George K. Kaufman

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

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.

Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

This paper reports that the acetylation of lysine ε‐NH3+ groups of α‐amylase—one of the most important hydrolytic enzymes used in industry—produces highly negatively charged variants that are enzymatically active, thermostable, and more resistant than the wild‐type enzyme to irreversible inactivation on exposure to denaturing conditions (e.g., 1 h at 90°C in solutions containing 100‐mM sodium dodecyl sulfate). Acetylation also protected the enzyme against irreversible inactivation by the neutral surfactant TRITON X‐100 (polyethylene glycol p‐(1,1,3,3‐tetramethylbutyl)phenyl ether), but not by the cationic surfactant, dodecyltrimethylammonium bromide (DTAB). The increased resistance of acetylated α‐amylase toward inactivation is attributed to the increased net negative charge of α‐amylase that resulted from the acetylation of lysine ammonium groups (lysine ε‐NH3+ → ε‐NHCOCH3). Increases in the net negative charge of proteins can decrease the rate of unfolding by anionic surfactants, and can also decrease the rate of protein aggregation. The acetylation of lysine represents a simple, inexpensive method for stabilizing bacterial α‐amylase against irreversible inactivation in the presence of the anionic and neutral surfactants that are commonly used in industrial applications.

Patterns of Electrostatic Charge and Discharge in Contact Electrification

Here we describe a study of the charging and discharging of solids in a system comprising a metal sphere that rolls across an electrically insulating plate. There are two kinetically distinct processes: 1) charging at a constant rate; 2) abrupt discharging, when the potential difference between sphere and surface reaches a critical value determined by the dielectric strength of air. This work has two objectives: 1) to develop a procedure for examining the rate of charging and discharging as a function of a range of relevant variables; 2) to use this information to test the hypotheses that charge separation involved ions and that discharge of the potential produced involved a breakdown of air. In published work, we have described this system; this study demonstrates the wealth of quantitative information it can provide as a tool for studying the atomic/molecular mechanisms of contact electrification. These mechanisms are relevant to processes ranging from lightning to xerography, and are a subject of active controversy. Three mechanisms appear to contribute to contact electrification: 1) ion transfer between surfaces having mobile ions, 2) partitioning of ions from adsorbed water onto the surfaces of non-ionic insulators, and 3) electron transfer between conductors and semiconductors (materials with mobile electrons and well-defined Fermi surfaces). We have concluded—in agreement with a hypothesis by Diaz —that the transfer of ions between the contacting surfaces is the most common mechanism for charge separation when organic materials are involved. The data we present here are consistent with contact charging by the slow transfer of ions, interrupted by episodic, rapid discharge events involving ionized plasmas when the difference in electrical potential between the surfaces exceeds the breakdown limit of air. These experiments used the rolling sphere tool (RST, Figure 1) developed by Grzybowski et al. We investigated contact electrification between stainless steel spheres (d= 3.2 mm) and three different surfaces (relative humidity, RH = 20–25 %, T 22 8C, w = 80 rpm). The Supporting Information contains the experimental procedures we followed for preparing the insulating surfaces: 1) glass (a 1.0 mm thick, 76 mm diameter wafer of low-alkali glass); 2) glass silanized with N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride; 3) glass silanized with 3-(trihydroxysilyl)-1-propane-sulfonic acid. When the sphere was far (more than ca. 2.5 cm) from the electrode (width 5 mm, 0.2 radians), the electrometer reported only the charge on the portion of the insulator (the glass plate) to which the electrode coupled (Qw). When the sphere passed over the electrode, the electrometer registered a peak in the charge, the height of which was the sum (Qs+w) of the charges that the electrode sensed on the sphere (Qs) and Qw. Figure 2a shows the charge the electrometer recorded for one revolution of the sphere. The fullwidth at half-maximum of the peak was about 0.63 radians. Figure 2b shows a representative plot of the complex pattern of charge (Q, in picocoulomb, pC: 1 pC = 6.2 ; 10 elementary charges) the electrometer reported as the sphere rolled on a glass wafer. When the sphere was directly over the electrode, the electrometer measured a fraction of the charge on the sphere (80–90 %; see the Supporting Information). Qw (grey dot-dash guidelines) and Qs+w (black dashed guidelines) increased linearly with time. Sharp discontinuities— discharge events through air—interrupted the charging. Subtracting Qw from Qs+w gave Qs—the charge that the electrometer sensed on the rolling sphere alone—as a function of time (Figure 2c). We have observed qualitatively similar behavior on a variety of materials, including organic polymers; we will detail these experiments in a full paper. The polarity of charge separation was invariant when a steel sphere (positive) rolled on a clean glass wafer (negative) (Figure 3a). When the sphere rolled on a surface with bound Figure 1. Illustration of the “rolling sphere tool” to measure the kinetics of contact electrification between rolling stainless steel spheres and insulating surfaces. The Supporting Information contains additional graphical representations.

A Non-Chromatographic Method for the Purification of a Bivalently Active Monoclonal IgG Antibody from Biological Fluids

This paper describes a method for the purification of monoclonal antibodies (rat anti-2,4-dinitrophenyl IgG: IgG(DNP); and mouse antidigoxin IgG: IgG(Dgn)) from ascites fluid. This procedure (for IgG(DNP)) has three steps: (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation of cyclic complexes of IgG(DNP) by causing it to bind to synthetic multivalent haptens containing multiple DNP groups; (iii) selective precipitation of these dimers, trimers, and higher oligomers of the target antibody, followed by regeneration of the free antibody. This procedure separates the targeted antibody from a mixture of antibodies, as well as from other proteins and globulins in a biological fluid. This method is applicable to antibodies with a wide range of monovalent binding constants (0.1 microM to 0.1 nM). The multivalent ligands we used (derivatives of DNP and digoxin) isolated IgG(DNP) and IgG(Dgn) from ascites fluid in yields of >80% and with >95% purity. This technique has two advantages over conventional chromatographic methods for purifying antibodies: (i) it is selective for antibodies with two active Fab binding sites (both sites are required to form the cyclic complexes) over antibodies with one or zero active Fab binding sites; (ii) it does not require chromatographic separation. It has the disadvantage that the structure of the hapten must be compatible with the synthesis of bi- and/or trivalent analogues.

Controlling the Kinetics of Contact Electrification with Patterned Surfaces

This communication describes a new approach for controlling static charging (contact electrification), and resulting electrical discharging, that occurs when two contacting materials separate. The prevention of contact electrification is an important problem; unwanted adhesion between oppositely charged materials, spark-initiated explosions, and damage to microelectronic circuitry are some of the deleterious effects of static charging. Current strategies for controlling contact electrification rely upon dissipating an accumulated charge by making contacting surfaces conductive and, therefore, can be difficult to implement with electrically insulating materials. Specifically, using our understanding of the ion-transfer mechanism of contact electrification, we patterned glass slides with negatively charging areas (clean glass) and positively charging areas (glass silanized with a cationic siloxane terminated with a quaternary ammonium group). The rate of charge separation due to a steel sphere rolling on the patterned glass surface correlated linearly with the percentage of the glass surface that was silanized; the rate of charge transfer was minimal when 50% of the glass surface area was silanized. Patterned surfaces also prevented electrical discharges between electrically conducting (bare steel) or insulating (acrylate-coated steel) spheres rolling on the glass, because the rate of charging was sufficiently slow to prevent electric fields greater than the dielectric strength of air to develop. This strategy for preventing static charging therefore does not require one of the two contacting surfaces to be electrically conductive. More generally, these results show that our enhanced understanding of the ion-transfer mechanism of contact electrification enables the rational design of chemically tailored surfaces for functional electrets.

Phase separation of 2D meso-scale Coulombic crystals from meso-scale polarizable “solvent”

This paper describes the phase separation of millimetre-scale spheres based on electrostatic charge. Initially, polymeric (Teflon, T; Nylon-6,6, N) and metallic (gold-coated Nylon-6,6, Au(N)) spheres are uniformly mixed in a two-dimensional (2D) monolayer on a gold-coated plate. Oscillating the plate vertically caused the spheres to charge by contact electrification (tribocharging). Positively charged N and negatively charged T spheres attracted each other more strongly than they attracted the capacitively charged, Au(N) spheres. The T and N spheres formed 2D Coulombic crystals, and these crystals separated from the Au(N) spheres. The extent and rate of separation increased with increasing amplitude of agitation during tribocharging, and with decreasing density of spheres on the surface. At high surface density, the T and N spheres did not separate from the Au(N) spheres. This system models the 2D nucleation of an ionic crystal from a polarizable liquid.

Phase separation of two-dimensional Coulombic crystals of mesoscale dipolar particles from mesoscale polarizable “solvent”

This letter describes the formation of two-dimensional (2D) crystals of dipolar particles (TN) made of electrostatically charged, joined, millimeter-scale Teflon (T) and nylon-6,6 (N) spheres, and the separation of these crystals, as a distinct phase, from a mixture of TN and similar, capacitively charged particles that were coated with gold (Au2). The extent of separation increased with increasing amplitude of agitation, and with decreasing density of particles. Above a threshold in the amplitude of agitation, the crystals broke apart and the particles remixed. This system is a 2D model of the nucleation of crystals of polar molecules in a polarizable liquid.

The Epic of Evolution as a Framework for Human Orientation in Life

This article sketches what is required of a world picture (religious or nonreligious) that is intended to provide orientation in the world for ongoing human life today. How do we move from conceptions and theories prominent in the modern sciences-such as cosmic and biological evolution-to an overall picture or cosmology which can orient us for the effective address of todays deepest human problems? A biohistorical conception of the human is proposed in answer to this question.