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BIAcore: a microchip-based system for analyzing the formation of macromolecular complexes

1995; Elsevier BV; Volume: 3; Issue: 4 Linguagem: Inglês

10.1016/s0969-2126(01)00164-2

1878-4186

Malini Raghavan, Pamela J. Björkman,

Microfluidic and Capillary Electrophoresis Applications

The formation of macromolecular complexes, for example the complex between an antibody and an antigen, is essential to many biological processes. In order to gain a better insight into the biological processes involved, the formation of complexes is often measured and their interaction kinetics and affinities analyzed. Considerable effort has been expended in developing techniques to measure the formation of complexes, the most promising being electronic microchip-based biosensors. Such analytical systems have come into increasing use recently for the characterization of interactions between biological macromolecules and ligands. One such commercially available system, called BIAcore (Pharmacia Biosensor, Uppsala, Sweden), uses biosensor-based technology and offers a method for rapid analysis of interaction kinetics and affinities [1Fägerstam L.G. Frostell-Karlsson A. Karlsson R. Persson B. Ronnberg I. Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.J. Chromatogr. 1992; 597 (92388323): 397-410Crossref PubMed Scopus (425) Google Scholar, 2Malmqvist M. Biospecific interaction analysis using biosensor technology.Nature. 1993; 361 (93133326): 186-187Crossref PubMed Scopus (525) Google Scholar, 3Jönsson U. Malmqvist M. Real time biospecific interaction analysis: the integration of surface plasmon resonance detection, general biospecific interface chemistry, and microfluidics into one analytical system.in: Turner A Advances in Biosensors. JAI Press, London1992: 291-336Google Scholar]. Such systems have the advantage of replacing tedious binding assays utilizing radiolabelled or fluorescent components and potentially unstable cell lines, and can be used where biological activity assays are not available. The BIAcore system contains a sensor microchip, a laser light source emitting polarized light, an automated fluid-handling system [[4]Sjölander S. Urbaniczky C. Integrated fluid handling system for biomolecular interaction analysis.Anal. Chem. 1991; 63 (92102036): 2338-2345Crossref PubMed Scopus (235) Google Scholar], and a diode-array position-sensitive detector (Figure 1). The sensor chip consists of a glass support, an overlaid gold film and a carboxymethylated dextran matrix [[5]Löfås S. Jönsson B. A novel hydrogel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilization of ligands.J. Chem. Soc. Chem. Commun. 1990; 21: 1526-1528Crossref Google Scholar] to which biomolecules can be coupled (Figure 1). To study interactions between biomolecules, this system uses a surface plasmon resonance (SPR)-based assay, an optical technique that measures changes in the refractive index at the sensor chip surface. When plane-polarized light is incident on the sensor chip at an angle greater than a defined critical angle, and the intensity of the reflected light is monitored using a position-sensitive detector, SPR is observed as a decrease in light intensity for a specific angle of incidence (the SPR angle) [1Fägerstam L.G. Frostell-Karlsson A. Karlsson R. Persson B. Ronnberg I. Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.J. Chromatogr. 1992; 597 (92388323): 397-410Crossref PubMed Scopus (425) Google Scholar, 2Malmqvist M. Biospecific interaction analysis using biosensor technology.Nature. 1993; 361 (93133326): 186-187Crossref PubMed Scopus (525) Google Scholar]. The angle at which the decrease in light intensity occurs is proportional to the refractive index at the sensor chip; this is in turn proportional to surface mass changes. To study interactions between two molecules, one of the molecules is covalently immobilized on the sensor chip and the second molecule is injected over the chip surface. Interactions between the two molecules result in mass changes at the chip surface, which translate to refractive index changes and changes in the SPR angle. Changes in SPR angles are monitored as resonance units (RU, where 1000 RU corresponds to a 0.10° change in SPR angle, a 1.0 ng mm-2 change in surface mass, and a bulk refractive index change of 0.001) [[1]Fägerstam L.G. Frostell-Karlsson A. Karlsson R. Persson B. Ronnberg I. Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.J. Chromatogr. 1992; 597 (92388323): 397-410Crossref PubMed Scopus (425) Google Scholar]. RU value shifts may occur either in response to specific binding to the immobilized molecule, or in response to differences in buffer compositions. Nonspecific RU value changes can be distinguished from a specific response by carrying out blank injections, of buffer only or buffer plus a non-specific control protein, over the chip surface. The coupling of biomolecules to the sensor chip surface is accomplished using amine, thiol or aldehyde-based immobilizations [6Jönsson B. Löfås S. Lindqvist G. Immobilization of proteins to a carboxymethyldextran modified gold surface for biospecific interaction analysis in surface plasmon resonance.Anal. Biochem. 1991; 198 (92189154): 268-277Crossref PubMed Scopus (1202) Google Scholar, 7O'Shannessy D.J. Brigham-Burke M. Peck K. Immobilization chemistries suitable for use in the BIAcore surface plasmon resonance detector.Anal. Biochem. 1992; 205 (93072845): 132-136Crossref PubMed Scopus (224) Google Scholar]. When a defined orientation of a protein molecule is required, cysteine residues can be introduced at specific sites in the protein to allow oriented coupling. A knowledge of the isoelectric point (pI) of the sample to be immobilized is useful before attempting coupling to the sensor chip. It is necessary to prepare the sample of macromolecule to be coupled in a buffer with a pH value below the pI, in order to enhance electrostatic interactions with carboxyl groups on the sensor chip. Various parameters in addition to pH can influence protein immobilization levels. These include the ionic strength of the buffer, the protein concentration and the duration of the coupling reaction. For proteins that are refractive to coupling using the methods described above, an alternative immobilization strategy involves biotinylation followed by binding to commercially available streptavidin-coated sensor chips. The data derived from the BIAcore system are in the form of sensorgrams, which are plots of RU versus time. Progress of association and dissociation events between molecules can thus be followed in real time. As an example, Figure 2 shows representative sensorgrams derived for the binding of two concentrations of IgG to a soluble form of the neonatal constant framework of antibody Fc receptor (FcRn) [[8]Gastinel L.N. Simister N.E. Bjorkman P.J. Expression and crystallization of a soluble and functional form of an Fc receptor related to class I histocompatibility molecules.Proc. Natl. Acad. Sci. USA. 1992; 89 (92115716): 638-642Crossref PubMed Scopus (97) Google Scholar], which was immobilized on a sensor chip. The data are shown as sensorgrams resulting from the difference in RU between identical injections onto the flow cells of a sensor chip, to which were coupled either FcRn or buffer. The end of the flow of sample through the cell (association phase) is at about 450 s. The complex dissociates in two steps: the first dissociation step is in a buffer of pH 6.0, and the second dissociation step, initiated at about 950 s, is in buffer of pH 7.5. This experiment illustrates the marked instability of the FcRn-IgG interaction at pH 7.5, a well-known feature of this receptor [[9]Rodewald R. pH-dependent binding of immunoglobulins to intestinal cells of the neonatal rat.J. Cell Biol. 1976; 71: 666-670Crossref PubMed Scopus (206) Google Scholar] that is important physiologically for the efficient unidirectional transport of IgG from the gut (pH 6.0–6.5) into the blood (pH 7.0–7.5). Visual inspection of the sensorgrams illustrates that the dissociation rate of the FcRn-IgG complex is significantly faster at pH 7.5 than at pH 6.0. Estimates of association and dissociation rate constants can be obtained from sensorgrams, using either linearized transformations of primary data [[10]Karlsson R. Michaelsson A. Mattson L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor-based analytical system.J. Immunol. Methods. 1991; 145 (92113306): 229-240Crossref PubMed Scopus (1011) Google Scholar] or non-linear curve-fitting methods [[11]O'Shannessy D.J. Brigham-Burke M. Soneson K.K. Hensley P. Brooks I. Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: use of nonlinear least squares analysis methods.Anal. Biochem. 1993; 212 (94027939): 457-468Crossref PubMed Scopus (519) Google Scholar]. Use of the recently developed BIAevaluation 2.0 software package enables rapid non-linear analysis of BIAcore-generated data using the Marquardt-Levenberg curve-fitting routine [[12]Marquardt D.W. An algorithm for least squares estimation of non-linear parameters.J. Soc. Ind. Appl. Math. 1963; 11: 431-441Crossref Google Scholar]. Affinity constants from experiments in which the interaction reaches equilibrium during the sample injection can also be determined by a method similar to Scatchard analysis (see BIAcore methods manual or [[13]House-Pompeo K. Boles J.O. Höök M. Characterization of bacterial adhesin interactions with extracellular matrix components using biosensor technology.in: Methods: A Companion to Methods in Enzymology vol 6. Academic Press, Orlando, USA1994: 134-142Google Scholar]). Equilibrium analyses are especially useful when the association and/or dissociation kinetics are too rapid to be measured accurately, as for the dissociation rate of the FcRn-IgG complex at pH 7.5 (Figure 2). A look at the literature reveals diverse uses of BIAcore-biosensor technology, including identification of specific protein-protein interactions in complex systems [14Schuster S.C. Swanson R.V. Alex L.A. Bourret R.B. Simon M.I. Assembly and function of a quarternary signal transduction complex monitored by surface plasmon resonance.Nature. 1993; 365 (93390615): 343-347Crossref PubMed Scopus (229) Google Scholar, 15Calakos N. Bennett M.K. Peterson K.E. Scheller R.H. Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking.Science. 1994; 263 (94151686): 1146-1149Crossref PubMed Scopus (365) Google Scholar, 16Seth A. Wiley D.C. et al.Binary and ternary complexes between T-cell receptor, class II MHC and superantigen in vitro..Nature. 1994; 369 (94239529): 324-327Crossref PubMed Scopus (157) Google Scholar, 17Matsui K. Boniface J.J. Steffner P. Reay P.A. Davis M.M. Kinetics of T cell receptor binding to peptide-MHC complexes: correlation of the dissociation rate with T cell responsiveness.Proc. Natl. Acad. Sci. USA. 1994; 91 (95108058): 12862-12866Crossref PubMed Scopus (366) Google Scholar], mapping of binding sites [18Patti J.M. Boles J.O. Höök M. Identification and biochemical characterization of the ligand binding domain of the collagen adhesin from Staphylococcus aureus..Biochemistry. 1993; 32 (94032261): 11428-11435Crossref PubMed Scopus (118) Google Scholar, 19Raghavan M. Chen M.Y. Gastinel L.N. Bjorkman P.J. Identification of interaction sites in the class I MHC-related Fc receptor/immunoglobulin G complex.Immunity. 1994; 1: 303-315Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 20Marengere L.E.M. Pawson T. et al.SH2 domain specificity and activity modified by a single residue.Nature. 1994; 369 (94261187): 502-505Crossref PubMed Scopus (161) Google Scholar], identification of ligands for receptors from crude cellular supernatants [[21]Bartley T.D. Fox G.M. et al.B61 is a ligand for the ECK receptor protein-tyrosine kinase.Nature. 1994; 368 (94187881): 558-560Crossref PubMed Scopus (229) Google Scholar], and investigations of kinetic parameters for previously characterized interactions [[22]Ponzetto C. Comoglio P.M. et al.A multifunctional docking site mediates signalling and transformation by the hepatocyte growth factor/scatter factor receptor family.Cell. 1994; 77 (94221643): 261-271Abstract Full Text PDF PubMed Scopus (892) Google Scholar]. From the perspective of a structural biologist, the BIAcore system offers a convenient tool with which to dissect intermolecular interfaces using proteins engineered with site-directed mutations. Cunningham and Wells [[23]Cunningham B.C. Wells J.A. Comparison of a structural and a functional epitope.J. Mol. Biol. 1993; 234 (94076335): 554-563Crossref PubMed Scopus (485) Google Scholar] have carried out a comprehensive analysis of the energetic importance of 31 residues buried at the interface between human growth hormone (hGH) and the extracellular region of its receptor (hGHbp), deduced from the crystal structure of the complex [[24]De Vos A.M. Ultsch M. Kossiakoff A. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex.Science. 1992; 255 (92196577): 306-312Crossref PubMed Scopus (2022) Google Scholar]. Each of the 31 side chains in hGH was changed to alanine and the kinetics and affinities of interaction with hGHbp were measured using BIAcore assays. The study revealed that as few as a quarter of the side chains, clustered near the center of the interface, accounted for most of the binding energy [[23]Cunningham B.C. Wells J.A. Comparison of a structural and a functional epitope.J. Mol. Biol. 1993; 234 (94076335): 554-563Crossref PubMed Scopus (485) Google Scholar]. These results, and more recent studies with alanine mutants of hGHbp [[25]Clackson T. Wells J.A. A hot spot of binding energy in a hormone-receptor interface.Science. 1995; 267 (95125446): 383-386Crossref PubMed Scopus (1779) Google Scholar], suggest a differentiation between a structural epitope (those residues at the intermolecular interface determined from a structural analysis) and a functional epitope (those contact residues that provide the majority of the stabilization energy for the complex). The conclusion from the structural and functional analysis of the hGH-hGHbp complex was that only a small and complementary subset of the total contact residues was required for maintaining binding affinity [[25]Clackson T. Wells J.A. A hot spot of binding energy in a hormone-receptor interface.Science. 1995; 267 (95125446): 383-386Crossref PubMed Scopus (1779) Google Scholar]. Further studies with other systems should verify the generality of these conclusions, but the authors [[25]Clackson T. Wells J.A. A hot spot of binding energy in a hormone-receptor interface.Science. 1995; 267 (95125446): 383-386Crossref PubMed Scopus (1779) Google Scholar] suggest that these results may also be characteristic of other protein-protein interfaces. Crystal structure characterization can reveal higher-order structures that may turn out to be either functionally important, or an artifact of crystallization conditions. Further experiments using BIAcore technology can be designed to test the relevance of such structures. We have tested the biological relevance of an Fc receptor dimer that was crystallographically observed [[26]Burmeister W.P. Gastinel L.N. Simister N.E. Blum M.L. Bjorkman P.J. The 2.2 å crystal structure of the MHC-related neonatal Fc receptor.Nature. 1994; 372: 336-343Crossref PubMed Scopus (275) Google Scholar], by designing a receptor whose ability to dimerize was impaired when coupled via a cysteine residue that was introduced at the dimer interface. Ligand binding by such a dimerization-impaired receptor was compared with ligand binding by a dimerization-competent receptor. It was deduced that the crystallographically observed Fc receptor dimer was important for high-affinity ligand binding (M Raghavan, YP Wang and PJ Bjorkman, unpublished data). In combination with recombinant DNA technology for generating mutant proteins, biosensor-based functional assays are becoming essential tools in the structural biology laboratory. BIAcore is one such system that can be used conveniently to test structural predictions of possible contact sites, to examine contributions of individual residues at intermolecular interfaces to the overall binding energy, and to demonstrate the validity of proposed functional models. The diversity of applications of such systems will no doubt continue to expand in the coming months. We thank Shirley Demer of Pharmacia Biosensor for technical advice and critical reading of this manuscript. Some of the work described here was supported by the Howard Hughes Medical Institute (PJB) and by a post-doctoral fellowship from the Cancer Research Institute (MR). Malini Raghavan, Division of Biology 156–29, Pasadena. Pamela J Bjorkman, Division of Biology 156–29, Pasadena and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA.