Equilibrium And Dynamic Design Principles For Binding Molecules Engineered For Reagentless Biosensors
ABSTRACT Reagentless biosensors rely on the interaction of a binding partner and its target to generate a change in fluorescent signal using an environment sensitive fluorophore or Förster Resonance Energy Transfer. Binding affinity can exert a significant influence on both the equilibrium and the dynamic response characteristics of such a biosensor. We here develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. These concepts are broadly relevant to reagentless biosensor modalities.
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ABSTRACT: Barnase, an extracellular ribonuclease of Bacillus amyloliquefaciens, forms a very tight complex with its intracellular polypeptide inhibitor barstar. At pH 8, the values for the rate constants k1 (association) and k-1 (dissociation) are 6.0 x 10(8) s-1 M-1 and 8.0 x 10(-6) s-1, respectively. The value of Ki, the dissociation constant of barstar and barnase, calculated from the ratio k-1/k1 is 1.3 x 10(-14) M, which corresponds to a delta G of -18.9 kcal/mol at 25 degrees C. The dissociation constant increases with decreasing pH according to the ionization of an acid in free barnase of pKa 6.4, with very weak, if any, binding to the protonated form. This pH dependence for dissociation of the complex can be attributed almost entirely to residue His102 in barnase, as determined by a His102-->Ala mutation. Analysis of the pH dependence of the kinetic constants indicates that binding is, at least, a two-step process. The first, and rate-determining, step is association at close to the diffusion-controlled rate. There is then the precise docking of the complex. The value of Ki increases to 2.4 x 10(-11) M in the presence of 500 mM NaCl, and to 1.6 x 10(-11) M at pH 5 (100 mM NaCl). The binding site of barstar on barnase was mapped by measuring the values of Ki for a broad range of site-specific mutants of barnase. Mutagenesis of residues Lys27, Arg59, Arg87, and His102 to Ala increases the values of Ki by a factor of 10(4).(ABSTRACT TRUNCATED AT 250 WORDS)Biochemistry 05/1993; 32(19):5145-50. DOI:10.1021/bi00070a025 · 3.01 Impact Factor
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ABSTRACT: To explore the role that surface and active center charges play in electrostatic attraction of ligands to the active center gorge of acetylcholinesterase (AChE), and the influence of charge on the reactive orientation of the ligand, we have studied the kinetics of association of cationic and neutral ligands with the active center and peripheral site of AChE. Electrostatic influences were reduced by sequential mutations of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three residues within the active center gorge (Asp-74 at the rim and Glu-202 and Glu-450 at the base). The peripheral site ligand, fasciculin 2 (FAS2), a peptide of 6.5 kDa with a net charge of +4, shows a marked enhancement of rate of association with reduction in ionic strength, and this ionic strength dependence can be markedly reduced by progressive neutralization of surface and active center gorge anionic residues. By contrast, neutralization of surface residues only has a modest influence on the rate of cationic m-trimethylammoniotrifluoroacetophenone (TFK+) association with the active serine, whereas neutralization of residues in the active center gorge has a marked influence on the rate but with little change in the ionic strength dependence. Brownian dynamics calculations for approach of a small cationic ligand to the entrance of the gorge show the influence of individual charges to be in quantitative accord with that found for the surface residues. Anionic residues in the gorge may help to orient the ligand for reaction or to trap the ligand. Bound FAS2 on AChE not only reduces the rate of TFK+ reaction with the active center but inverts the ionic strength dependence for the cationic TFK+ association with AChE. Hence it appears that TFK+ must traverse an electrostatic barrier at the gorge entry imparted by the bound FAS2 with its net charge of +4.Journal of Biological Chemistry 10/1997; 272(37):23265-77. DOI:10.1074/jbc.272.37.23265 · 4.57 Impact Factor
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ABSTRACT: The possibility of obtaining from any antibody a fluorescent conjugate which responds to the binding of the antigen by a variation of its fluorescence, would be of great interest in the analytical sciences and for the construction of protein chips. This possibility was explored with antibody mAbD1.3 directed against hen egg white lysozyme. Rules of design were developed to identify the residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. These rules were based on: the target residue belonging to a topological neighbourhood of the antigen in the structure of the complex between antibody and antigen; its absence of functional importance for the interaction with the antigen; and its solvent accessibility in the structure of the free antibody. Seventeen conjugates between the single-chain variable fragment scFv of mAbD1.3 and an environment-sensitive fluorophore were constructed. For six of the ten residues which fully satisfied the design rules, the relative variation of the fluorescence intensity between the free and bound states of the conjugate was comprised between 12 and 75% (in non-optimal buffer), and the affinity of the conjugate for lysozyme remained unchanged relative to the parental scFv. In contrast, such results were true for only one of the seven residues which failed to satisfy one of the rules and were used as controls. One of the conjugates was studied in more detail. Its fluorescence increased proportionally to the concentration of lysozyme in a nanomolar range, up to 90% in a defined buffer, and 40% in serum. This increase was specific for hen egg lysozyme and it was not observed with a closely related protein, turkey egg lysozyme. The residues which gave operational conjugates (six in V(L) and one in V(H)), were located in the immediate vicinity of residues which are functionally important, along the sequence of FvD1.3. The results suggest rules of design for constructing antigen-sensitive fluorescent conjugates from any antibody, in the absence of structural data.Journal of Molecular Biology 05/2002; 318(2):429-42. DOI:10.1016/S0022-2836(02)00023-2 · 4.33 Impact Factor