463.3 Protein complexes 473.4 Mobility studies 503.5 Determination of time-dependent structures 523.6 Drug discovery 534. The future of NMR 544.1 The ease of structure determination 544.2 The ease of making recombinant protein 554.3 Post-translationally modified proteins 554.4 Approaches to large and/or membrane-bound proteins 564.5 NMR in structural genomics 564.6 Synergy of NMR and crystallography in protein structure determination 565. Conclusion 576. Acknowledgements 577. References 57Since the publication of the first complete solution structure of a protein in 1985 (Williamson et al. 1985), tremendous technological advances have brought nuclear magnetic resonance spectroscopy to the forefront of structural biology. Innovations in magnet design, electronics, pulse sequences, data analysis, and computational methods have combined to make NMR an extremely powerful technique for studying biological macromolecules at atomic resolution (Clore & Gronenborn, 1998). Most recently, new labeling and pulse techniques have been developed that push the fundamental line-width limit for resolution in NMR spectroscopy, making it possible to obtain high-field spectra with better resolution than ever before (Dotsch & Wagner, 1998). These methods are facilitating the study of systems of ever-increasing complexity and molecular weight.

Impact of Transverse Relaxation Optimized Spectroscopy (TROSY) on NMR as a technique in structural biology

Abstract: 1. Transverse relaxation and the molecular size limit in liquid state NMR 1612. TROSY: how does it work? 1632.1 Transverse relaxation in coupled spin systems 1632.2 The TROSY effect, relaxation due to remote protons and 2H isotope labeling 1653. Direct heteronuclear chemical shift correlations 1683.1 Single-Quantum [15N,1H]-TROSY 1683.2 Zero-Quantum [15N,1H]-TROSY 1713.3 Single-Quantum TROSY with aromatic 13C–1H moieties 1764. Resonance assignment and NOE spectroscopy of large biomolecules 1804.1 TROSY-based triple resonance experiments for 13C, 15N and 1HN backbone resonance assignment in uniformly 2H, 13C, 15N labeled proteins 1804.2 TROSY-type NOE spectroscopy 1865. Scalar coupling across hydrogen bonds observed by TROSY 1876. The use of TROSY for measurements of residual dipolar coupling constants 1907. Conclusions 1918. Acknowledgements 1919. References 191The application of nuclear magnetic resonance (NMR) spectroscopy for structure determination of proteins and nucleic acids (Wuthrich, 1986) with molecular mass exceeding 30 kDa is largely constrained by two factors, fast transverse relaxation of spins of interest and complexity of NMR spectra, both of which increase with increasing molecular size (Wagner, 1993b; Clore & Gronenborn, 1997, 1998b; Kay & Gardner, 1997). The good news is that neither of these factors represent a fundamental limit for the application of NMR techniques to protein structure determination in solution (Clore & Gronenborn, 1998a; Wuthrich, 1998; Wider & Wuthrich, 1999). In fact, in the past few years the size limitations imposed by these factors have been pushed up to 50–70 kDa by the use of 13C, 15N and 2H isotope labeling combined with selective reprotonation of individual chemical groups in conjunction with the use of triple-resonance experiments (Bax, 1994; Gardner et al. 1997; Gardner & Kay, 1998) and heteronuclear-resolved NMR (Fesik & Zuiderweg, 1988; Marion et al. 1989a; Otting & Wuthrich, 1990). Among the largest biomolecules whose 3D structure was solved by NMR are the 44 kDa trimeric ectodomain of simian immunodeficiency virus (SIV) gp41 (Caffrey et al. 1998) and 40–60 kDa particles of the elongation initiation factor 4E solubilized in CHAPS micelles (Matsuo et al. 1997; McGuire et al. 1998).

Thermodynamics of nucleic acids and their interactions with ligands.

Abstract: 1. Introduction 2551.1 General thermodynamics 2562. Nucleic acid thermodynamics 2602.1 DNA duplexes 2612.2 RNA duplexes 2632.3 Hybrid DNA–RNA duplexes 2642.4 Hydration 2672.5 Conformational flexibility 2692.6 Thermodynamics 2723. Nucleic acid–ligand interactions 2773.1 Minor groove binders 2783.2 DNA intercalators 2843.3 Triple-helical systems 2883.3.1 Structures 2883.3.2 Hydration 2913.3.3 Thermodynamics 2914. Conclusions 2955. Acknowledgements 2986. References 298In recent years the availability of large quantities of pure synthetic DNA and RNA has revolutionised the study of nucleic acids, such that it is now possible to study their conformations, dynamics and large-scale properties, and their interactions with small ligands, proteins and other nucleic acids in unprecedented detail. This has led to the (re)discovery of higher order structures such as triple helices and quartets, and also the catalytic activity of RNA contingent on three-dimensional folding, and the extraordinary specificity possible with DNA and RNA aptamers.Nucleic acids are quite different from proteins, even though they are both linear polymers formed from a small number of monomeric units. The major difference reflects the nature of the linkage between the monomers. The 5′–3′ phosphodiester linkage in nucleic acids carries a permanent negative charge, and affords a relatively large number of degrees of freedom, whereas the essentially rigid planar peptide linkage in proteins is neutral and provides only two degrees of torsional freedom per backbone residue. These two properties conspire to make nucleic acids relatively flexible and less likely to form extensive folded structures. Even when true 3D folded structures are formed from nucleic acids, the topology remains simple, with the anionic phosphates forming the surface of the molecule. Nevertheless, nucleic acids do occur in a variety of structures that includes single strands and high-order duplex, triplex or tetraplex (‘quadruplex’) forms. The principles of biological recognition and the related problem of understanding the forces that stabilise such folded structures are in some respects more straightforward than for proteins, making them attractive model systems for understanding general biophysical problems. This view is aided by the relatively facile chemical synthesis of pure nucleic acids of any desired size and defined sequence, and the ease of incorporation of a wide spectrum of chemically modified bases, sugars and backbone linkers. Such modifications are considerably more difficult to achieve with oligopeptides or proteins.

CCD detectors in high-resolution biological electron microscopy.

Abstract: 1. Introduction 11.1 The ‘band gap’ in silicon 22. Principles of CCD detector operation 32.1 Direct detection 32.2 Electron energy conversion into light 42.3 Optical coupling: lens or fibre optics? 62.4 Readout speed and comparison with film 83. Practical considerations for electron microscopic applications 93.1 Sources of noise 93.1.1 Dark current noise 93.1.2 Readout noise 93.1.3 Spurious events due to X-rays or cosmic rays 103.2 Efficiency of detection 113.3 Spatial resolution and modulation transfer function 123.4 Interface to electron microscope 143.5 Electron diffraction applications 154. Prospects for high-resolution imaging with CCD detectors 185. Alternative technologies for electronic detection 235.1 Image plates 235.2 Hybrid pixel detectors 246. References 26During the past decade charge-coupled device (CCD) detectors have increasingly become the preferred choice of medium for recording data in the electron microscope. The CCD detector itself can be likened to a new type of television camera with superior properties, which makes it an ideal detector for recording very low exposure images. The success of CCD detectors for electron microscopy, however, also relies on a number of other factors, which include its fast response, low noise electronics, the ease of interfacing them to the electron microscope, and the improvements in computing that have made possible the storage and processing of large images.CCD detectors have already begun to be routinely used in a number of important biological applications such as tomography of cellular organelles (reviewed by Baumeister, 1999), where the resolution requirements are relatively modest. However, in most high- resolution microscopic applications, especially where the goal of the microscopy is to obtain structural information at near-atomic resolution, photographic film has continued to remain the medium of choice. With the increasing interest and demand for high-throughput structure determination of important macromolecular assemblies, it is clearly important to have tools for electronic data collection that bypass the slow and tedious process of processing images recorded on photographic film.In this review, we present an analysis of the potential of CCD-based detectors to fully replace photographic film for high-resolution electron crystallographic applications.

How does light regulate chloroplast enzymes? Structure-function studies of the ferredoxin/thioredoxin system.

Abstract: 1. Introduction 682. Ferredoxin reduction by photosystem I 723. Ferredoxins 734. Ferredoxin[ratio ]thioredoxin reductase 734.1 Spectroscopic investigations of FTR 764.2 The three-dimensional structure of FTR from the cyanobacterium Synechocystis sp. PCC6803 774.2.1 The variable subunit 774.2.2 The catalytic subunit 814.2.3 The iron–sulfur center and active site disulfide bridge 824.2.4 The dimer 844.3 Thioredoxin f and m 854.4 Ferredoxin and thioredoxin interactions 864.5 Mechanism of action 884.6 Comparison with other chloroplast FTRs 925. Target enzymes 955.1 NADP-dependent malate dehydrogenase 955.1.1 Regulatory role of the N-terminal extension 975.1.2 Regulatory role of the C-terminal extension 995.1.3 Thioredoxin interactions 1015.2 Fructose-1,6-bisphosphatase 1015.3 Redox regulation of chloroplast target enzymes 1036. Conclusion 1037. Acknowledgements 1048. References 104A pre-requisite for life on earth is the conversion of solar energy into chemical energy by photosynthetic organisms. Plants and photosynthetic oxygenic microorganisms trap the energy from sunlight with their photosynthetic machinery and use it to produce reducing equivalents, NADPH, and ATP, both necessary for the reduction of carbon dioxide to carbohydrates, which are then further used in the cellular metabolism as building blocks and energy source. Thus, plants can satisfy their energy needs directly via the light reactions of photosynthesis during light periods. The situation is quite different in the dark, when these organisms must use normal catabolic processes like non-photosynthetic organisms to obtain the necessary energy by degrading carbohydrates, like starch, accumulated in the chloroplasts during daylight. The chloroplast stroma contains both assimilatory enzymes of the Calvin cycle and dissimilatory enzymes of the pentose phosphate cycle and glycolysis. This necessitates a strict, light-sensitive control that switches between assimilatory and dissimilatory pathways to avoid futile cycling (Buchanan, 1980, 1991; Buchanan et al. 1994; Jacquot et al. 1997; Schurmann & Buchanan, 2000).