Analytical biotechnology Editorial overview Preston Hensley* and Lloyd M Smith?

Addresses *Department of Macromolecular Sciences, UE-0447B, Smithkline Beecham, 709 Swedeland Road, King of Prussia, PA 19406-0939, USA; e-mail: c_preston_hensley@sbphrd.com -1. Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706.1396, USA; e-mail: smith@chem.wisc.edu

Current Opinion in Biotechnology 1997, 8:1-5

Electronic identifier: 0958-l 669-008-00001

0 Current Biology Ltd ISSN 0958-l 669

Abbreviations

IL interleukin ITC isothermal titration calorimetry LS light scattering MS mass spectrometry SPR surface plasmon resonance TIRF total internal reflection fluorescence

New and classical approaches to the measurement of affinity for ‘well-characterized biopharmaceuticals It is clear that the single most important aspect of biologi- cal macro- and micromolecules is that they self-assemble. At the heart of any important cellular biological process is some specific molecular assembly event. The specificity of these events is a result of the intrinsic three-dimensional structure of the molecules. This specificity may be modulated by changes in solvent conditions (e.g. pH, temperature and ionic strength), by ligand binding or by specific changes in covalent structure, for example, phosphorylation or glycosylation, amongst others. We now have at our disposal powerful and well-developed analytical approaches for the determination of covalent structure (e.g. chemical methods and mass spectrometry) and for the determination of three-dimensional structure (e.g. NMR and crystallography).

As scientists, we feel satisfied if we can give a molecular description of basic cellular processes such as specific gene regulation or signal transduction. Complementary to the description of the structures, or the changes in structure, of the relevant biomolecules is a description of the dynamics of their interactions. Which molecules bind to which and with what stoichiometry? How tight is the complex? How fast does it form? What are the discrete steps in the assembly process? These determinations define function on a molecular level and give us the ability to define and test kinetic and thermodynamic models for the assembly process. With the advent of molecular biology we have access to large amounts of practically any protein or macromolecule. Now, with access to a wide range of biophysical methodologies to quantitate discrete molecular

interactions that are robust, accessible and that almost anyone (with proper training, some experience and a little hard work) can use, the ability to construct a true molecular cell biology is a dream that is beginning to come true on a large scale.

These biophysical methodologies have been a mainstay of academic research and are also employed in the drug- discovery departments of major pharmaceutical companies to define the molecular function of biopharmaceutical agents and targets. As a result of the changing policies of drug regulatory agencies, however, their use is going to be extended into drug-development departments, whose responsibilities include the large-scale manufacture of these agents. As a result, methods for quantitating affinity are likely to see even wider application.

The shift in the pharmaceutical industry towards the production of biopharmaceuticals is having an impact on the regulatory agencies that oversee the bringing of these new agents to the market. Historically, biopharmaceuticals were agents like vaccines with no defined chemical com- position. Consequently, it was the process of manufacture that was regulated. Now, the products of biotechnology are macromolecules of a highly resolved composition of matter. As a result, regulatory agencies are beginning to employ practices developed for small-molecule chemical agents. We are beginning to see a shift in regulations from those based on the process of manufacture to those based on the properties of the products of manufacture. This shift has given rise to a new class of pharmaceuticals called ‘well-characterized biopharmaceuticals’. This is more applicable in the US, but Europe and Japan are watching closely.

With regard to these regulatory processes, the application of analytical biotechnology in the pharmaceutical industry is a classical one: to define the identity, purity, quantity and potency of the product. In contrast to identity, purity and quantity, which can now be defined by reliable physical and chemical methods, potency is usually and properly defined biologically using cell-based or animal-based stud- ies. However, this is a time-consuming and costly process. Because affinity is a necessary condition for potency, a first view to potency may be obtained from its measurement. At the moment, the affinity of pharmaceutical agents is often measured using immunochemical (ELISA) approaches. These are familiar, medium-resolution methods. With the increasing need to define the functional properties of pharmaceutical agents with high resolution and at earlier stages in the development process (making sure that these macromolecules are ‘well-characterized’ and that their

2 Analytical biotechnology

properties do not change with changes in, or location of, the manufacturing process), the accurate determination of affinity is likely to play a more important role.

One focus of this issue of Current Opinion in Biotechnology is to review the major tools for characterizing macromolecular interactions in solution. Because it is important to use these tools together, it is useful to review them together. In the first article, Roepstorff (pp 6-13) reviews advances in mass spectrometry (MS). Although this approach is not an affinity-determining methodology, the precise knowledge of molecular mass and the extent and nature of covalent modification is an absolute prerequisite to accurate affinity measurements. As he points out, the covalent structure of a biological macromolecule can now be determined almost completely using MS approaches. This is as a result of in- strumentation (matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF] and electrospray ionization [ESI]) that has increased in sensitivity to the low attomole level with improved resolution and mass accuracy. MS can now be used for the identification of individual proteins from the cellular proteome. The article includes a useful table summarizing various approaches. He ends with a discussion of methods for the determination of the nature and extent of protein covalent modification and protein folding, as well as methods for characterizing higher order structure in proteins. A useful additional review in the latter area can be found in [l].

Analytical ultracentrifugation was developed in the late 1920s by Svedberg and was commercialized in the early 1950s. Along with light scattering and osmotic pressure, it was one of the early solution mass determining approaches and it remains one of the most practical, thermodynamically rigorous approaches to the character- ization of solution molecular weight and assembly state. In the early 1990s the instrument was reintroduced by Beckman Instruments (Palo Alto, USA) with improved optics (absorption and interference) and computerized data acquisition. In his review, Stafford (pp 14-24) focuses on the most significant analytical advance in the field in the last three decades; namely, the analysis of sedimentation velocity data using whole boundary approaches. These approaches fall into two classes. In the first, extrapolation methods are used to remove the effects of diffusion. These methods have found application in the study of chromatin structure by Hansen’s laboratory [a]. In the second, both the sedimentation and translational diffusion coefficients can be explicitly solved for. These two constants, in combination with the Svedberg equation, allow the determination of molecular mass with an accuracy of a few percent of that determined by mass spectrometry. Stafford presents an clear review of one of these methods, the time-derivative approach, developed in his laboratory. These approaches have the advantage that they can be used to precisely define molecular assembly processes, stoichiometries and/or affinities, depending on the strength of the interactions for complex reversibly

interacting and noninteracting systems at a level of complexity not tractable by other solution methods. The future of instrumentation, including fluorescence-based instruments, whose feasibility has been demonstrated by Schmidt and Riesner [3] and TM Laue (personal communication), is also discussed.

Murphy (pp 25-30) then discusses a second classical approach, static and dynamic light scattering (LS). After a useful review of the basic principles of dynamic and static light scattering, she points out the complementarity of LS and other methods in the analysis of some complex systems (see, for example, [4]). She then reviews applications of LS in the analysis of protein-protein inter- actions, polysaccharide gelation, and the characterization of lipid vesicle size. The kinetics of macromolecular interactions may be studied by LS and this approach may be especially useful for kinetic processes occurring over long time periods. A study by Shen and Murphy [5] nicely demonstrates this approach in an analysis of the self-assembly of B-amyloid peptide. With the availability of commercial, computerized LS instruments, this approach should experience renewed interest and wide application.

Isothermal titration calorimetry (ITC) is another approach that is beginning to have wide application in the biotechnology industry by virtue of its ease of use, its wide applicability and its high accuracy and precision. Historically the tool of the academic, it is rapidly becoming one of the tools of choice in the pharmaceutical industry for the characterization of macromolecule-macromolecule and macromolecule-small molecule interactions. In his re- view, Doyle (pp 31-35) cites a wide variety of biologically relevant molecular interactions that have been studied by ITC. As with most of the methods discussed here, this is due to the availability of high-sensitivity computerized instrumentation. He reviews the determination of affinity by direct and indirect methods and the determination of stoichiometry. One of the most interesting new applications of ITC is in the interpretation of heat capacity changes in terms of the determination of the extent of buried surface area upon macromolecular association [6], and in interpreting the origins of entropy changes [7]. Both approaches may give insight into conformational changes that result from macromolecular association. The demonstration of these changes can be made by few other solution methods and, depending on the system, such conformational changes may have significant biological relevance. The study of Johanson et al. [8] is an illustrative example of these approaches.

A class of macromolecular interactions that lends itself to a particularly powerful binding analysis approach is protein-nucleic acid interactions. The technique is called quantitative nucleic acids footprinting, and the approach was developed by Shea and Ackers in the mid 1980s [9-l l] and now is finding wide application. The approach takes

Editorial overview Hensley and Smith 3

advantage of the fact that when proteins (or drugs) are bound to nucleic acids, the binding site is resistant to nuclease cleavage. Because this is the case, the binding of ligands to unique sites can be quantitated, making it possible to resolve intrinsic and potential cooperative Gibbs free energy changes. This is in contrast to more usual thermodynamic analyses where only the moles of ligand bound to moles of receptor are known. Petri and Brenowitz (pp 36-44) present a concise introduction to the methodology and cite applications of the approach to a number of DNA-binding proteins both cooperative and noncooperative in prokaryotic and eukaryotic systems. Exciting new applications of the approach are its use to visualize sequence-dependent structural changes in DNA upon protein binding (significantly extended by the use of chemical nucleases), RNA footprinting and kinetic analysis of footprinting. The latter discussion includes applications from the authors’ laboratory using synchrotron radiation to generate the cleavage agents allowing the analysis of events on the millisecond timescale with single-base resolution.

Of the non-NMR spectroscopic methods for the charac- terization of macromolecular interactions, fluorescence is one of the richest in terms of the number of different approaches that are available, its sensitivity to solvent polarity and the fact that complementary steady-state and time-resolved techniques exist. In their review, Brown and Royer (pp 4549) also note that fluorescence studies may be carried out at subnanomolar concentrations, making true equilibrium affinity measurements possible. Studies with intrinsic and extrinsic fluorophores give access to a wide range of protein-small molecule, protein-protein and protein-nucleic acid interactions. A good example is work from Millar’s group [l&13], who have used fluorescence studies to dissect the proofreading function of the Klenow fragment of DNA polymerase I. Protein-membrane and even lipid-lipid interactions yield to fluorescence analysis. An intriguing study is that of Soekarjo et nl. [14] who were the first to be able to determine the free energy of insertion of a protein into a membrane.

Surface plasmon resonance (SPR), like ITC, has had a major impact on the quantitative characterization of macromolecular interactions in solution. The first com- mercial SPR instruments were introduced in 1990 and since then there has been a wealth of applications. The instrument is robust and, together with the analytical package provided, has evolved significantly since its introduction with the result that equilibrium dissociation constants in the range lOA-lo-1zM and rate constants that range from near 108 s-1 to near 104 s-1 may be confidently determined. In his review, Myszka (pp 50-57) develops the thesis that high-precision measurements require two important considerations. The first is careful attention to experimental design. In this, the operation of the instrument in a dual-beam mode is critical for the reduction of systematic error, fast flow rates and low

surface densities to reduce or eliminate the mass transport problem, and the specific attachment of the ligand to the SPR surface all combine to maximize the signal to noise ratio. The second critical point is to acquire and analyze data under a variety of conditions (e.g. surface densities and analyte concentrations). In an important paper, Morton et nl. [15] demonstrated the power of the global analysis of data in discriminating between models that could not be differentiated using single data sets. A recent application of all of these principles is a study from Ciardelli’s group [ 161, who were able to able to characterize the binding of interleukin (IL)-2 to a complex of the receptor a and B subunits, each separately attached to the SPR surface. Here again, the global analysis of data allowed the discrimination of models in which the c@ complex was preformed from one where IL-Z binding promoted receptor subunit association.

With the exception of the review just discussed, the reviews mentioned above have concerned themselves primarily with macromolecular interactions in solution. However, arguably much of interesting biology occurs on membrane surfaces. Until recently, however, biophysical approaches to the study of molecular interactions on mem- branes have been less well developed. A new and powerful approach for this area, total internal reflection fluorescence (TIRF), is reviewed here by Thompson and Lagerholm (pp 58-64). TIRF and its various hyphenated extensions can bring us a wealth of information about a wide variety of membrane-associated phenomena. These include the kinetics and thermodynamics of protein binding to mem- branes (TIRF and total internal reflection fluorescence photobleaching recovery [TIR-FPR]), the translational diffusion of membrane-bound proteins (total internal re- flection with fluorescence pattern photobleaching recovery [TIR-FPPR]), the effects of flow on protein-membrane interactions (TIRF), the molecular orientation of lipids in membranes, receptor clustering, analysis of cell-substrate contact regions, time-resolved fluorescence studies in membranes (TIRF and PREWIF), studies of liquid-liquid interfaces and spectroelectrochemical measurements. A particularly dramatic example is in the area of low background TIRF for the imaging of single fluorescent molecules. With this approach, Funatsu and colleagues [17] were able to visualize the hydrolysis of single ATP molecules by fluorescently labeled myosin molecules, an impressive achievement.

In the next review we are reminded of a very important point. Although most of the techniques discussed above allow the kinetics and thermodynamics of macromolecular interactions to be viewed at high resolution and with precision and accuracy, they are done for the most part under very dilute conditions with respect to the concentrations of macromolecules. This point is usually cited as a measure of the robustness of the approach. Physiological buffers are often used, but this usually refers to ionic strength and pH. What are missing, to

4 Analytical biotechnology

be really relevant to biology, are the high concentrations of other proteins. As Minton (pp 65-69) points out, biological reactions and interactions occur at protein concentrations in the range of a hundred to many hundreds of milligrams per milliliter. The concentration of protein inside a mitochondrion is so high (400-500 mgml-1) that the concept of concentration may have no clear meaning. Under these conditions, there may, in fact, be no free water. The resulting excluded volume effect has two significant consequences. First, the kinetics and thermodynamics of macromolecular interactions may be significantly affected. Association rates are increased and thermodynamically favorable interactions become more favorable. Second, there are a host of interactions with equilibrium dissociation constants in the range lOA-10-Z M which, under excluded volume conditions, are likely to play important roles in specific biologically relevant interactions. In his review, Minton discusses the effects of excluded volume on protein-protein interac- tions, protein-DNA interactions, cytoskeletal structure and assembly and diffusional transport.

In summary, with the new focus on the quantitative description of molecular interactions, it is time to reaquaint ourselves with the classical approaches to measuring affinity, now updated with modern optics, solid state electronics and, thankfully, computer interfacing, and exciting newly developed approaches. In the process, we should also remind ourselves that these approaches rarely work well alone. To describe a molecular interaction, we must first define the assembly process. What is the aggregation state of reactants and products? What is the stoichiometry of reactants in the complex? What are the kinetics of assembly? What are the thermodynamics of assembly? No one approach can answer all these questions. Most methods are complementary and view different aspects of the assembly process. Where they view the same aspect, they can act as powerful controls for each other [18].

New frontiers in the analysis of nucleic acids We are in the midst of a continuing revolution expanding the power and versatility of nucleic acid analytical techniques. This era was ushered in during the 1970s with the invention of molecular cloning and recombinant DNA, and was bolstered by the parallel emergence of restriction enzymes, DNA sequencing methods and hybridization techniques as critical analytical tools. In the 1980s the development of PCR and automated DNA sequencing further expanded the arsenal of tools available to molecular biologists, and saw the new molecular techniques begin to penetrate into an unprecedented breadth of formerly disparate fields such as anthropology, forensics and epidemiology. Now, in the 1990s the Human Genome Project is in full swing as the first serious efforts at large-scale DNA sequencing in humans begin, and a new

generation of previously unimagined analytical techniques has appeared on the scene.

In this issue of Current Opinion in Biotechnology, several of these new breakthrough technologies are presented. Schwartz and Samad (pp 70-74) describe optical mapping, a new technique for the rapid generation of DNA restriction maps at the single molecule level. The review by Szybalski (pp 75-81) describes an important synergistic technology, RecA-AC or RARE, which permits restriction enzyme cleavage to be targeted to any single desired site within a large target DNA molecule of interest.

A core technology that permeates all of molecular biology is electrophoresis. Yager, Dunn and Stevens (pp 107-113) provide an overview of bioseparations by electrophoresis, and are followed by Quesada (pp 82-93) who describes an important breakthrough in this area, the development of liquid-like entangled polymer networks as sieving media. These media have the potential to obviate the presently ubiquitous agarose and cross-linked polyacrylamide, elim- inating the laborious manual preparation of gels that has characterized molecular biology for decades, essentially making the use of gel media ‘transparent’ to the user.

Fluorescence detection has evolved into the standard ap- proach for the detection of nucleic acids in electrophoresis; in their article, Glazer and Mathies (pp 94-102) describe the development of a new generation of ‘designer’ fluorescent dyes that use elegant chemical principles to greatly extend their sensitivity and specificity.

Finally, Gibbons, Amos and Hodgson (pp 103-106) pro- vide a peek into a possible future, exploring whether the massive combinatorial complexity possible with nucleic acids (for example, the set of all possible lOO-mers of DNA consists of 1060 distinct molecules) could serve as the basis for the next generation of supercomputer.

Together, these reviews illustrate both the tremendous breadth and power of modern nucleic acid technologies as they penetrate into ever-new areas of science, and the dynamism and vigor of the scientific enterprise, which seems able to produce unforeseen and unimagined new breakthroughs with regularity. It is a pleasure to be a scientist in these exciting times, and it is our hope that in this issue some of this is conveyed to the reader.

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