Irina Gitlin

Components for integrated poly(dimethylsiloxane) microfluidic systems.

This review describes the design and fabrication of microfluidic systems in poly(dimethylsiloxane) (PDMS). PDMS is a soft polymer with attractive physical and chemical properties: elasticity, optical transparency, flexible surface chemistry, low permeability to water, and low electrical conductivity. Soft lithography makes fabrication of microfluidic systems in PDMS particularly easy. Integration of components, and interfacing of devices with the user, is also convenient and simpler in PDMS than in systems made in hard materials. Fabrication of both single and multilayer microfluidic systems is straightforward in PDMS. Several components are described in detail: a passive chaotic mixer, pneumatically actuated switches and valves, a magnetic filter, functional membranes, and optical components.

Formation of monodisperse bubbles in a microfluidic flow-focusing device

This letter describes a method for generating monodisperse gaseous bubbles in a microfluidic flow-focusing device. The bubbles can be obtained in a range of diameters from 10 to 1000μm. The volume Vb of the bubbles scales with the flow rate q and the viscosity μ of the liquid, and the pressure p of the gas stream as Vb∝p∕qμ. This method allows simultaneous, independent control of the size of the individual bubbles and volume fraction of the dispersed phase. Under appropriate conditions, bubbles self-assemble into highly ordered, flowing lattices. Structures of these lattices can be adjusted dynamically by changing the flow parameters.

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.

Using Ion Channel-Forming Peptides to Quantify Protein−Ligand Interactions

This paper proposes a method for sensing affinity interactions by triggering disruption of self-assembly of ion channel-forming peptides in planar lipid bilayers. It shows that the binding of a derivative of alamethicin carrying a covalently attached sulfonamide ligand to carbonic anhydrase II (CA II) resulted in the inhibition of ion channel conductance through the bilayer. We propose that the binding of the bulky CA II protein (MW approximately 30 kD) to the ion channel-forming peptides (MW approximately 2.5 kD) either reduced the tendency of these peptides to self-assemble into a pore or extracted them from the bilayer altogether. In both outcomes, the interactions between the protein and the ligand lead to a disruption of self-assembled pores. Addition of a competitive inhibitor, 4-carboxybenzenesulfonamide, to the solution released CA II from the alamethicin-sulfonamide conjugate and restored the current flow across the bilayer by allowing reassembly of the ion channels in the bilayer. Time-averaged recordings of the current over discrete time intervals made it possible to quantify this monovalent ligand binding interaction. This method gave a dissociation constant of approximately 2 microM for the binding of CA II to alamethicin-sulfonamide in the bilayer recording chamber: this value is consistent with a value obtained independently with CA II and a related sulfonamide derivative by isothermal titration calorimetry.

Pumping based on transverse electrokinetic effects

This work presents a strategy for microfluidic pumping based on transverse electro-osmotic flow in a channel with topographical features on one wall. In this channel, flow along the long axis is generated by an electric field applied across the channel. The pump operates at low (5–10 V) voltage and achieves pumping speeds up to ∼100 μm/s in submillimeter channels. The pump is straightforward to fabricate, contains no moving parts, and provides local control of the direction and strength of pumping. The performance of the pump scales favorably with decreasing size of a microchannel.

Effects of Surface Charge on Denaturation of Bovine Carbonic Anhydrase

This work compares the denaturation of two proteins—bovine carbonic anhydrase II (BCA) and its derivative with all lysine groups acetylated (BCA‐Ac18)—by urea, guanidinium chloride (GuHCl), heat, and sodium dodecyl sulfate (SDS). It demonstrates that increasing the net negative charge of the protein by acetylation of lysines reduces its stability to urea, GuHCl, and heat, but increases its kinetic stability (its thermodynamic stability cannot be measured) towards denaturation by SDS. Increasing the ionic strength of the buffer improves the stability of BCA‐Ac18 to urea and heat, but still leaves it less stable than unacetylated BCA to those denaturants. In urea, the large change in electrostatic interactions not only modifies the free energy of denaturation, but also introduces a stable intermediate into the unfolding pathway. This work shows that modifications of charges on the surfaces of proteins can have a large effect—positive or negative, depending on the denaturant—on the stability of the proteins despite the exposure of these charges to high dielectric solvent and buffer ions.