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Role of uncoupled and non-coupled oxidations in maintenance of safely low levels of oxygen and its one-electron reductants
- Vladimir P. Skulachev
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- 17 March 2009, pp. 169-202
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To proceed at a high rate, phosphorylating respiration requires ADP to be available. In the resting state, when the energy consumption is low, the ADP concentration decreases so that phosphorylating respiration ceases. This may result in an increase in the intracellular concentrations of O2 as well as of one-electron O2 reductants such as These two events should dramatically enhance non-enzymatic formation of reactive oxygen species, i.e. of , and OHׁ, and, hence, the probability of oxidative damage to cellular components. In this paper, a concept is put forward proposing that non-phosphorylating (uncoupled or non-coupled) respiration takes part in maintenance of low levels of both O2 and the O2 reductants when phosphorylating respiration fails to do this job due to lack of ADP.
In particular, it is proposed that some increase in the H+ leak of mitochondrial membrane in State 4 lowers , stimulates O2 consumption and decreases the level of which otherwise accumulates and serves as one-electron O2 reductant. In this connection, the role of natural uncouplers (thyroid hormones), recouplers (male sex hormones and progesterone), non-specific pore in the inner mitochondrial membrane, and apoptosis, as well as of non-coupled electron transfer chains in plants and bacteria will be considered.
- Cited by 552
Control of muscle contraction
- Setsuro Ebashi, Makoto Endo, Iwao Ohtsuki
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- 17 March 2009, pp. 351-384
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As is well known, the memorable discovery of Galvani (1791) was followed by the development of two new fields of science, electrochemistry and electrophysiology. During the course of this development, the most remarkable feature of the original finding, i.e. ‘contraction of muscle induced by a piece of metal’, gradually came to be ignored. As a consequence, the simple question as to how electrical stimulation might induce muscle contraction was left unanswered until the middle of this century, when several physiologists became aware of the crucial nature of the problem and tried to attack it from various directions. This resulted in a marked progress of physiological and morphological studies which were intentionally or unintentionally concerned with the mechanism of the link between excitation, that is the electrical phenomenon at the surface membrane, and the contractile process.
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Intermolecular forces in biology
- Deborah Leckband, Jacob Israelachvili
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- 10 December 2001, pp. 105-267
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0. Abbreviations 106
1. Introduction: overview of forces in biology 108
1.1 Subtleties of biological forces and interactions 108
1.2 Specific and non-specific forces and interactions 113
1.3 van der Waals (VDW) forces 114
1.4 Electrostatic and ’double-layer‘ forces (DLVO theory) 122
1.4.1 Electrostatic and double-layer interactions at very small separation 126
1.5 Hydration and hydrophobic forces (structural forces in water) 131
1.6 Steric, bridging and depletion forces (polymer-mediated and tethering forces) 137
1.7 Thermal fluctuation forces: entropic protrusion and undulation forces 142
1.8 Comparison of the magnitudes of the major non-specific forces 146
1.9 Bio-recognition 146
1.10 Equilibrium and non-equilibrium forces and interactions 150
1.10.1 Multiple bonds in parallel 153
1.10.2 Multiple bonds in series 155
2. Experimental techniques for measuring forces between biological molecules and surfaces 156
2.1 Different force-measuring techniques 156
2.2 Measuring forces between surfaces 161
2.3 Measuring force–distance functions, F(D) 161
2.4 Relating the forces between different geometries: the ‘Derjaguin Approximation’ 162
2.5 Adhesion forces and energies 164
2.5.1 An example of the application of adhesion mechanics of biological adhesion 166
2.6 Measuring forces between macroscopic surfaces: the surface forces apparatus (SFA) 167
2.7 The atomic force microscope (AFM) and microfiber cantilever (MC) techniques 173
2.8 Micropipette aspiration (MPA) and the bioforce probe (BFP) 177
2.9 Osmotic stress (OS) and osmotic pressure (OP) techniques 179
2.10 Optical trapping and the optical tweezers (OT) 181
2.11 Other optical microscopy techniques: TIRM and RICM 184
2.12 Shear flow detachment (SFD) measurements 187
2.13 Cell locomotion on elastically deformable substrates 189
3. Measurements of equilibrium (time-independent) interactions 191
3.1 Long-range VDW and electrostatic forces (the two DVLO forces) between biosurfaces 191
3.2 Repulsive short-range steric–hydration forces 197
3.3 Adhesion forces due to VDW forces and electrostatic complementarity 200
3.4 Attractive forces between surfaces due to hydrophobic interactions: membrane adhesion and fusion 209
3.4.1 Hydrophobic interactions at the nano- and sub-molecular levels 211
3.4.2 Hydrophobic interactions and membrane fusion 212
3.5 Attractive depletion forces 213
3.6 Solvation (hydration) forces in water: forces associated with water structure 215
3.7 Forces between ‘soft-supported’ membranes and proteins 218
3.8 Equilibrium energies between biological surfaces 219
4. Non-equilibrium and time-dependent interactions: sequential events that evolve in space and time 221
4.1 Equilibrium and non-equilibrium time-dependent interactions 221
4.2 Adhesion energy hysteresis 223
4.3 Dynamic forces between biomolecules and biomolecular aggregates 226
4.3.1 Strengths of isolated, noncovalent bonds 227
4.3.2 The strengths of isolated bonds depend on the activation energy for unbinding 229
4.4 Simulations of forced chemical transformations 232
4.5 Forced extensions of biological macromolecules 235
4.6 Force-induced versus thermally induced chemical transformations 239
4.7 The rupture of bonds in series and in parallel 242
4.7.1 Bonds in series 242
4.7.2 Bonds in parallel 244
4.8 Dynamic interactions between membrane surfaces 246
4.8.1 Lateral mobility on membrane surfaces 246
4.8.2 Intersurface forces depend on the rate of approach and separation 249
4.9 Concluding remarks 253
5. Acknowledgements 255
6. References 255
While the intermolecular forces between biological molecules are no different from those that arise between any other types of molecules, a ‘biological interaction’ is usually very different from a simple chemical reaction or physical change of a system. This is due in part to the higher complexity of biological macromolecules and systems that typically exhibit a hierarchy of self-assembling structures ranging in size from proteins to membranes and cells, to tissues and organs, and finally to whole organisms. Moreover, interactions do not occur in a linear, stepwise fashion, but involve competing interactions, branching pathways, feedback loops, and regulatory mechanisms.
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Electron tunneling through proteins
- Harry B. Gray, Jay R. Winkler
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- 26 January 2004, pp. 341-372
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1. History 342
2. Activation barriers 343
2.1 Redox potentials 344
2.2 Reorganization energy 344
3. Electronic coupling 345
4. Ru-modified proteins 348
4.1 Reorganization energy 349
4.1.1 Cyt c 349
4.1.2 Azurin 350
4.2 Tunneling timetables 352
5. Multistep tunneling 357
6. Protein–protein reactions 359
6.1 Hemoglobin (Hb) hybrids 359
6.2 Cyt c/cyt b5 complexes 360
6.3 Cyt c/cyt c peroxidase complexes 360
6.4 Zn–cyt c/Fe–cyt c crystals 361
7. Photosynthesis and respiration 362
7.1 Photosynthetic reaction centers (PRCs) 362
7.2 Cyt c oxidase (CcO) 364
8. Concluding remarks 365
9. Acknowledgments 366
10. References 366
Electron transfer processes are vital elements of energy transduction pathways in living cells. More than a half century of research has produced a remarkably detailed understanding of the factors that regulate these ‘currents of life’. We review investigations of Ru-modified proteins that have delineated the distance- and driving-force dependences of intra-protein electron-transfer rates. We also discuss electron transfer across protein–protein interfaces that has been probed both in solution and in structurally characterized crystals. It is now clear that electrons tunnel between sites in biological redox chains, and that protein structures tune thermodynamic properties and electronic coupling interactions to facilitate these reactions. Our work has produced an experimentally validated timetable for electron tunneling across specified distances in proteins. Many electron tunneling rates in cytochrome c oxidase and photosynthetic reaction centers agree well with timetable predictions, indicating that the natural reactions are highly optimized, both in terms of thermodynamics and electronic coupling. The rates of some reactions, however, significantly exceed timetable predictions; it is likely that multistep tunneling is responsible for these anomalously rapid charge transfer events.
- Cited by 504
Mechanisms of cooperativity and allosteric regulation in proteins
- M. F. Perutz
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- 17 March 2009, pp. 139-237
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AUosteric proteins control and coordinate chemical events in the living cell. When Monod conceived that idea he said that he had discovered the second secret of life. The first was the structure of DNA. The theory as published by Monod et al. (1963) was concerned chiefly with cooperativity and feedback inhibition of enzymes, such as the inhibition of threonine deaminase, the first enzyme in the pathway of the synthesis of isoleucine, by isoleucine, and its activation by valine. Two years later the theory was formalized by Monod et al. (1965).
- Cited by 501
Soft X-ray microscopes and their biological applications
- Janos Kirz, Chris Jacobsen, Malcolm Howells
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- 17 March 2009, pp. 33-130
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In this review we propose to address the question: for the life-science researcher, what does X-ray microscopy have to offer that is not otherwise easily available?
We will see that the answer depends on a combination of resolution, penetrating power, analytical sensitivity, compatibility with wet specimens, and the ease of image interpretation.
- Cited by 492
Hydrodynamic properties of complex, rigid, biological macromolecules: theory and applications
- José García de la Torre, Victor A. Bloomfield
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- 17 March 2009, pp. 81-139
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Among the Various methods for characterizing macromolecules in solution, hydrodynamic techniques play a major role. Since the advent of the ultracentrifuge and the development of viscometric apparatus, sedimentation coefficients and intrinsic viscosities have been extensively used to learn about the size and shape of synthetic and biological polymers. More recently, refined techniques such as quasielastic light scattering, transient electric birefringence and fluorescence anisotropy decay have made it possible to obtain in a simple and rapid way quantitative information of high precision on the translational and rotational brownian dynamics of dissolved macromolecules.
- Cited by 488
Membrane fusion proteins of enveloped animal viruses
- Judith White, Margaret Kielian, Ari Helenius
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- 17 March 2009, pp. 151-195
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In a living cell membrane-bound compartments are continuously either separated or united through fusion reactions, and literally thousands of such reactions take place every minute. The formation of membrane vesicles from pre-existing membranes, and their fusion with specific acceptor membranes, constitute a prerequisite for the transport of most impermeant molecules and macromolecules into the cell by endocytosis, out of the cell by exocytosis, and between the cellular organelles (Palade, 1975; Silverstein, 1978; Evered & Collins, 1982). Less frequent, but equally crucial, are fusion events in fertilization, cell division, polykaryon formation, enucleation, etc. (for reviews see Poste & Nicholson, 1978). Although a great deal is known about the properties and consequences of individual forms of membrane fusion in cellular systems, and about fusion in artificial lipid membranes, the molecular basis for the reactions remain largely unclear.
- Cited by 480
Long-range distance determinations in biomacromolecules by EPR spectroscopy
- Olav Schiemann, Thomas F. Prisner
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- 13 June 2007, pp. 1-53
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Electron paramagnetic resonance (EPR) spectroscopy provides a variety of tools to study structures and structural changes of large biomolecules or complexes thereof. In order to unravel secondary structure elements, domain arrangements or complex formation, continuous wave and pulsed EPR methods capable of measuring the magnetic dipole coupling between two unpaired electrons can be used to obtain long-range distance constraints on the nanometer scale. Such methods yield reliably and precisely distances of up to 80 Å, can be applied to biomolecules in aqueous buffer solutions or membranes, and are not size limited. They can be applied either at cryogenic or physiological temperatures and down to amounts of a few nanomoles. Spin centers may be metal ions, metal clusters, cofactor radicals, amino acid radicals, or spin labels. In this review, we discuss the advantages and limitations of the different EPR spectroscopic methods, briefly describe their theoretical background, and summarize important biological applications. The main focus of this article will be on pulsed EPR methods like pulsed electron–electron double resonance (PELDOR) and their applications to spin-labeled biosystems.
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Molecular structure of amyloid fibrils: insights from solid-state NMR
- Robert Tycko
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- Published online by Cambridge University Press:
- 13 June 2006, pp. 1-55
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1. Introduction 2
2. Sources of structural information in solid-state NMR data 5
2.1 General remarks 5
2.2 Chemical shifts, linewidths, and magic-angle spinning 6
2.3 Dipole–dipole couplings and dipolar recoupling 8
2.4 Tensor correlation techniques 12
2.5 Solid-state NMR of aligned samples 14
2.6 Indirect sources of structural information 15
2.7 Sample preparation for solid-state NMR 15
3. Levels of structure in amyloid fibrils 18
4. Molecular structure of β-amyloid fibrils 25
4.1 Self-propagating, molecular-level polymorphism in Aβ1–40 fibrils 25
4.2 Structural model for Aβ1-40 fibrils 28
4.3 Staggering of β-strands in Aβ1-40 fibrils 32
4.4 Structure of Aβ1-42 fibrils 34
4.5 Structure of fibrils formed by short β-amyloid fragments 34
4.6 Structures of non-fibrillar aggregates 35
5. Molecular structure of other amyloid fibrils 36
5.1 Ure2p10–39 and full-length Ure2p fibrils 36
5.2 TTR105–115 fibrils 38
5.3 HET-s fibrils 38
5.4 Amylin fibrils 39
5.5 PrP fibrils 39
5.6 ccβ fibrils 40
5.7 α-synuclein fibrils 40
5.8 Calcitonin fibrils 41
6. Data relevant to various proposals regarding amyloid structure 41
6.1 β-helical models for amyloid fibrils 41
6.2 Amyloid fibrils as water-filled nanotubes 42
6.3 Domain swapping in amyloid fibrils 42
6.4 The parallel superpleated β-structure model 43
6.5 α-sheet structures in amyloid fibrils 43
7. Conclusions 44
8. Acknowledgments 46
9. References 46
Solid-state nuclear magnetic resonance (NMR) measurements have made major contributions to our understanding of the molecular structures of amyloid fibrils, including fibrils formed by the β-amyloid peptide associated with Alzheimer's disease, by proteins associated with fungal prions, and by a variety of other polypeptides. Because solid-state NMR techniques can be used to determine interatomic distances (both intramolecular and intermolecular), place constraints on backbone and side-chain torsion angles, and identify tertiary and quaternary contacts, full molecular models for amyloid fibrils can be developed from solid-state NMR data, especially when supplemented by lower-resolution structural constraints from electron microscopy and other sources. In addition, solid-state NMR data can be used as experimental tests of various proposals and hypotheses regarding the mechanisms of amyloid formation, the nature of intermediate structures, and the common structural features within amyloid fibrils. This review introduces the basic experimental and conceptual principles behind solid-state NMR methods that are applicable to amyloid fibrils, reviews the information about amyloid structures that has been obtained to date with these methods, and discusses how solid-state NMR data provide insights into the molecular interactions that stabilize amyloid structures, the generic propensity of polypeptide chains to form amyloid fibrils, and a number of related issues that are of current interest in the amyloid field.
- Cited by 459
Single-particle electron cryo-microscopy: towards atomic resolution
- Marin van Heel, Brent Gowen, Rishi Matadeen, Elena V. Orlova, Robert Finn, Tillmann Pape, Dana Cohen, Holger Stark, Ralf Schmidt, Michael Schatz, Ardan Patwardhan
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- 01 March 2001, pp. 307-369
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1. Introduction 308
2. Electron microscopy 311
2.1 Specimen preparation 311
2.2 The electron microscope 311
2.3 Acceleration voltage, defocus, and the electron gun 312
2.4 Magnification and data collection 313
3. Digitisation and CTF correction 317
3.1 The patchwork densitometer 318
3.2 Particle selection 320
3.3 Position dependent CTF correction 321
3.4 Precision of CTF determination 321
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
5. Computational hardware/software aspects 341
5.1 The (IMAGIC) image processing workstation 342
5.2 Operating systems and GUIs 342
5.3 Computational logistics 344
5.4 Shared memory machines 344
5.5 Farming on loosely coupled computers 346
5.6 Implementation using MPI protocol 347
5.7 Software is what it's all about 347
6. Interpretation of results 348
6.1 Assessing resolution: the Fourier Shell Correlation 348
6.2 Influence of filtering 351
6.3 Rendering 351
6.4 Searching for known sub-structures 352
6.5 Interpretation 353
7. Examples 353
7.1 Icosahedral symmetry: TBSV at 5·9 Å resolution 354
7.2 The D6 symmetrical worm hemoglobin at 13 Å resolution 356
7.3 Functional states of the 70S E. coli ribosome 357
7.4 The 50S E. coli ribosomal subunit at 7·5 Å resolution 359
8. Perspectives 361
9. Acknowledgements 364
10. References 364
In the past few years, electron microscopy (EM) has established itself as an important – still upcoming – technique for studying the structures of large biological macromolecules. EM is a very direct method of structure determination that complements the well-established techniques of X-ray crystallography and NMR spectroscopy. Electron micrographs record images of the object and not just their diffraction patterns and thus the classical ‘phase’ problem of X-ray crystallography does not exist in EM. Modern microscopes may reach resolution levels better than ∼ 1·5 Å, which is more than sufficient to elucidate the polypeptide backbone in proteins directly. X-ray structures at such resolution levels are considered ‘excellent’. The fundamental problem in biological EM is not so much the instrumental resolution of the microscopes, but rather the radiation sensitivity of the biological material one wants to investigate. Information about the specimen is collected in the photographic emulsion with the arrival of individual electrons that have (elastically) interacted with the specimen. However, many electrons will damage the specimen by non-elastic interactions. By the time enough electrons have passed through the object to produce a single good signal-to-noise (SNR) image, the biological sample will have been reduced to ashes. In contrast, stable inorganic specimens in material science often show interpretable details down to the highest possible instrumental resolution.
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Nucleases: diversity of structure, function and mechanism
- Wei Yang
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- 21 September 2010, pp. 1-93
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Nucleases cleave the phosphodiester bonds of nucleic acids and may be endo or exo, DNase or RNase, topoisomerases, recombinases, ribozymes, or RNA splicing enzymes. In this review, I survey nuclease activities with known structures and catalytic machinery and classify them by reaction mechanism and metal-ion dependence and by their biological function ranging from DNA replication, recombination, repair, RNA maturation, processing, interference, to defense, nutrient regeneration or cell death. Several general principles emerge from this analysis. There is little correlation between catalytic mechanism and biological function. A single catalytic mechanism can be adapted in a variety of reactions and biological pathways. Conversely, a single biological process can often be accomplished by multiple tertiary and quaternary folds and by more than one catalytic mechanism. Two-metal-ion-dependent nucleases comprise the largest number of different tertiary folds and mediate the most diverse set of biological functions. Metal-ion-dependent cleavage is exclusively associated with exonucleases producing mononucleotides and endonucleases that cleave double- or single-stranded substrates in helical and base-stacked conformations. All metal-ion-independent RNases generate 2′,3′-cyclic phosphate products, and all metal-ion-independent DNases form phospho-protein intermediates. I also find several previously unnoted relationships between different nucleases and shared catalytic configurations.
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Small-angle scattering: a view on the properties, structures and structural changes of biological macromolecules in solution
- Michel H. J. Koch, Patrice Vachette, Dmitri I. Svergun
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- Published online by Cambridge University Press:
- 23 October 2003, pp. 147-227
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1. Introduction 148
2. Basics of X-ray and neutron scattering 149
2.1 Elastic scattering of electromagnetic radiation by a single electron 149
2.2 Scattering by assemblies of electrons 151
2.3 Anomalous scattering and long wavelengths 153
2.4 Neutron scattering 153
2.5 Transmission and attenuation 155
3. Small-angle scattering from solutions 156
3.1 Instrumentation 156
3.2 The experimental scattering pattern 157
3.3 Basic scattering functions 159
3.4 Global structural parameters 161
3.4.1 Monodisperse systems 161
3.4.2 Polydisperse systems and mixtures 163
3.5 Characteristic functions 164
4. Modelling 166
4.1 Spherical harmonics 166
4.2 Shannon sampling 169
4.3 Shape determination 170
4.3.1 Modelling with few parameters: molecular envelopes 171
4.3.2 Modelling with many parameters: bead models 173
4.4 Modelling domain structure and missing parts of high-resolution models 178
4.5 Computing scattering patterns from atomic models 184
4.6 Rigid-body refinement 187
5. Applications 190
5.1 Contrast variation studies of ribosomes 190
5.2 Structural changes and catalytic activity of the allosteric enzyme ATCase 191
6. Interactions between molecules in solution 203
6.1 Linearizing the problem for moderate interactions: the second virial coefficient 204
6.2 Determination of the structure factor 205
7. Time-resolved measurements 211
8. Conclusions 215
9. Acknowledgements 216
10. References 216
A self-contained presentation of the main concepts and methods for interpretation of X-ray and neutron-scattering patterns of biological macromolecules in solution, including a reminder of the basics of X-ray and neutron scattering and a brief overview of relevant aspects of modern instrumentation, is given. For monodisperse solutions the experimental data yield the scattering intensity of the macromolecules, which depends on the contrast between the solvent and the particles as well as on their shape and internal scattering density fluctuations, and the structure factor, which is related to the interactions between macromolecules. After a brief analysis of the information content of the scattering intensity, the two main approaches for modelling the shape and/or structure of macromolecules and the global minimization schemes used in the calculations are presented. The first approach is based, in its more advanced version, on the spherical harmonics approximation and relies on few parameters, whereas the second one uses bead models with thousands of parameters. Extensions of bead modelling can be used to model domain structure and missing parts in high-resolution structures. Methods for computing the scattering patterns from atomic models including the contribution of the hydration shell are discussed and examples are given, which also illustrate that significant differences sometimes exist between crystal and solution structures. These differences are in some cases explainable in terms of rigid-body motions of parts of the structures. Results of two extensive studies – on ribosomes and on the allosteric protein aspartate transcarbamoylase – illustrate the application of the various methods. The unique bridge between equilibrium structures and thermodynamic or kinetic aspects provided by scattering techniques is illustrated by modelling of intermolecular interactions, including crystallization, based on an analysis of the structure factor and recent time-resolved work on assembly and protein folding.
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Structural studies of protein–nucleic acid interaction: the sources of sequence-specific binding
- Thomas A. Steitz
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- 17 March 2009, pp. 205-280
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Structural studies of DNA-binding proteins and their complexes with DNA have proceeded at an accelerating pace in recent years due to important technical advances in molecular genetics, DNA synthesis, protein crystallography and nuclear magnetic resonance. The last major review on this subject by Pabo & Sauer (1984) summarized the structural and functional studies of the three sequence-specific DNA-binding proteins whose crystal structures were then known, the E. coli catabolite gene activator protein (CAP) (McKay & Steitz, 1981; McKay et al. 1982; Weber & Steitz, 1987), a cro repressor from phage λ (Anderson et al. 1981), and the DNA-binding proteolytic fragment of λcI repressor protein (Pabo & Lewis, 1982) Although crystallographic studies of the E. coli lac repressor protein were initiated as early as 1971 when it was the only regulatory protein available in sufficient quantities for structural studies (Steitz et al. 1974), little was established about the structural aspects of DNA-binding proteins until the structure of CAP was determined in 1980 followed shortly thereafter by the structure of λcro repressor and subsequently that of the λ repressor fragment. There are now determined at high resolution the crystal structures of seven prokaryotic gene regulatory proteins or fragments [CAP, λcro, λcI repressor fragment, 434 repressor fragment (Anderson et al. 1987), 434 cro repressor (Wolberger et al. 1988), E. coli trp repressor (Schevitz et al. 1985), E. coli met repressor (Rafferty et al. 1989)], EcoR I restriction endonuclease (McClarin et al. 1986), DNAse I (Suck & Ofner, 1986), the catalytic domain of γδ resolvase (Hatfull et al. 1989) and two sequence-independent double-stranded DNA-binding proteins [the Klenow fragment of E. coli DNA polymerase I (Ollis et al. 1985) and the E. coli Hu protein (Tanaka et al., 1984)].
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Physics and chemistry of spin labels
- Harden M. McConnell, Betty Gaffney McFarland
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- 17 March 2009, pp. 91-136
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Biological systems provide the physical chemist with an abundance of interesting, challenging and significant problems. One example is the problem of the molecular basis of co-operative or allosteric interactions between distant ligand or substrate binding sites in hemoglobin and in enzymes. This problem has been discussed recently in This Journal by Eigen (1968) and by Wyman (1968). Another particularly challenging problem is the molecular organization of biological membranes. Such problems tend to be particularly resistant to solution by the straight-forward application of most spectroscopic techniques, in large part because of the enormous chemical and spectroscopic complexity of biological macromolecules. This spectroscopic complexity has stimulated the use of various ‘probes’ that can be introduced into selected sites in complex systems to provide spectroscopic signals that are comparatively free from interference. The use of heavy metal atoms (‘isomorphous replacement’) in X-ray studies of protein crystals (Green, Ingram & Perutz, 1954), and fluorescent dyes in the study of proteins in solutions (Weber, 1953; Steiner & Edelhoch, 1962) are early examples. Spin labels represent a new member of the family of spectroscopic structural probes. A spin label is a synthetic paramagnetic organic free radical, usually having a molecular structure and/or chemical reactivity that results in its attachment or incorporation at some particular target site in a biological macromolecule, or assemblage of macromolecules (Ohnishi & McConnell, 1965; Stone et al. 1965). This type of probe is being used in our laboratory to study allosteric interactions in proteins, and molecular dynamics and organization in membranes.
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The effect of high pressure upon proteins and other biomolecules
- Gregorio Weber, Harry G. Drickamer
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- 17 March 2009, pp. 89-112
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We shall not attempt here to enumerate the results or review in a systematic way the significant literature dealing with the use of high pressure in studies of proteins and other molecules of biological interest. Two recent reviews on this subject, one by MOrild (1981) and another by Heremans (1982), and a further article by Jaenicke (1981) on enzymes under extreme environmental conditions contain expositions and references that would render redundatn such a task. Rather we concentrate here on the examination of othe conceptual framework employed in the interpretation of high pressure experiments and in the critical discussion of our knowledge of selected areas of present interest and likely future significance.
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Coupling of quanta, electrons, fields, ions and phosphorylation in the functional membrane of photosynthesis. Results by pulse spectroscopic methods
- H. T. Witt
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- 17 March 2009, pp. 365-477
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From the globular to the fibrous state: protein structure and structural conversion in amyloid formation
- MARGARET SUNDE, COLIN C. F. BLAKE
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- 01 February 1998, pp. 1-39
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The term ‘amyloid’ was used originally to describe certain deposits found post- mortem in organs and tissues, which gave a positive reaction when stained with iodine (Virchow, 1854). Only later was it realized that the material was in fact predominantly proteinaceous, although it is known to be associated with carbohydrates, particularly glucosoaminoglycans, when obtained from many ex vivo sources. With the increasing precision in the definition of amyloid, initially from its characteristic green birefringence when stained with the dye Congo Red (Missmahl & Hartwig, 1953), and later from its particular appearance under the electron microscope (Cohen & Calkins, 1959) and its X-ray diffraction pattern (Eanes & Glenner, 1968), it has become evident that it is a specific fibrillar protein state, which can also be formed by some proteins when denatured in vitro (Burke & Rougvie, 1972), and by synthetic oligopeptides (Bradbury et al. 1960) that may form amyloid spontaneously when placed in pure aqueous medium (Serpell, 1996). Although these latter may form useful experimental systems for the study of amyloid, its major interest at present is that it is associated with a number of prominent lethal diseases (Benson & Wallace, 1989; Pepys, 1994).
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The moving parts of voltage-gated ion channels
- GARY YELLEN
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- 01 August 1998, pp. 239-295
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Ion channels, like many other proteins, have moving parts that perform useful functions. The channel proteins contain an aqueous, ion-selective pore that crosses the plasma membrane, and they use a number of distinct ‘gating’ mechanisms to open and close this pore in response to biological stimuli such as the binding of a ligand or a change in the transmembrane voltage.
This review is written at a watershed in our understanding of ion channels.
1. INTRODUCTION 240
1.1 Basic structure of voltage-activated channels 241
1.2 What are the physical motions of the channel protein during gating? 243
1.3 Gating involves several distinct mechanisms of activation and inactivation 246
2. ACTIVATION GATING 246
2.1 Early evidence for an activation gate at the intracellular mouth 247
2.1.1 Open channel blockade 247
2.1.2 The ‘ foot-in-the-door’ effect 249
2.1.3 Trapping of blockers behind closed activation gates 249
2.2 Site-directed mutagenesis and the difficulty of inferring structural roles from functional effects 250
2.3 State-dependent cysteine modification as a reporter of position and motion 251
2.4 Localization of activation gating 254
2.4.1 The trapping cavity 254
2.4.2 The activation gate 255
2.4.3 Is there more than one site of activation gating? 258
3. INACTIVATION GATING 259
3.1 Ball-and-chain (N-type) inactivation 261
3.1.1 Nature of the ‘ball’ – a tethered blocking particle 262
3.1.2 The ball receptor 263
3.1.3 The chain 264
3.1.4 Variations on the N-type inactivation theme: multiple balls, foreign balls, anti-balls 265
3.2 C-type inactivation 266
3.2.1 C-type inactivation and the outer mouth of the K+channel 266
3.2.2 The selectivity filter participates in C-type inactivation 267
3.2.3 A consistent structural picture of C-type inactivation 269
3.3 By what mechanism do other voltage-gated channels inactivate? 272
4. THE VOLTAGE SENSOR 273
4.1 Quantitative principles of voltage-dependent gating 276
4.2 S4 (and its neighbours) as the principal voltage sensor 277
4.2.1 Mutational effects on voltage-dependence and charge movement 277
4.2.2 Evidence for the translocation of S4 279
4.2.3 Real-time monitoring of S4motion by fluorescence 282
4.3 Coupling between the voltage sensor and gating 283
5. CONCLUSION 284
6. ACKNOWLEDGEMENTS 287
7. REFERENCES 287
- Cited by 367
NMR structures of biomolecules using field oriented media and residual dipolar couplings
- J. H. Prestegard, H. M. Al-Hashimi, J. R. Tolman
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- Published online by Cambridge University Press:
- 01 March 2001, pp. 371-424
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1. Introduction 372
1.1 Residual dipolar couplings as a route to structure and dynamics 372
1.2 A brief history of oriented phase high resolution NMR 374
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
3. Inducing molecular order in high resolution NMR 383
3.1 Tensorial interactions between the magnetic field and anisotropic magnetic susceptibilities 383
3.2 Dilute liquid crystal media: a tunable source of order 384
3.2.1 Bicelles : from membrane mimics to aligning media 385
3.2.2 Filamentous phage 387
3.2.3 Transfer of alignment from ordered media to macromolecules 388
3.3 Magnetic field alignment 389
3.3.1 Paramagnetic assisted alignment 389
3.3.2 Advantages of using magnetic alignment 389
4. The measurement of residual dipolar couplings 391
4.1 Introduction 391
4.2 Frequency based methods 392
4.2.1 Coupling enhanced pulse schemes 392
4.2.2 In phase anti-phase methods (IPAP): 1DNH couplings in proteins 393
4.2.3 Exclusive correlated spectroscopy (E-COSY): 1DNH, 1DNC′ and 2DHNC′ 395
4.2.4 Extraction of splitting values from the frequency domain 396
4.3 Intensity based experiments 397
4.3.1 J-Modulated experiments: the measurement of 1DCαHα in proteins 397
4.3.2 Phase modulated methods 399
4.3.3 Constant time COSY – the measurement of DHH couplings 399
4.3.4 Systematic errors in intensity based experiments 400
5. Interpretation of residual dipolar coupling data 401
5.1 Structure determination protocols utilizing orientational constraints 401
5.1.1 The simulated annealing approach 401
5.1.2 Order matrix analysis of dipolar couplings 402
5.1.3 A discussion of the two approaches 402
5.2 Reducing orientational degeneracy 403
5.2.1 Multiple alignment media in the simulated annealing approach 404
5.2.2 Multiple alignment media in the order matrix approach 405
5.3 Simplifying effects arising due to molecular symmetry 406
5.4 Database approaches for determining protein structure 407
6. Applications to the characterization of macromolecular systems 408
6.1 Protein structure refinement 408
6.2 Protein domain orientation 409
6.3 Oligosaccharides 413
6.4 Biomolecular complexes 415
6.5 Exchanging systems 416
7. Acknowledgements 418
8. References 419
Within its relatively short history, nuclear magnetic resonance (NMR) spectroscopy has managed to play an important role in the characterization of biomolecular structure. However, the methods on which most of this characterization has been based, Nuclear Overhauser Effect (NOE) measurements for short-range distance constraints and scalar couplings measurements for torsional constraints, have limitations (Wüthrich, 1986). For extended structures, such as DNA helices, for example, propagation of errors in the short distance constraints derived from NOEs leaves the relative orientation of remote parts of the structures poorly defined. Also, the low density of observable protons in contact regions of molecules held together by factors other than hydrophobic packing, leads to poorly defined structures. This is especially true in carbohydrate containing complexes where hydrogen bonds often mediate contacts, and in multi-domain proteins where the area involved in domain–domain contact can also be small. Moreover, most NMR based structural applications are concerned with the characterization of a single, rigid conformer for the final structure. This can leave out important mechanistic information that depends on dynamic aspects and, when motion is present, this can lead to incorrect structural representations. This review focuses on one approach to alleviating some of the existing limitations in NMR based structure determination: the use of constraints derived from the measurement of residual dipolar couplings (D).