Reversible Ligand Binding E-Book

Andrea Bellelli

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 638

Veröffentlichungsjahr: 2017

Beschreibung

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.

Leseprobe

Reversible Ligand Binding

Theory and Experiment

Andrea Bellelli

Department of Biochemical Sciences, Sapienza University of Rome, Italy

Jannette Carey

Department of Chemistry, Princeton University, USA

This edition first published 2018© 2018 John Wiley & Sons Ltd

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Library of Congress Cataloging‐in‐Publication Data

Names: Bellelli, Andrea, 1958– author. | Carey, Jannette, author.Title: Reversible ligand binding : theory and experiment / by Andrea Bellelli, Jannette Carey.Description: First edition. | Hoboken, NJ : John Wiley & Sons, 2018. | Includes bibliographical references and index. |Identifiers: LCCN 2017026931 (print) | LCCN 2017040990 (ebook) | ISBN 9781119238478 (pdf) | ISBN 9781119238492 (epub) | ISBN 9781119238485 (cloth)Subjects: LCSH: Ligand binding (Biochemistry)Classification: LCC QP517.L54 (ebook) | LCC QP517.L54 B45 2017 (print) | DDC 572/.33–dc23LC record available at https://lccn.loc.gov/2017026931

Cover design by WileyCover Image: Courtesy of RCSB Protein Data Bank (entry 1vyf, deposited by Angelucci et al. 2004)

A.B. dedicates this book to Maurizio Brunori, teacher, mentor and friend for thirty‐eight years until now and with more to come.

Preface

Ligand binding is a crucial event in virtually every biological phenomenon. Detailed understanding of many biologically relevant events including enzymatic catalysis, transport, and molecular recognition requires quantitative description of ligand binding. Such description may prove exquisitely complex because biological macromolecules may bind multiple ligands at once or alternatively and their reactions may present several types of thermodynamic linkage. The scope of this book is to provide a comprehensive view of the various biochemical considerations that govern reversible as well as irreversible ligand binding. Special attention is devoted to enzymology, a field usually treated separately from ligand binding, but actually governed by identical thermodynamic relationships.

This book is intended for PhD students and researchers, and aims at providing the understanding necessary to interpret ligand‐binding experiments, formulate plausible reaction schemes, and analyze the data according to the chosen model(s). Attention is given to the design of the experiment because a properly designed experiment may provide clear evidence of biochemical features that can otherwise escape notice. Classical experiments are reviewed in order to further highlight the importance of the design of the experiment.

The book includes treatment of thermodynamic relationships that are most often left to the specialized literature, for example, ligand‐linked dissociation. To make the book accessible to a general audience, we simplified the analysis of these relationships to the maximum possible extent, for example, whenever possible we adopted homodimeric proteins as model systems instead of more complex macromolecular assemblies. This choice allowed us to explore a large range of effects with minimally complex equations. Our scope throughout the book has been to present all the essential and distinguishing aspects of the phenomena we describe rigorously, but at the same time in the simplest possible form. Indeed, we are confident that every reader having competence in elementary algebra may take advantage of our work.

Acknowledgments

The authors of scientific books always contract debts with many people who, directly or indirectly, contributed ideas, suggestions, and comments. This book contains considerations and ideas that we elaborated over many years, long before our decision to write, and the list of people we would like to thank is very long. Moreover, the background of the authors being different, our two lists sum without overlapping.

We acknowledge the innumerable hours of education on the subject matters discussed in this book by our teachers and mentors. A.B. expresses his thanks to Maurizio Brunori, Eraldo Antonini, Bruno Giardina, Gino Amiconi, and Quentin H. Gibson.

We enjoyed many long and fruitful discussions with our colleagues, that often shaped our views as expressed in this book. Thanks are due to W.A. Eaton, S.J. Gill, T. Yonetani, M. Coletta, P. Ascenzi, G. Antonini, P. Sarti, F. Malatesta, E. Henry, P. Brzezinski, J.S. Olson, W.F. Xue, S. Linse, G.K. Ackers, and many others.

We express our thanks to our marvelous students, whose questions made us grow. Many of them are now well reputed researchers and close friends: R. Ippoliti, E. Lendaro, G. Boumis, A. Brancaccio, S. Santanché, A. Arcovito, F. Angelucci, L. Jin, B. Harish and R. Grandori. Special thanks are due to F. Saccoccia and A. Di Matteo who read the manuscript more than once and pointed out errors and unclear passages: their contribution has been invaluable.

Part ILigand Binding to Single Binding Site Targets

1Theory of Ligand Binding to Monomeric Proteins

1.1 Importance of Ligand‐Binding Phenomena in Biology

Reversible interactions between or among molecules underlie nearly every aspect of biology. To understand these interactions in a chemical way means to describe them quantitatively. To do so we must be able to determine their affinity, stoichiometry, and cooperativity by carrying out ligand‐binding experiments. We use the term “ligand” in a way distinct from its use to discuss coordination complexes within inorganic chemistry. In ligand‐binding theory we use this term to mean any interacting partner. Although many people consider a ligand to be a small molecule that interacts with a macromolecule, in fact, either partner can be considered to be the ligand of the other. In a typical experiment the concentration of one partner is held fixed while the concentration of the other partner is incremented. In ligand‐binding theory and practice we define the ligand operationally as the partner whose concentration is incremented during the experiment. Such experiments resemble pH titrations both practically and theoretically, and thus are referred to as titrations. The partner whose concentration is held fixed is referred to as the “target,” and again the definition is strictly operational, that is, either a small molecule or a macromolecule can be the target, depending on how the titration is set up.

As we will show later, there are good reasons to carry out experiments with first one partner, then the other, treated as the ligand; however, depending on the chemical properties of the two partners in the reaction, there may be practical limitations to, or conceptual difficulties in, the possibility of interchanging their respective roles as the ligand and the target. In particular, if either of the reaction partners has multiple binding sites for the other, one may not obtain superimposable binding isotherms when exchanging the target and the ligand.

Affinity refers to the strength of interaction between partners. Affinity is quantitatively expressed by an equilibrium constant that we measure in our experiment or, equivalently, a free energy difference between the bound and free states of the system that we calculate from the equilibrium constant.

Stoichiometry refers to the number of molecules of each partner that participate in the binding process, and it must also be determined by our measurements. In practice, what we really mean by stoichiometry is more often molar ratio. For example, in a process involving four molecules of one kind with two of another kind, the stoichiometry is 4:2 but the molar ratio is 2:1. The determination of true stoichiometries usually requires additional information from sources other than a binding experiment (e.g., molecular weights, and state of aggregation of the target and ligand in solution).

Thermodynamic linkage is a general term that applies to ligand‐binding experiments in which the same target binds two or more molecules of the same or different ligands, and each ligand modulates the affinity of the target for the other. There are at least three different types of linkage, called identical, homotropic, and heterotropic (Wyman and Gill, 1990). Identical linkage occurs when two different ligands compete for the same binding site on the target, and their binding is mutually exclusive. This type of linkage is discussed in Section 1.8 for single binding site targets, and in Section 4.8 for multiple binding site targets. Competitive enzyme inhibition is a very important case of identical linkage and is described in Section 8.6. Homotropic linkage occurs when the target can bind more than a single molecule of the same ligand, with different affinity. Homotropic linkage can occur only in targets with multiple binding sites, thus its analysis is deferred to Chapter 4. Finally, heterotropic linkage occurs when the target can bind two different ligands in a non‐exclusive manner and the binding of one ligand alters the affinity of the other. It is described in Section 1.9, and for targets with multiple binding sites in Section 4.9. Important examples of heterotropic linkage are uncompetitive enzyme‐inhibition (Section 8.7), and regulation of the oxygen affinity of hemoglobin by effectors, including protons (Bohr effect), diphosphoglycerate, or inositol hexaphosphate, dealt with in Chapter 7.

Homotropic and heterotropic linkage are typically regarded as an emergent property unique to proteins, but some non‐protein molecules of ~500–1000 Da have been shown to exhibit cooperative binding of their ligands (Rebek, 1985). An interesting and biologically relevant example is provided by the axial ligands of iron‐porphyrins (Traylor and Sharma, 1992).

Homotropic and heterotropic linkages may be either positive (if each ligand increases the affinity of the other) or negative (if each ligand decreases the affinity of the other). Cooperativity has often been used as a synonym of linkage, but unfortunately not always with the necessary precision. Often, cooperativity, or positive cooperativity, is used to indicate positive homotropic linkage, but the terms of negative cooperativity or anti‐cooperativity may be used to indicate a negative homotropic or heterotropic linkage. The definition of cooperativity is sufficiently general to encompass cases in which even monomeric proteins can respond cooperatively to two different ligands, for example, the physiological ligand and an ionic component of the solution (Weber, 1992). Note that in such cases the ion must be also considered as a physiological effector. Because the general applicability of a definition is inversely related to its precision, in this book, we shall prefer the terms positive or negative homotropic or heterotropic linkage whenever precision is required. Positive cooperativity occurs in several proteins and has special relevance in physiology. For example, the binding of oxygen to hemoglobin is cooperative in that oxygen affinity becomes stronger as binding progresses, as described in detail in Chapter 7.

In this chapter we describe the theoretical bases of ligand binding under equilibrium conditions for protein:ligand complexes with 1:1 stoichiometry; in the next chapter we discuss the kinetics of the same system, and in Chapter 3 we consider some practical aspects of experimental design, and some common sources of errors.

1.2 Preliminary Requirements for Ligand‐Binding Study