This volume commemorates the 50th anniversary of the appearance in Volume 4 in 1948 of Dr. Jeffries Wyman's famous paper in which he "laid down" the foundations of linkage thermodynamics. Experts in this area contribute articles on the state-of-the-art of this important field and on new developments of the original theory. Among the topics covered in this volume are electrostatic contributions to molecular free energies in solution; site-specific analysis of mutational effects in proteins; allosteric transitions of the acetylcholine receptor; and deciphering the molecular code of hemoglobin allostery.

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.

Binding involves two steps, desolvation and association. While water is ubiquitous and occurs at high concentration, it is typically ignored. In vitro experiments typically use infinite dilution conditions, while in vivo, the concentration of water is decreased due to the presence of high concentrations of molecules in the cellular milieu. Our paper discusses isothermal titration calorimetry approaches that address the role of water in binding. For example, use of D2O allows the contribution of solvent reorganization to the enthalpy component to be assessed. Furthermore, the addition of osmolytes will decrease the water activity of a solution and allow effects on Ka to be determined. In most cases, binding becomes tighter in the presence of osmolytes as the desolvation penalty associated with binding is minimized. In other cases, the osmolytes prefer to interact with the ligand or protein, and if their removal is more difficult than shedding water, then binding can be weakened. Lastly, these complicating layers can be discerned by different slopes in ln(Ka) vs osmolality plots and by differential scanning calorimetry in the presence of the osmolyte.

The spontaneous assembly of polypeptides through non-covalent interactions at physiological conditions is the main focus of the presented work and will be discussed from two different perspectives: (i) the interaction of peptide chains with themselves leading to formation of higher order structures (self-assembling peptides); (ii) the interaction of polypeptides with nano-sized surfaces (protein-nanoparticle interactions). Although self-assembling peptides are an important growing class of biomaterials, most of the works in this field have focused upon their various biomedical applications without highlighting the molecular mechanisms which result in their self-assembly into supra-molecular structures inside the body. Herein, through an in-depth thermodynamic analysis utilizing Isothermal Titration Calorimtry technique, the driving forces for self-assembly of ionic self-complementary peptide RADA4 and its variants were identified implying great contribution of molecular hydration and charge to the self-assembly process. Furthermore, the interfacial molecules involved in self-assembly of these molecules was experimentally quantified. It was found that appending five serine residues to C-terminus of RADA4 can overshadow the hydrophobic contribution of RADA segment leading to hydrogen bonding being the main driving force for self-assembly; while presence of 5 lysine residues inhibited RADA4 self-assembly. Secondly, the interaction of proteins with zwitterionic-modified nanoparticles (NPs) was investigated. Although widely studied, the underlying mechanism for the protein-repellent behavior of zwitterionic polymers is largely unknown. A set of thermodynamic investigations was performed to study the interaction of two model proteins (with distinctly different adsorption behaviour) with the surface of zwitterionic-modified silica nanoparticles. The nature of the interaction between proteins and polymer-modified nanoparticle was identified along with highlighting the main driving forces leading to their adsorption onto the nanoparticle's surface. Moreover, the impact of zwitterion's spacer length and end-group chemistry on thermodynamics of protein adsorption was analyzed. Overall, our results indicated that the main advantage of zwitterionic polymer modification of surfaces are: i) an increase in water molecules at the interface, ii) lack of counter-ion release from surfaces and iii) lower structural reorganization of the system upon protein-surface interaction. The findings presented in this work will fundamentally impact our understanding of nano-bio interfaces leading to development of more optimum nano-biomaterials in future.

Macromolecules in the body form noncovalent associations, such as DNA-protein or protein-protein complexes, that control and regulate numerous cellular functions. Understanding how changes in the concentration and conformation of these macromolecules can trigger physiological responses is essential for researchers developing drug therapies to treat

This volume successfully and clearly examines how biophysical approaches can be used to study complex systems of reversibly interacting proteins. It deals with the methodology behind the research and shows how to synergistically incorporate several methodologies for use. Each chapter treats and introduces the reader to different biological systems, includes a brief summary of the physical principles, and mentions practical requirements.

Ligand binding by macromolecules represents a core event of broad relevance to a range of systems, including catalytic systems alongside noncatalytic systems such as nucleic acid binding by transcription factors or extracellular ligand binding by proteins involved in signaling pathways. The scope of this primer is constrained to introduce only foundational models without significant discussion of more advanced topics such as allosteric or linkage effects. Linkage occurs when the binding of a ligand is influenced by the binding of another molecule of the same ligand (homotropic linkage), the binding of a different ligand (heterotropic linkage), physical variables such as temperature or pressure (physical linkage), or changes in macromolecular assembly state (polysteric linkage). Taking this into account, the foundational themes presented in this primer can be used to describe any macromolecule–ligand interaction either by direct use of the models and techniques described here or by applying them to develop more advanced models to explain additional complexities such as those allosteric or linkage effects just mentioned. The target audience of this primer is the senior undergraduate or junior graduate student who lacks a foundation in ligand-binding thermodynamics. As such, we have focused primarily on foundational thermodynamic treatments and presented only general discussions of relevant experimental designs. Readers of this primer will learn how to build a working understanding of common factors that promote energetic favorability for ligand binding; develop a functional toolbox to understand ligand binding from the perspective of collecting, plotting, and interpreting ligand-binding data; enhance proficiency in deriving thermodynamic mechanisms for ligand binding; and become comfortable in interpreting binding data reported in the literature and independently expanding knowledge beyond the scope introduced in this primer.