Philip Gribbon, Boon C. Heng, and Timothy E. Hardingham 1. Introduction
Extracellular matrices (ECMs) contain a mixture of fibrillar and nonfibrillar mac-romolecular components, which interact through a range of covalent and noncovalent associations to form a composite structure (1-3). It is the ECM that defines the architecture, the form, and the biomechanical properties of many tissues. In order to perform their functional roles, many of the important noncollagenous components of extracellular matrices are required to be immobilized or focally located within tissues (4,5). Positioning of macromolecules within tissues occurs as a consequence of specific protein-protein and protein-carbohydrate interactions. Aggrecan, the large aggregating proteoglycan, is noncovalently associated with hyaluronan via its N-ter-minal domain and this association is further stabilised by link protein (6). A large immobilized hydrated aggregate structure is formed, with up to 50 aggrecan molecules per hyaluronan chain, which gives articular cartilage the ability to resist the high compressive loads generated during joint articulation. Other examples of specific intermolecular associations include the binding of cell surface integrins to fibronectins and collagenous proteins (7) and growth factors to heparan sulphate (S). A variety of methods have been developed to investigate the affinity and specificity of these associations and their sensitivity to environmental factors such as pH and metal ion concentration. Many techniques for analyzing binding equilibria or kinetics require either the ligand or substrate to be linked to a solid support. As a consequence of derivitization and high local concentrations, binding equilibria can be significantly perturbed.
Confocal fluorescence recovery after photobleaching (confocal FRAP) offers a method for investigating the interactions of molecules at high concentrations, the formation of molecular networks, and the permeability of such networks to "reporter"
From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ
molecules of different sizes. Additionally, confocal FRAP can be used to quantify protein-protein binding and protein-carbohydrate binding under true equilibrium conditions with a minimum of chemical modifications (9-13).
Experimentally, confocal FRAP involves viewing a solution of a fluorescently labeled molecule with a confocal microscope, locally creating a bleached area in the solution, followed by observing the subsequent redistribution of fluorescence (see Notes 1 and 2). The bleaching can be achieved rapidly by brief high-power laser illumination. In simple solutions of monodisperse macromolecules, the measurement of the recovery of fluorescence yields a long time translational lateral self-diffusion coefficient. This describes the movement of the macromolecule through a matrix of similar macromolecules and can be used to investigate self-association and molecule entanglement at high concentration (11). The lateral tracer diffusion coefficient describes the movement of a macromolecule within a matrix composed of a high concentration of another macromolecule. Self and tracer diffusion coefficients are a function of the hydrodynamic friction caused by the solvent and the hindrances, steric or otherwise, offered by other components in the solution. If a macromolecule associates specifically with one or more components in a matrix, and its diffusion coefficient is sufficiently changed in the process, then confocal FRAP can be used to determine the affinity and specificity of the interaction (13).
The application of confocal FRAP to characterize the specific binding of a low-molecular-weight protein to a high-molecular-weight ligand will be discussed in detail here. The movement of a fluorescently labeled macromolecule A, diffusing within a matrix of an unlabeled macromolecule B, will be considered. The transla-tional diffusion coefficients of A, B, and the complex AB are DA, DB and DAB, respectively. The binding equilibrium between A and B is defined as
and at equilibrium, the dissociation constant (Kd) is given by:
where [ ] represents concentration. In a confocal FRAP experiment only the fluorescence recovery of component A is measured. At equilibrium, A exchanges rapidly between bound and free states that have characteristic lifetimes of xbound and Tfree respectively. If the fluorescence recovery of A is observed over time t, where t >> (Tbound + Tfree), then the redistribution of A results from diffusion during multiple free and bound phases. The measured diffusion coefficient (Dm) can then be defined as
where (Tfree) is the fraction of time t that macromolecule A was free during time t. Since t >> (tbound + tfree), then (Tfree) — Afree, the fraction of free A at equilibrium. Similarly, if DA >> DB, then DB — DAB and Eq. 3 can be re-expressed as feD (4)
The expression for the inverse Scatchard plot is then
where [B]bs is the concentration of binding sites on B, and n is the number of molecules of A bound per molecule of B. Values for Kd are determined by measuring Dm as a function of the concentration of substrate B. Magnitudes of DA and DB are determined independently.
Confocal FRAP can be used to characterise competitive binding. Consider an equilibrium between A and B, in the presence of a competitor, C:
This equilibrium can be analyzed by confocal FRAP if (1) the translational diffusion coefficient of C, (Dc), is very different from that of B, and (2) binding between B and C is minimal, that is, [BC] _ 0.
Confocal FRAP has recently been used to analyze the association between the G1 domain of aggrecan and hyaluronan. This interaction is ideally suited to analysis by this technique because (1) the interaction is noncovalent, (2) the diffusion coefficients of G1 and HA differ considerably, and (3) the diffusion coefficient of the G1-HA complex is similar to that of hyaluronan (HA) (14). The G1 domain of aggrecan was labeled with fluorescein isothiocyanate (FITC) at 2.5 mol FITC per mole of G1 (see Subheading 3.1.1.) and its diffusion coefficient in increasing concentrations of hyaluronan (800 kDa) at pH 7.4 was determined (see Fig. 1). The diffusion coefficient of the FITC-G1 was reduced with increasing hyaluronan concentration. Tracer diffusion studies on nonbinding FITC-G1, which had been reduced and alkylated, showed the reduction in FITC-G1 mobility was not due to the steric effect of the hyaluronan (14). The self-diffusion of fluoresceinamine (FA)-labeled hyaluronan (800 kDa) was then determined independently by conventional confocal FRAP. An inverse Scatchard plot was constructed from the FITC-Gl-binding isotherm (see Fig. 2). This gave Ku as 4 x 10-8 M, and n _ 1, (see Fig. 2) which agrees well with dissociation constants determined by solid-phase-type assays (15,16).
The confocal FRAP technique was used to determine the minimum length of hyaluronan oligosaccharide required for binding to a single G1 domain. The low molecular weight of the competitor oligosaccharides compared to the FITC-G1 and the high-molecular-weight hyaluronan makes this an ideal system for analysis by confocal FRAP. A mixture of FITC-G1 (5.5 ^M) and 800-kDa hyaluronan (12.5 mM of HA10 binding sites) was prepared and >99% of the FITC-G1 was found to be bound to the hyaluronan. Hyaluronan oligosaccharides (21 mM) of length 4-14 monosaccharides were added (17), and the diffusion coefficient of the FITC-G1 was determined (see Fig. 3). It can be seen that hyaluronan oligosaccharides compete effectively with full-length hyaluronan and cause an increase in the diffusion coefficient of FITC-G1. The method shows that a minimum length of 10 hyaluronan monosaccharides is needed for maximal binding to FITC-G1 (15).
The confocal FRAP technique is a novel and direct method for investigating the many protein-protein and protein-carbohydrate interactions that contribute greatly to the organization and function of extracellular matrices. It can be applied over a
wide range of conditions, including high concentrations, and in the presence of multiple interacting components. Confocal FRAP also has the major advantage that interactions are analyzed under near-equilibrium conditions, which allows the analysis of the weak interactions that are potentially important in defining matrix properties.
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