▪ Abstract Proton-coupled electron transfer (PCET) is an important mechanism for charge transfer in a wide variety of systems including biology- and materials-oriented venues. We review several are...

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Proton-Coupled Electron Transfer

Hydrogels have applications in surgery and drug delivery, but are never considered alongside polymers and composites as materials for mechanical design. This is because synthetic hydrogels are in general very weak. In contrast, many biological gel composites, such as cartilage, are quite strong, and function as tough, shock-absorbing structural solids. The recent development of strong hydrogels suggests that it may be possible to design new families of strong gels that would allow the design of soft biomimetic machines, which have not previously been possible.

Hydrogels for Soft Machines

1. Introduction: Overview of CA as a Model
Carbonic anhydrase (CA, EC 4.2.1.1) is a protein that is especially well-suited to serve as a model in many types of studies in biophysics, bioanalysis, the physical-organic chemistry of inhibitor design, and medicinal chemistry. In vivo, this enzyme catalyzes the hydration of CO2 and the dehydration of bicarbonate (eq 1).


CO2+H2O⇌HCO3−+H+

(1)


The active site of α-CAs comprises a catalytic ZnII ion coordinated by three imidazole groups of histidines and by one hydroxide ion (or water molecule), all in a distorted tetrahedral geometry. This grouping is located at the base of a cone-shaped amphiphilic depression, one wall of which is dominated by hydrophobic residues and the other of which is dominated by hydrophilic residues.1 Unless otherwise stated, “CA” in this review refers to (i) various isozymes of α-CAs or (ii) the specific α-CAs human carbonic anhydrases I and II (HCA I and HCA II) and bovine carbonic anhydrase II (BCA II); “HCA” refers to HCA I and HCA II; and “CA II” refers to HCA II and BCA II.

CA is particularly attractive for biophysical studies of protein–ligand binding for many reasons. (i) CA is a monomeric, single-chain protein of intermediate molecular weight (~30 kDa), and it has no pendant sugar or phosphate groups and no disulfide bonds. (ii) It is inexpensive and widely available. (iii) It is relatively easy to handle and purify, due in large part to its excellent stability under standard laboratory conditions. (iv) Amino acid sequences are available for most of its known isozymes. (v) The structure of CA, and of its active site, has been defined in detail by X-ray diffraction, and the mechanism of its catalytic activity is well-understood. (vi) As an enzyme, CA behaves not only as a hydratase/anhydrase with a high turnover number but also as an esterase (a reaction that is easy to follow experimentally). (vii) The mechanism of inhibition of CA by ligands that bind to the ZnII ion is fairly simple and well-characterized; it is, therefore, easy to screen inhibitors and to examine designed inhibitors that test theories of protein–ligand interactions. (viii) It is possible to prepare and study the metal-free apoenzyme and the numerous variants of CA in which the ZnII ion is replaced by other divalent ions. (ix) Charge ladders of CA II—sets of derivatives in which acylation of lysine amino groups (−NH3+ → −NHAc) changes the net charge of the protein—allow the influence of charge on properties to be examined by capillary electrophoresis. Some disadvantages of using CA include the following: (i) the presence of the ZnII cofactor, which can complicate biophysical and physical-organic analyses; (ii) a structure that is more stable than a representative globular protein and, thus, slightly suspect as a model system for certain studies of stability; (iii) a function—interconversion of carbon dioxide and carbonate—that does not involve the types of enzyme/substrate interactions that are most interesting in design of drugs; (iv) a catalytic reaction that is, in a sense, too simple (determining the mechanism of a reaction is, in practice, usually made easier if the reactants and products have an intermediate level of complexity); and (v) the absence of a solution structure of CA (by NMR spectroscopy). The ample X-ray data, however, paint an excellent picture of the changes (which are generally small) in the structure of CA that occur on binding ligands or introducing mutations.

The most important class of inhibitors of CA, the aryl-sulfonamides, has several characteristics that also make it particularly suitable for physical-organic studies of inhibitor binding and in drug design: (i) arylsulfonamides are easily synthesized; (ii) they bind with high affinity to CA (1 μM to sub-nM); (iii) they share one common structural feature; and (iv) they share a common, narrowly defined geometry of binding that exposes a part of the ligand that can be easily modified synthetically. There are also many non-sulfonamide, organic inhibitors of CA, as well as anionic, inorganic inhibitors.

We divide this review into five parts, all with the goal of using CA as a model system for biophysical studies: (I) an overview of the enzymatic activity and medical relevance of CA; (II) the structure and structure–function relationships of CA and its engineered mutants; (III) the thermodynamics and kinetics of the binding of ligands to CA; (IV) the effect of electrostatics on the binding of ligands to and the denaturation of CA; and (V) what makes CA a good model for studying protein–ligand binding and protein stability.



1.1. Value of Models 
CA serves as a good model system for the study of enzymes. That is, it is a protein having some characteristics representative of enzymes as a class, but with other characteristics that make it especially easy to study. It is a moderately important target in current medicinal chemistry: its inhibition is important in the treatment of glaucoma, altitude sickness, and obesity; its overexpression has recently been implicated in tumor growth; and its inhibition in pathogenic organisms might lead to further interesting drugs.2,3 More than its medical relevance, its tractability and simplicity are what make CA a particularly attractive model enzyme.

The importance of models in science is often underestimated. Models represent more complex classes of related systems and contribute to the study of those classes by focusing research on particular, tractable problems. The development of useful, widely accepted models is a critical function of scientific research: many of the techniques (both experimental and analytical) and concepts of science are developed in terms of models; they are thoroughly engrained in our system of research and analysis.

Examples of models abound in successful areas of science: in biology, E. coli, S. cerevisiae, Drosophila mela-nogaster, C. elegans, Brachydanio rerio (zebrafish), and the mouse; in chemistry, the hydrogen atom, octanol as a hydrophobic medium, benzene as an aromatic molecule, the 2-norbornyl carbocation as a nonclassical ion, substituted cyclohexanes for the study of steric effects, p-substituted benzoic acids for the study of electronic effects, cyclodextrins for ligand–receptor interactions; in physics, a vibrating string as an oscillator and a particle in a box as a model for electrons in orbitals.

Science needs models for many reasons:


Focus: Models allow a community of researchers to study a common subject. Solving any significant problem in science requires a substantial effort, with contributions from many individuals and techniques. Models are often the systems chosen to make this productive, cooperative focus possible.


Research Overhead: Development of a system to the point where many details are scientifically tractable is the product of a range of contributions: for enzymes, these contributions are protocols for preparations, development of assays, determination of structures, preparation of mutants, definition of substrate specificity, study of rates, and development of mechanistic models. In a well-developed model system, the accumulation of this information makes it relatively easy to carry out research, since before new experiments begin, much of the background work–the fundamental research in a new system–has already been carried out.


Recruiting and Interdisciplinarity: The availability of good model systems makes it relatively easy for a neophyte to enter an area of research and to test ideas efficiently. This ease of entry recruits new research groups, who use, augment, and improve the model system. It is especially important to have model systems to encourage participation by researchers in other disciplines, for whom even the elementary technical procedures in a new field may appear daunting.


Comparability: A well-established model allows researchers in different laboratories to calibrate their experiments, by reproducing well-characterized experiments.


Community: The most important end result of a good model system is often the generation of a scientific community–that is, a group of researchers examining a common problem from different perspectives and pooling information relevant to common objectives.



One of the goals of this review is to summarize many experimental and theoretical studies of CA that have established it as a model protein. We hope that this summary will make it easier for others to use this protein to study fundamentals of two of the most important questions in current chemistry: (i) Why do a protein and ligand associate selectively? (ii) How can one design an inhibitor to bind to a protein selectively and tightly? We believe that the summary of studies of folding and stability of CA will be useful to biophysicists who study protein folding. In addition, we hope that the compilation of data relevant to CA in one review will ease the search for information for those who are beginning to work with this protein.

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Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein–Ligand Binding

A new type of physically linked double-network hydrogel is synthesized by a simple, time-saving, facile, easily controlled, one-pot method. The resulting agar/polyacrylamide double-network hydrogels exhibit good mechanical properties, excellent recoverability, and a unique free-shapeable property, which makes them very promising hydrogels for load-bearing soft tissues.

A Robust, One‐Pot Synthesis of Highly Mechanical and Recoverable Double Network Hydrogels Using Thermoreversible Sol‐Gel Polysaccharide

Strategies and Molecular Design Criteria for 3D Printable Hydrogels

New insight into agarose gel mechanical properties.

The structure and internal dynamics of β-lactoglobulin aggregates formed after heat-induced denaturation at pH 2 and different ionic strengths were investigated using light, neutron, and X-ray scattering. Polydisperse aggregates are formed with a rigid rodlike local structure with mass per unit length close to that of a string of β-lactoglobulin monomers but with a somewhat larger diameter. The persistence length decreases with increasing ionic strength from more than 600 nm at 0.013 M to 38 nm at 0.1 M. At ionic strengths of 0.1 and 0.2 M, a self-similar structure with fractal dimensions of 1.8 and 2.0 is seen by using light scattering. The concentration dependence of the static structure factor and the internal dynamics are close to those of flexible linear chains. In contrast, a rigid behavior is observed at lower ionic strength (0.03 and 0.013 M). The persistence length of aggregates formed at 0.013 M is reduced after dilution in 0.1 and 0.2 M ionic strength solvents but remains larger than that of ag...

Static and Dynamic Scattering of β-Lactoglobulin Aggregates Formed after Heat-Induced Denaturation at pH 2

The effect of temperature and ionic strength on the dimerisation of β-lactoglobulin

Using a multitechnique approach, two temperature domains have been identified in agarose gelation Below 35°C, fast gelation results in strong, homogeneous and weakly turbid networks The correlation length, evaluated from the wavelength dependence of the turbidity, is close to values of pore size reported in the literature Above 35°C, gelation is much slower and is associated with the formation of large-scale heterogeneities that can be monitored by a marked change in the wavelength dependence of turbidity and visualised by transmission electron microscopy Curing agarose gels at temperatures above 35°C, and then cooling them to 20°C, produces much weaker gels than those formed directly at 20°C Dramatic reductions in the elastic modulus and failure strain and stress are found in this case as a result of demixing during cure An interpretation, based on the kinetic competition between osmotic forces (in favor of phase separation) and elastic forces (that prevent it) is proposed © 2001 John Wiley & Sons, Inc Biopolymers 59: 131–144, 2001

Influence of thermal history on the structural and mechanical properties of agarose gels.

A two-step mechanism for β-lactoglobulin aggregation at pH7 has been reported in the literature. The first aggregation step is difficult to characterise, as small particles are formed with a strong tendency to further associate in bigger aggregates. Experimental evidence for a two-step aggregation process, using scattering techniques and size exclusion chromatography, is shown here. The effect of the ionic strength, the protein concentration and the heating temperature is discussed. The results show that the first step consists in the formation of small globular aggregates, whose size are only weakly, if at all, influenced by the parameters mentioned above.

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Pierre Aymard

Papers

New insight into agarose gel mechanical properties.

Static and Dynamic Scattering of β-Lactoglobulin Aggregates Formed after Heat-Induced Denaturation at pH 2

The effect of temperature and ionic strength on the dimerisation of β-lactoglobulin

Influence of thermal history on the structural and mechanical properties of agarose gels.

Experimental evidence for a two-step process in the aggregation of β-lactoglobulin at pH 7