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Conformational dynamics is often essential for a protein's function. For example, proteins are able to communicate the effect of binding at one site to a distal region of the molecule through changes in its conformational dynamics. This so called allosteric coupling fine tunes the sensitivity of ligand binding to changes in concentration. A conformational change between a "closed" (apo) and an "open" (holo) conformation upon ligation often produces this coupling between binding sites. Enhanced sensitivity between the unbound and bound ensembles leads to a sharper binding curve. There are two basic conceptual frameworks that guide our visualization about ligand binding mechanisms. First, a ligand can stabilize the unstable "open" state from a dynamic ensemble of conformations within the unbound basin. This binding mechanism is called conformational selection. Second, a ligand can weakly bind to the low-affinity "closed" state followed by a conformational transition to the "open" state. This is called induced fit binding.In this dissertation, I focus on molecular dynamics simulations to understand microscopic origins of ligand binding cooperativity. A minimal model of allosteric binding transitions must include ligand binding/unbinding events, while capturing the transition mechanism between two distinct meta-stable free energy basins. Due in part to computational timescales limitations, work in this dissertation describes large-scale conformational transitions through a simplified, coarse-grained model based on the energy basins defined by the open and closed conformations of the protein Calmodulin (CaM). CaM is a ubiquitous calcium-binding protein consisting of two structurally similar globular domains connected by a flexible linker. The two domains of CaM, N-terminal domain (nCaM) and C-terminal domain (cCaM) consists of two helix-loop-helix motifs (the EF-hands) connected by a flexible linker. Each domain of CaM consists of two binding loops and binds 2 calcium ions each. The intact domain binds up to 4 calcium ions. The simulations use a coupled molecular dynamics/monte carlo scheme where the protein dynamics is simulated explicitly, while ligand binding/unbinding are treated implicitly. This helps us characterize complementary thermodynamic and kinetic description of calcium binding mechanism to CaM.In the model, ligand binding/unbinding events coupled with a conformational change of the protein within the grand canonical ensemble. Here, ligand concentration is controlled through the chemical potential (μ). This allows us to use a simple thermodynamic model to analyze the simulated data and quantify binding cooperativity. Simulated binding titration curves are calculated through equilibrium simulations at different values of μ.First, I study domain opening transitions of isolated nCaM and cCaM in the absence of calcium. This work is motivated by results from a recent analytic variational model that predicts distinct domain opening transition mechanism for the domains of CaM. This is a surprising result because the domains have the same folded state topology. In the simulations, I find the two domains of CaM have distinct transition mechanism over a broad range of temperature, in harmony with the analytic predictions. In particular, the simulated transition mechanism of nCaM follows a two-state behavior, while domain opening in cCaM involves global unfolding and refolding of the tertiary structure. The unfolded intermediate also appears in the landscape of nCaM, but at a higher temperature than it appears in cCaM's energy landscape. This is consistent with nCaM's higher thermal stability. Under approximate physiological conditions, majority of the sampled transitions in cCaM involves unfolding and refolding during conformational change. Kinetically, the transient unfolding and refolding in cCaM significantly slows the domain opening and closing rates in cCaM.Second, I investigate the structural origins of binding affinity and allosteric cooperativity of binding 2 calcium-ions to each domain of CaM. In my work, I predict the order of binding strength of CaM's loops. I analyze simulated binding curves within the framework of the classic Monod-Wyman-Changeux (MWC) model of allostery to extract the binding free energies to the closed and open ensembles. The simulations predict that cCaM binds calcium with higher affinity and greater cooperativity than nCaM. Where it is possible to compare, these predictions are in good agreement with experimental results. The analysis of the simulations offers a rationale for why the two domains differ in cooperativity: the higher cooperativity of cCaM is due to larger difference in affinity of its binding loops.Third, I extend the work to investigate structural origins of binding cooperativity of 4 calcium-ions to intact CaM. I characterize the microscopic cooperativities of each ligation state and provide a kinetic description of the binding mechanism. Due to the heterogeneous nature of CaM's loops, as predicted in our simulations of isolated domains, I focus on investigating the influence of this heterogeneity on the kinetic flux of binding pathways as a function of concentration. The formalism developed for Network Models of protein folding kinetics, is used to evaluate the directed flux of all possible pathways between unligated and fully loaded CaM.
Presents the physical background of ligand binding and instructs on how experiments should be designed and analyzed Reversible Ligand Binding: Theory and Experiment discusses the physical background of protein-ligand interactions—providing a comprehensive view of the various biochemical considerations that govern reversible, as well as irreversible, ligand binding. Special consideration is devoted to enzymology, a field usually treated separately from ligand binding, but actually governed by identical thermodynamic relationships. Attention is given to the design of the experiment, which aids in showing clear evidence of biochemical features that may otherwise escape notice. Classical experiments are reviewed in order to further highlight the importance of the design of the experiment. Overall, the book supplies students with the understanding that is necessary for interpreting ligand binding experiments, formulating plausible reaction schemes, and analyzing the data according to the chosen model(s). Topics covered include: theory of ligand binding to monomeric proteins; practical considerations and commonly encountered problems; oligomeric proteins with multiple binding sites; ligand binding kinetics; hemoglobin and its ligands; single-substrate enzymes and their inhibitors; two-substrate enzymes and their inhibitors; and rapid kinetic methods for studying enzyme reactions. Bridges theory of ligand binding and allostery with experiments Applies historical and physical insight to provide a clear understanding of ligand binding Written by a renowned author with long-standing research and teaching expertise in the area of ligand binding and allostery Based on FEBS Advanced Course lectures on the topic Reversible Ligand Binding: Theory and Experiment is an ideal text reference for students and scientists involved in biophysical chemistry, physical biochemistry, biophysics, molecular biology, protein engineering, drug design, pharmacology, physiology, biotechnology, and bioengineering.
Ligand-macromolecule interactions are of fundamental importance in the control of biological processes. This book applies the principles of linkage thermodynamics to polyfunctional macromolecular systems under equilibrium conditions, and describes the binding, linkage, and feedback phenomena that lead to control of complex metabolic processes. The first chapter sets out the different processes (conformational changes, changes in state of aggregation, phase changes) involving biological macromolecules which are affected by chemical variables (such as ligands) or physical variables (such as temperature and pressure). The general effects of ligands on micromolecular conformations and interactions are illustrated with specific examples from the respiratory proteins, electron-transport proteins, and nucleic acid binding proteins. Subsequent chapters develop these themes, and describe in detail how the mathematics of regulation and control can be applied to macromolecules in biological system.
Computational neurosciences and systems biology are among the main domains of life science research where mathematical modeling made a difference. This book introduces the many different types of computational studies one can develop to study neuronal systems. It is aimed at undergraduate students starting their research in computational neurobiology or more senior researchers who would like, or need, to move towards computational approaches. Based on their specific project, the readers would then move to one of the more specialized excellent textbooks available in the field. The first part of the book deals with molecular systems biology. Functional genomics is introduced through examples of transcriptomics and proteomics studies of neurobiological interest. Quantitative modelling of biochemical systems is presented in homogeneous compartments and using spatial descriptions. A second part deals with the various approaches to model single neuron physiology, and naturally moves to neuronal networks. A division is focused on the development of neurons and neuronal systems and the book closes on a series of methodological chapters. From the molecules to the organ, thinking at the level of systems is transforming biology and its impact on society. This book will help the reader to hop on the train directly in the tank engine.
A recent review of one of my grant applications commented on the ‘rediscovery of tropomyosin’. I was tempted to write back in my rebuttal to the reviewer that I didn’t realise it had been lost. Uncharacteristic maturity prevailed and I resisted the temptation, but I was struck by the underlying observation that research on the str- ture and function of tropomyosin has been somewhat invisible, particularly in terms of the cytoskeleton isoforms. So, how can it be that one of the two major components of the actin filament has been so thoroughly overlooked? I suspect that the answer is disappointingly pedestrian. Whereas the biochemistry of the 1980s revealed the potential of tropomyosin isoforms to diversify the function of actin filaments, the subsequent disenchantment with isoform biology in general in the 1990s inhibited growth of this field. With the development of more sophisticated experimental - proaches we are now seeing a growing realisation of the importance of tropomyosin in regulating actin filaments beyond its pivotal role in muscle contraction. The opportunity to edit this book came at a time when we had written several reviews on different aspects of tropomyosin function and I had just finished the background reading for a comprehensive review of tropomyosin biology. I realised that the field was simply beyond the capacity of any one person to do the field j- tice.
Drug-Acceptor Interactions: Modeling theoretical tools to test and evaluate experimental equilibrium effects suggests novel theoretical tools to test and evaluate drug interactions seen with combinatorial drug therapy. The book provides an in-depth, yet controversial, exploration of existing tools for analysis of dose-response studies at equilibrium or steady state. The book is recommended reading for post-graduate students and researchers engaged in the study of systems biology, networks, and the pharmacodynamics of natural or industrial drugs, as well as for medical clinicians interested in drug application and combinatorial drug therapy. Even people without mathematical skills will be able to follow the pros and cons of reaction schemes and their related distribution equations. Chapter 9 is a hands-on guide for software to plot, fit and analyze one’s own data.
Molecular and Cellular Biophysics provides advanced undergraduate and graduate students with a foundation in the basic concepts of biophysics. Students who have taken physical chemistry and calculus courses will find this book an accessible and valuable aid in learning how these concepts can be used in biological research. The text provides a rigorous treatment of the fundamental theories in biophysics and illustrates their application with examples. Conformational transitions of proteins are studied first using thermodynamics, and subsequently with kinetics. Allosteric theory is developed as the synthesis of conformational transitions and association reactions. Basic ideas of thermodynamics and kinetics are applied to topics such as protein folding, enzyme catalysis and ion channel permeation. These concepts are then used as the building blocks in a treatment of membrane excitability. Through these examples, students will gain an understanding of the general importance and broad applicability of biophysical principles to biological problems.
Connecting past, present, and future instrument development and use, Biocalorimetry: Foundations and Contemporary Approaches explores biocalorimetry's history, fundamentals, methodologies, and applications. Some of the most prominent calorimeter developers and users share invaluable personal accounts of discovery, discussing innovative techniques a