Most cited
This page lists all time most cited articles for this title. Please use the publication date filters on the left if you would like to restrict this list to recently published content, for example to articles published in the last three years. The number of times each article was cited is displayed to the right of its title and can be clicked to access a list of all titles this article has been cited by.
- Cited by 242
Two-photon fluorescence excitation and related techniques in biological microscopy
- Alberto Diaspro, Giuseppe Chirico, Maddalena Collini
-
- Published online by Cambridge University Press:
- 15 February 2006, pp. 97-166
-
- Article
- Export citation
-
1. Introduction 98
2. Historical background of two-photon effects 99
2.1 2PE 100
2.2 Harmonic generation 100
2.3 Fluorescence correlation spectroscopy 100
3. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 101
3.1 General considerations 101
3.2 Fluorescence intensity under the 2PE condition 103
3.3 Optical consequences of 2PE 104
3.4 Saturation effects in 2PE 108
3.5 Fluorescence correlation spectroscopy 109
3.5.1 Autocorrelation analysis 110
3.5.2 Photon-counting histogram analysis 112
4. Two-photon-excited probes 115
5. Design considerations for a 2PE fluorescence microscope 119
5.1 General aspects 119
5.2 Descanned and non-descanned 2PE imaging 121
5.3 Lens objectives and pulse broadening 122
5.4 Laser sources 125
5.5 Example of a practical realization 127
6. Applications 134
6.1 Biological applications of 2PE 134
6.1.1 Brain images 134
6.1.2 Applications on the kidney 139
6.1.3 Mammalian embryos 139
6.1.4 Applications to immuno-response 141
6.1.5 Myocytes 141
6.1.6 Retina 142
6.1.7 DNA imaging 143
6.1.8 FISH applications 144
6.2 2PE imaging of single molecules 144
6.3 FCS applications 148
6.4 Signals from nonlinear interactions 151
7. Conclusions 153
8. Acknowledgements 154
9. References 155
This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.
- Cited by 241
Conformational dynamics of the molecular chaperone Hsp90
- Kristin A. Krukenberg, Timothy O. Street, Laura A. Lavery, David A. Agard
-
- Published online by Cambridge University Press:
- 18 March 2011, pp. 229-255
-
- Article
- Export citation
-
The ubiquitous molecular chaperone Hsp90 makes up 1–2% of cytosolic proteins and is required for viability in eukaryotes. Hsp90 affects the folding and activation of a wide variety of substrate proteins including many involved in signaling and regulatory processes. Some of these substrates are implicated in cancer and other diseases, making Hsp90 an attractive drug target. Structural analyses have shown that Hsp90 is a highly dynamic and flexible molecule that can adopt a wide variety of structurally distinct states. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis only shift the equilibria between a pre-existing set of conformational states. For bacterial, yeast and human Hsp90, there is a conserved three-state (apo–ATP–ADP) conformational cycle; however; the equilibria between states are species specific. In eukaryotes, cytosolic co-chaperones regulate the in vivo dynamic behavior of Hsp90 by shifting conformational equilibria and affecting the kinetics of structural changes and ATP hydrolysis. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90, as well as the roles that nucleotide, co-chaperones, post-translational modification and substrates play. This view of Hsp90's conformational dynamics was enabled by the use of multiple complementary structural methods including, crystallography, small-angle X-ray scattering (SAXS), electron microscopy, Förster resonance energy transfer (FRET) and NMR. Finally, we discuss the effects of Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics.
- Cited by 240
The experimental survey of protein-folding energy landscapes
- Mikael Oliveberg, Peter G. Wolynes
-
- Published online by Cambridge University Press:
- 19 June 2006, pp. 245-288
-
- Article
- Export citation
-
1. Introduction 2
2. The macroscopic and microscopic views of protein folding 2
2.1 The macroscopic view: the experimental folding free-energy profile 2
2.2 The microscopic view: an underlying energy landscape 3
3. The micro to macro projection: from an energy landscape to a free-energy profile 6
4. Global features of the protein folding transition-state ensemble 12
4.1 Overall transition state location β[Dagger]: a measure of compactness 12
4.2 What makes folding so robust ? 13
5. Structural characterization of the transition-state ensemble 16
5.1 Insights from ϕ-value analysis 16
6. Deviations from ideality 20
6.1 β[Dagger] shifts along seemingly robust trajectories 21
6.2 Anomalous ϕ values, frustration and inhomogeneities 25
7. Intermediates 28
8. Detours, traps and frustration 29
8.1 Premature collapse and non-native trapping 29
9. Diffusion on the energy landscape and the elementary events of protein folding 30
10. Malleability of folding routes: changes of the dominant collective coordinates for folding 33
11. The evolution of the shape of the energy landscape 35
11.1 Negative design: the hidden dimension of the folding code 35
12. Mechanistic multiplicity and evolutionary choice 36
13. Acknowledgements 37
14. References 38
We review what has been learned about the protein-folding problem from experimental kinetic studies. These studies reveal patterns of both great richness and surprising simplicity. The patterns can be interpreted in terms of proteins possessing an energy landscape which is largely, but not completely, funnel-like. Issues such as speed limitations of folding, the robustness of folding, the origin of barriers and cooperativity and the ensemble nature of transition states, intermediate and traps are assessed using the results from several experimental groups highlighting energy-landscape ideas as an interpretive framework.
- Cited by 235
Dynamics of biochemical and biophysical reactions: insight from computer simulations
- Arieh Warshel, William W. Parson
-
- Published online by Cambridge University Press:
- 30 January 2002, pp. 563-679
-
- Article
- Export citation
-
1. Introduction 563
2. Obtaining rate constants from molecular-dynamics simulations 564
2.1 General relationships between quantum electronic structures and reaction rates 564
2.2 The transition-state theory (TST) 569
2.3 The transmission coefficient 572
3. Simulating biological electron-transfer reactions 575
3.1 Semi-classical surface-hopping and the Marcus equation 575
3.2 Treating quantum mechanical nuclear tunneling by the dispersed-polaron/spin-boson method 580
3.3 Density-matrix treatments 583
3.4 Charge separation in photosynthetic bacterial reaction centers 584
4. Light-induced photoisomerizations in rhodopsin and bacteriorhodopsin 596
5. Energetics and dynamics of enzyme reactions 614
5.1 The empirical-valence-bond treatment and free-energy perturbation methods 614
5.2 Activation energies are decreased in enzymes relative to solution, often by electrostatic effects that stabilize the transition state 620
5.3 Entropic effects in enzyme catalysis 627
5.4 What is meant by dynamical contributions to catalysis? 634
5.5 Transmission coefficients are similar for corresponding reactions in enzymes and water 636
5.6 Non-equilibrium solvation effects contribute to catalysis mainly through Δg[Dagger], not the transmission coefficient 641
5.7 Vibrationally assisted nuclear tunneling in enzyme catalysis 648
5.8 Diffusive processes in enzyme reactions and transmembrane channels 651
6. Concluding remarks 658
7. Acknowledgements 658
8. References 658
Obtaining a detailed understanding of the dynamics of a biochemical reaction is a formidable challenge. Indeed, it might appear at first sight that reactions in proteins are too complex to analyze microscopically. At room temperature, even a relatively small protein can have as many as 1034 accessible conformational states (Dill, 1985). In many cases, however, we have detailed structural information about the active site of an enzyme, whereas such information is missing for corresponding chemical systems in solution. The atomic coordinates of the chromophore in bacteriorhodopsin, for example, are known to a resolution of 1–2 Å. In addition, experimental studies of biological processes such as photoisomerization and electron transfer have provided a wealth of detailed information that eventually may make some of these processes classical problems in chemical physics as well as biology.
- Cited by 234
Ionic pores, gates, and gating currents
- Clay M. Armstrong
-
- Published online by Cambridge University Press:
- 17 March 2009, pp. 179-209
-
- Article
- Export citation
-
The current phase of axon physiology began with the invention of the voltage clamp by Cole (1949) and its use by Hodgkin & Huxley (1952d) to produce an astonishingly complete analysis of the ionic permeabilities that are responsible for the action potential. Their description did notcontain much in the way of molecular detail, and left open such questions as whether ions cross the membrane by way of pores or carriers, and the nature of the ‘gating‘ processes that increase ordecrease ion permeability in response to changes of the membrane potential. In the last few years our picture of the ionicchannels has grown considerably more tangible, though it still falls far short of a detailed molecular description. This article describes this sharpened picture and reviews the evidence for it. The viewpoint expressed is a very personal one, andno attempt has been made to review the literature of axonology comprehensively.
- Cited by 234
In silico ADME/T modelling for rational drug design
- Yulan Wang, Jing Xing, Yuan Xu, Nannan Zhou, Jianlong Peng, Zhaoping Xiong, Xian Liu, Xiaomin Luo, Cheng Luo, Kaixian Chen, Mingyue Zheng, Hualiang Jiang
-
- Published online by Cambridge University Press:
- 02 September 2015, pp. 488-515
-
- Article
-
- You have access Access
- Open access
- HTML
- Export citation
-
In recent decades, in silico absorption, distribution, metabolism, excretion (ADME), and toxicity (T) modelling as a tool for rational drug design has received considerable attention from pharmaceutical scientists, and various ADME/T-related prediction models have been reported. The high-throughput and low-cost nature of these models permits a more streamlined drug development process in which the identification of hits or their structural optimization can be guided based on a parallel investigation of bioavailability and safety, along with activity. However, the effectiveness of these tools is highly dependent on their capacity to cope with needs at different stages, e.g. their use in candidate selection has been limited due to their lack of the required predictability. For some events or endpoints involving more complex mechanisms, the current in silico approaches still need further improvement. In this review, we will briefly introduce the development of in silico models for some physicochemical parameters, ADME properties and toxicity evaluation, with an emphasis on the modelling approaches thereof, their application in drug discovery, and the potential merits or deficiencies of these models. Finally, the outlook for future ADME/T modelling based on big data analysis and systems sciences will be discussed.
- Cited by 231
The sarcoplasmic Ca2+-ATPase: design of a perfect chemi-osmotic pump
- Jesper V. Møller, Claus Olesen, Anne-Marie L. Winther, Poul Nissen
-
- Published online by Cambridge University Press:
- 01 September 2010, pp. 501-566
-
- Article
- Export citation
-
The sarcoplasmic (SERCA 1a) Ca2+-ATPase is a membrane protein abundantly present in skeletal mucles where it functions as an indispensable component of the excitation–contraction coupling, being at the expense of ATP hydrolysis involved in Ca2+/H+ exchange with a high thermodynamic efficiency across the sarcoplasmic reticulum membrane. The transporter serves as a prototype of a whole family of cation transporters, the P-type ATPases, which in addition to Ca2+ transporting proteins count Na+, K+-ATPase and H+, K+-, proton- and heavy metal transporting ATPases as prominent members. The ability in recent years to produce and analyze at atomic (2·3–3 Å) resolution 3D-crystals of Ca2+-transport intermediates of SERCA 1a has meant a breakthrough in our understanding of the structural aspects of the transport mechanism. We describe here the detailed construction of the ATPase in terms of one membraneous and three cytosolic domains held together by a central core that mediates coupling between Ca2+-transport and ATP hydrolysis. During turnover, the pump is present in two different conformational states, E1 and E2, with a preference for the binding of Ca2+ and H+, respectively. We discuss how phosphorylated and non-phosphorylated forms of these conformational states with cytosolic, occluded or luminally exposed cation-binding sites are able to convert the chemical energy derived from ATP hydrolysis into an electrochemical gradient of Ca2+ across the sarcoplasmic reticulum membrane. In conjunction with these basic reactions which serve as a structural framework for the transport function of other P-type ATPases as well, we also review the role of the lipid phase and the regulatory and thermodynamic aspects of the transport mechanism.
- Cited by 230
Evolution of higher-organism DNA
- David E. Kohne
-
- Published online by Cambridge University Press:
- 17 March 2009, pp. 327-375
-
- Article
- Export citation
-
A great deal of information about evolutionary events and processes has been inferred from careful studies of fossil records. Other forms of evidence have also contributed greatly to the understanding of evolution. Comparative biochemistry (Florkin, 1949), immunology (Boyden, 1942), protein sequencing (Dayoff, 1969; Anfinsen, 1959), and early DNA studies (McCarthy & Bolton, 1963; Schildkraut, Marmur & Doty, 1961) have for the most part corroborated earlier evolutionary findings, and at the same time provided new understanding of molecular processes in evolution. Of these approaches the comparison of DNA seems most promising since a relatively precise quantitative comparison can be made of all of the genetic material of different species.
- Cited by 224
Photosystem II: the engine of life
- James Barber
-
- Published online by Cambridge University Press:
- 27 January 2003, pp. 71-89
-
- Article
- Export citation
-
1. Introduction 71
2. Electron transfer in PS II 72
3. (Mn)4cluster and mechanism of water oxidation 73
4. Organization and structure of the protein subunits 75
5. Organization of chlorophylls and redox active cofactors 81
6. Implications arising from the structural models 82
7. Perspectives 84
8. Acknowledgements 86
9. Addendum 86
10. References 87
Photosystem II (PS II) is a multisubunit membrane protein complex, which uses light energy to oxidize water and reduce plastoquinone. High-resolution electron cryomicroscopy and X-ray crystallography are revealing the structure of this important molecular machine. Both approaches have contributed to our understanding of the organization of the transmembrane helices of higher plant and cyanobacterial PS II and both indicate that PS II normally functions as a dimer. However the high-resolution electron density maps derived from X-ray crystallography currently at 3·7/3·8 Å, have allowed assignments to be made to the redox active cofactors involved in the light-driven water–plastoquinone oxidoreductase activity and to the chlorophyll molecules that absorb and transfer energy to the reaction centre. In particular the X-ray work has identified density that can accommodate the four manganese atoms which catalyse the water-oxidation process. The Mn cluster is located at the lumenal surface of the D1 protein and approximately 7 Å from the redox active tyrosine residue (YZ) which acts an electron/proton transfer link to the primary oxidant P680.+. The lower resolution electron microscopy studies, however, are providing structural models of larger PS II supercomplexes that are ideal frameworks in which to incorporate the X-ray derived structures.
- Cited by 220
Haemocyanins
- K. E. van Holde, Karen I. Miller
-
- Published online by Cambridge University Press:
- 17 March 2009, pp. 1-129
-
- Article
- Export citation
-
About ten years ago, one of the authors participated in a review of haemocyanin structure and function (van Holde & van Bruggen, 1971). At that time, it was possible to describe the field in terms of a limited amount of exciting new structural information, and a long list of unanswered questions. While the stoichiometry of oxygen binding was understood, virtually nothing was known about the active site. Even the oxidation state of the copper was a matter of conjecture. The size of the haemocyanin polypeptide chains was the subject of intense debate, with very little substantive knowledge available. While the haemocyanins were known to be allosteric proteins, there were virtually no experimental studies of oxygen binding on a level that could be meaningfully interpreted in terms of extant theories.
- Cited by 216
Frustration in biomolecules
- Diego U. Ferreiro, Elizabeth A. Komives, Peter G. Wolynes
-
- Published online by Cambridge University Press:
- 16 September 2014, pp. 285-363
-
- Article
-
- You have access Access
- HTML
- Export citation
-
Biomolecules are the prime information processing elements of living matter. Most of these inanimate systems are polymers that compute their own structures and dynamics using as input seemingly random character strings of their sequence, following which they coalesce and perform integrated cellular functions. In large computational systems with finite interaction-codes, the appearance of conflicting goals is inevitable. Simple conflicting forces can lead to quite complex structures and behaviors, leading to the concept of frustration in condensed matter. We present here some basic ideas about frustration in biomolecules and how the frustration concept leads to a better appreciation of many aspects of the architecture of biomolecules, and especially how biomolecular structure connects to function by means of localized frustration. These ideas are simultaneously both seductively simple and perilously subtle to grasp completely. The energy landscape theory of protein folding provides a framework for quantifying frustration in large systems and has been implemented at many levels of description. We first review the notion of frustration from the areas of abstract logic and its uses in simple condensed matter systems. We discuss then how the frustration concept applies specifically to heteropolymers, testing folding landscape theory in computer simulations of protein models and in experimentally accessible systems. Studying the aspects of frustration averaged over many proteins provides ways to infer energy functions useful for reliable structure prediction. We discuss how frustration affects folding mechanisms. We review here how the biological functions of proteins are related to subtle local physical frustration effects and how frustration influences the appearance of metastable states, the nature of binding processes, catalysis and allosteric transitions. In this review, we also emphasize that frustration, far from being always a bad thing, is an essential feature of biomolecules that allows dynamics to be harnessed for function. In this way, we hope to illustrate how Frustration is a fundamental concept in molecular biology.
- Cited by 213
Acceleration of reaction in charged microdroplets
- Jae Kyoo Lee, Shibdas Banerjee, Hong Gil Nam, Richard N. Zare
-
- Published online by Cambridge University Press:
- 16 July 2015, pp. 437-444
-
- Article
-
- You have access Access
- Open access
- HTML
- Export citation
-
Using high-resolution mass spectrometry, we have studied the synthesis of isoquinoline in a charged electrospray droplet and the complexation between cytochrome c and maltose in a fused droplet to investigate the feasibility of droplets to drive reactions (both covalent and noncovalent interactions) at a faster rate than that observed in conventional bulk solution. In both the cases we found marked acceleration of reaction, by a factor of a million or more in the former and a factor of a thousand or more in the latter. We believe that carrying out reactions in microdroplets (about 1–15 μm in diameter corresponding to 0·5 pl – 2 nl) is a general method for increasing reaction rates. The mechanism is not presently established but droplet evaporation and droplet confinement of reagents appear to be two important factors among others. In the case of fused water droplets, evaporation has been shown to be almost negligible during the flight time from where droplet fusion occurs and the droplets enter the heated capillary inlet of the mass spectrometer. This suggests that (1) evaporation is not responsible for the acceleration process in aqueous droplet fusion and (2) the droplet–air interface may play a significant role in accelerating the reaction. We argue that this ‘microdroplet chemistry’ could be a remarkable alternative to accelerate slow and difficult reactions, and in conjunction with mass spectrometry, it may provide a new arena to study chemical and biochemical reactions in a confined environment.
- Cited by 210
Structure and function of SNARE and SNARE-interacting proteins
- Axel T. Brunger
-
- Published online by Cambridge University Press:
- 09 February 2006, pp. 1-47
-
- Article
- Export citation
-
This review focuses on the so-called SNARE (soluble N-ethyl maleimide sensitive factor attachment protein receptor) proteins that are involved in exocytosis at the pre-synpatic plasma membrane. SNAREs play a role in docking and fusion of synaptic vesicles to the active zone, as well as in the Ca2+-triggering step itself, most likely in combination with the Ca2+ sensor synaptotagmin. Different SNARE domains are involved in different processes, such as regulation, docking, and fusion. SNAREs exhibit multiple configurational, conformational, and oliogomeric states. These different states allow SNAREs to interact with their matching SNARE partners, auxiliary proteins, or with other SNARE domains, often in a mutually exclusive fashion. SNARE core domains undergo progressive disorder to order transitions upon interactions with other proteins, culminating with the fully folded post-fusion (cis) SNARE complex. Physiological concentrations of neuronal SNAREs can juxtapose membranes, and promote fusion in vitro under certain conditions. However, significantly more work will be required to reconstitute an in vitro system that faithfully mimics the Ca2+-triggered fusion of a synaptic vesicle at the active zone.
- Cited by 206
The (Na+ +K+) activated enzyme system and its relationship to transport of sodium and potassium
- Jens Chr. Skou
-
- Published online by Cambridge University Press:
- 17 March 2009, pp. 401-434
-
- Article
- Export citation
-
It seems to be the membrane bound (Na++K +)-activated enzyme system which transforms the energy from a hydrolysis of ATP into a vectorial movement of sodium out and potassium into the cell against electrochemical gradients, i.e. this systems seems to be the transport system for sodium and potassium (see, for example, review by Skou, 1972; Hokin & Dahl, 1972).
- Cited by 206
RNA secondary structure: physical and computational aspects
- Paul G. Higgs
-
- Published online by Cambridge University Press:
- 12 January 2001, pp. 199-253
-
- Article
- Export citation
-
1. Background to RNA structure 200
1.1 Types of RNA 200
1.1.1 Transfer RNA (tRNA) 200
1.1.2 Messenger RNA (mRNA) 201
1.1.3 Ribosomal RNA (rRNA) 201
1.1.4 Other ribonucleoprotein particles 202
1.1.5 Viruses and viroids 202
1.1.6 Ribozymes 202
1.2 Elements of RNA secondary structure 203
1.3 Secondary structure versus tertiary structure 205
2. Theoretical and computational methods for RNA secondary structure determination 208
2.1 Dynamic programming algorithms 208
2.2 Kinetic folding algorithms 210
2.3 Genetic algorithms 212
2.4 Comparative methods 213
3. RNA thermodynamics and folding mechanisms 216
3.1 The reliability of minimum free energy structure prediction 216
3.2 The relevance of RNA folding kinetics 218
3.3 Examples of RNA folding kinetics simulations 221
3.4 RNA as a disordered system 227
4. Aspects of RNA evolution 233
4.1 The relevance of RNA for studies of molecular evolution 233
4.1.1 Molecular phylogenetics 234
4.1.2 tRNAs and the genetic code 234
4.1.3 Viruses and quasispecies 235
4.1.4 Fitness landscapes 235
4.2 The interaction between thermodynamics and sequence evolution 236
4.3 Theory of compensatory substitutions in RNA helices 238
4.4 Rates of compensatory substitutions obtained from sequence analysis 240
5. Conclusions 246
6. Acknowledgements 246
7. References 246
This 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.
- Cited by 202
Computational biology in the study of cardiac ion channels and cell electrophysiology
- Yoram Rudy, Jonathan R. Silva
-
- Published online by Cambridge University Press:
- 19 July 2006, pp. 57-116
-
- Article
- Export citation
-
1. Prologue 58
2. The Hodgkin–Huxley formalism for computing the action potential 59
2.1 The axon action potential model 59
2.2 Cardiac action potential models 62
3. Ion-channel based formulation of the action potential 65
3.1 Ion-channel structure 65
3.2 Markov models of ion-channel kinetics 66
3.3 Role of selected ion channels in rate dependence of the cardiac action potential 71
3.4 Physiological implications of IKs subunit interaction 77
3.5 Mechanism of cardiac action potential rate-adaptation is species dependent 78
4. Simulating ion-channel mutations and their electrophysiological consequences 81
4.1 Mutations in SCN5A, the gene that encodes the cardiac sodium channel 82
4.1.1 The ΔKPQ mutation and LQT3 82
4.1.2 SCN5A mutation that underlies a dual phenotype 87
4.2 Mutations in HERG, the gene that encodes IKr: re-examination of the ‘gain of function/loss of function’ concept 94
4.3 Role of IKs as ‘repolarization reserve’ 100
5. Modeling cell signaling in electrophysiology 102
5.1 CaMKII regulation of the Ca2+ transient 102
5.2 The β-adrenergic signaling cascade 105
6. Epilogue 107
7. Acknowledgments 108
8. References 109
The cardiac cell is a complex biological system where various processes interact to generate electrical excitation (the action potential, AP) and contraction. During AP generation, membrane ion channels interact nonlinearly with dynamically changing ionic concentrations and varying transmembrane voltage, and are subject to regulatory processes. In recent years, a large body of knowledge has accumulated on the molecular structure of cardiac ion channels, their function, and their modification by genetic mutations that are associated with cardiac arrhythmias and sudden death. However, ion channels are typically studied in isolation (in expression systems or isolated membrane patches), away from the physiological environment of the cell where they interact to generate the AP. A major challenge remains the integration of ion-channel properties into the functioning, complex and highly interactive cell system, with the objective to relate molecular-level processes and their modification by disease to whole-cell function and clinical phenotype. In this article we describe how computational biology can be used to achieve such integration. We explain how mathematical (Markov) models of ion-channel kinetics are incorporated into integrated models of cardiac cells to compute the AP. We provide examples of mathematical (computer) simulations of physiological and pathological phenomena, including AP adaptation to changes in heart rate, genetic mutations in SCN5A and HERG genes that are associated with fatal cardiac arrhythmias, and effects of the CaMKII regulatory pathway and β-adrenergic cascade on the cell electrophysiological function.
- Cited by 200
Kinetic Analysis of ATPase Mechanisms
- D. R. Trentham, J. F. Eccleston, C. R. Bagshaw
-
- Published online by Cambridge University Press:
- 17 March 2009, pp. 217-281
-
- Article
- Export citation
-
At even the simplest level we can expect an ATPase mechanism to comprise the following four steps: the binding of ATP, the reaction of ATP with water on the enzyme, and the release of the products ADP and P1. So at the outset techniques are needed to investigate these four processes. The range of techniques needed is soon extended once questions are asked about the role of protons and metal ions, the possibility of a multistep hydrolytic process, multistep substrate and product binding processes, and protein–lipid or protein–protein interactions. Since ATPases and ATP synthases are almost universally involved in some form of energy transduction there is a particular need in an ATPase or ATP synthase reaction to evaluate the equilibrium constants of the steps in the mechanism and to investigate the possibility of alternate reaction pathways. The nature of the coupling process by the protein of the chemical reactions of ATP to the other energetic process, be it muscle contraction, active transport, respiration or photosynthesis, is likewise of profound interest. Finally we would like to know as much as possible about the ATPase or ATP synthase mechanism during the period when the various forms of energy transduction are occurring.
- Cited by 200
NMR-based screening in drug discovery
- Philip J. Hajduk, Robert P. Meadows, Stephen W. Fesik
-
- Published online by Cambridge University Press:
- 01 August 1999, pp. 211-240
-
- Article
- Export citation
-
1. Introduction 211
2. Screening methods 213
2.1 Chemical shifts 213
2.2 Diffusion 214
2.3 Transverse relaxation 218
2.4 Nuclear Overhauser effects 218
3. Strategies for drug discovery and design 221
3.1 Fragment-based methods 221
3.1.1 Linked-fragment approach 221
3.1.2 Directed combinatorial libraries 222
3.1.3 Modification of high-affinity ligands 223
3.1.4 Solvent mapping techniques 223
3.2 High-throughput NMR-based screening 224
3.3 Enzymatic assays 226
4. Discovery of novel ligands 227
4.1 High-affinity ligands for FKBP 227
4.2 Potent inhibitors of stromelysin 229
4.3 Ligands for the DNA-binding domain of the E2 protein 233
4.4 Discovery of Erm methyltransferase inhibitors 233
4.5 Phosphotyrosine mimetics for SH2 domains 236
5. Conclusions 237
6. References 237
A critical step in the drug discovery process is the identification of high-affinity ligands for macromolecular targets. Traditionally, the identification of such lead compounds has been accomplished through the high-throughout screening (HTS) of corporate compound repositories. Conventional HTS methodology has enjoyed widespread application and success in the pharmaceutical industry and, through recent technological advances in screening (Fernandes, 1998; Oldenburg et al. 1998; Silverman et al. 1998) and combinatorial chemistry (Fauchere et al. 1998; Fecik et al. 1998), these programs will continue to have a prominent role in drug discovery. However, suitable leads cannot always be found using conventional methods. This is not surprising since typical corporate libraries contain fewer than 106 compounds compared with the estimated 1050–1080 universe of compounds (Martin, 1997). In addition, most conventional assays are limited to screening libraries of compounds against proteins with known function, excluding the large number of targets becoming available from genomics research.
Recently, a number of NMR-based screening methods have been employed to identify and design lead ligands for protein targets (see Table 1). These NMR-based strategies can augment ongoing conventional HTS for identifying leads and can be used to aid in lead optimization. All of these techniques take advantage of the fact that upon complex formation between a target molecule and a ligand, significant perturbations can be observed in NMR-sensitive parameters of either the target or the ligand. These perturbations can be used qualitatively to detect ligand binding or quantitatively to assess the strength of the binding interaction. In addition, some of the techniques allow the identification of the ligand binding site or which part of the ligand is responsible for interacting with the target. In this article, the current state of NMR-based screening is reviewed.
- Cited by 198
Protein kinase inhibitors: contributions from structure to clinical compounds
- Louise N. Johnson
-
- Published online by Cambridge University Press:
- 19 March 2009, pp. 1-40
-
- Article
- Export citation
-
Protein kinases catalyse key phosphorylation reactions in signalling cascades that affect every aspect of cell growth, differentiation and metabolism. The kinases have become prime targets for drug intervention in the diseased state, especially in cancer. There are currently 10 drugs that have been approved for clinical use and many more in clinical trials. This review summarises the structural basis for protein kinase inhibition and discusses the mode of action for each of the approved drugs in the light of structural results. All but one of the approved compounds target the ATP binding site on the kinase. Both the active and inactive conformations of protein kinases have been used in strategies to produce potent and selective compounds. Targeting the inactive conformation can give high specificity. Targeting the active conformation is favourable where the diseased state has arisen from activating mutations, but such inhibitors generally target several protein kinases. Drug resistance mutations are a potential risk for both conformational states, where drug-binding regions are not directly involved in catalysis. Imatinib (Glivec), the most successful of protein kinase inhibitors, targets the inactive conformation of ABL tyrosine kinase. Newer compounds, such as dasatinib, which targets the ABL active state, have been developed to increase potency and have proved effective for some, but not all, drug-resistant mutations. The first epidermal growth factor receptor (EGFR) inhibitors in clinical use [gefitinib (Iressa) and erlotinib (Tarceva)] targeted the active form of the kinase, and this proved advantageous for patients whose cancer was caused by mutations that resulted in a constitutively active EGFR kinase domain. Newer approved compounds, such as lapatinib (Tykerb), target the inactive conformation with high potency. A further compound that forms a covalent attachment to the kinase has been found to overcome one of the major drug resistance mutations, where the effectiveness of the drug in vivo is dependent on its ability to compete successfully in the presence of cellular concentrations of ATP. Inhibitors of vascular endothelial growth factor receptor (VEGFR) kinase against cancer angiogenesis show the advantage of some relaxation in specificity. Sorafenib, originally developed as RAF inhibitor, is now in clinical use as a VEGFR inhibitor. Temsirolimus (a derivative of rapamycin) is the only example of a drug in clinical use that does not target the kinase ATP site. Instead rapamycin, when in complex with the protein FKBP12, effectively targets mTOR kinase at a site located on a domain, the FRB domain, that appears to be involved in localisation or substrate docking.
- Cited by 198
Ionic regulation of egg activation
- M. J. Whitaker, R. A. Steinhardt
-
- Published online by Cambridge University Press:
- 17 March 2009, pp. 593-666
-
- Article
- Export citation
-
Developing cells have constantly to make decisions: when to proliferate and divide, when and how to differentiate. It is an increasingly attractive idea that these decisions involve changes in intracellular cation concentrations. Our ideas about the mechanisms of changes in intracellular cations come largely from the application of biophysical techniques in the study of excitable tissues. These ideas are proving very valuable to the investigation of the control of proliferation and cell development and it is evident that the ionic mechanisms which pertain in nerve and muscle have their counterparts in other cells. Just as alterations in intracellular ion concentrations serve a signalling function in excitable tissue, so too they act as signals during development. Since almost all the quantitative data on the ionic mechanisms of fertilization come from work on sea urchins we have confined our review to sea urchin eggs.