Investigations of ligand-receptor binding between bivalent antibodies and membrane-bound ligands are presented. The purpose of these studies was to explore binding as a function of hapten density in a two-dimensionally fluid environment. A novel microfluidic strategy in conjunction with total internal reflection fluorescence microscopy was designed to achieve this. The method allowed binding curves to be acquired with excellent signal-to-noise ratios while using only minute quantities of protein solution. The specific system investigated was the interaction between anti-DNP antibodies and phospholipid membranes containing DNP-conjugated lipids. Binding curves for ligand densities ranging from 0.1 to 5.0 mol % were obtained. Two individual dissociation constants could be extracted from the data corresponding to the two sequential binding events. The first dissociation constant, K(D1), was 2.46 x 10(-)(5) M, while the second was K(D2) = 1.37 x 10(-)(8) mol/m(2). This corresponded to a positively cooperative binding effect with an entropic difference between the two events of 62.3 +/- 2.7 J/(mol.K). Furthermore, the percentage of monovalently and bivalently bound protein was determined at each ligand density.

We describe a non-invasive technique for determining pH in biomolecular NMR sample using buffer components (formate, tris, piperazine, and imidazole) as internal pH indicators, whose (1)H NMR chemical shifts are sensitive to pH in a range from 2.5 to 9.8. This method is suitable for a wide range of applications where samples are handled intensively during NMR titrations or in high throughput analysis in structural genomics or metabolomics.

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SDF-1a is a member of the chemokine family implicated in various reactions in the immune system. The interaction of SDF-1a with its receptor, CXCR4, is responsible for metastasis of a variety of cancers. SDF-1a is also known to play a role in HIV-1 pathogenesis. The structures of SDF-1a determined by NMR spectroscopy have been shown to be monomeric while X-ray structures are dimeric. Biochemical data and in vivo studies suggest that dimerization is likely to be important for the function of chemokines. We report here the dynamics of SDF-1a determined through measurement of main chain 15 N NMR relaxation data. The data were obtained at several concentrations of SDF-1a and used to determine a dimerization constant of ;5 mM for a monomer-dimer equilibrium. The dimerization constant was subsequently used to extrapolate values for the relaxation data corresponding to monomeric SDF-1a. The experimental relaxation data and the extrapolated data for monomeric SDF1a were analyzed using the model free approach. The model free analysis indicated that SDF-1a is rigid on the nano-to picosecond timescale with flexible termini. Several residues involved in the dimer interface display slow micro-to millisecond timescale motions attributable to chemical exchange such as monomer-dimer equilibrium. NMR relaxation measurements are shown to be applicable for studying oligomerization processes such as the dimerization of SDF-1a.

The interaction of Cardiac Troponin C (cTnC) and Cardiac Troponin I (cTnI) plays a critical role in transmitting the Ca2+ signal to the other myofilament proteins in the activation of cardiac muscle contraction. As such, the cTnC−cTnI interface is a logical target for cardiotonic agents such as levosimendan that can modulate the Ca2+ sensitivity of the myofilaments. Evidence indicates that drug candidates may exert their effects by targeting a site formed by binding of the switch region of cTnI to the regulatory N domain of cTnC (cNTnC). In this study, we utilized two-dimensional 1H−15N HSQC NMR spectroscopy to monitor the binding of levosimendan and its analogues, CMDP, AMDP, CI-930, imazodan, and MPDP, to cNTnC·Ca2+ in complex with two versions of the switch region of cTnI (cTnI147−163 and cTnI144−163). Levosimendan, CMDP, AMDP, and CI-930 were found to bind to both cNTnC·Ca2+·cTnI147−163 and cNTnC·Ca2+·cTnI144−163 complexes. These compounds contain a methyl group that is absent in MPDP or imazodan. Thus, the methyl group is one of the pharmacophores responsible for the action of these pyridazinone drugs on cTnC. Furthermore, the results showed that the cNTnC·Ca2+·cTnI144−163 complex presents a higher-affinity binding site for these compounds than the cNTnC·Ca2+·cTnI147−163 complex. This is consistent with our observation that the affinity of cTnI144−163 for cNTnC·Ca2+ is ∼10-fold stronger than that of cTnI147−163, likely a result of electrostatic forces between the N-terminal RRV extension in cTnI144−163 and the acidic residues in the C and D helices of cNTnC. These results will help in the delineation of the mode of action of levosimendan on the important functional unit of cardiac troponin that constitutes the regulatory domain of cTnC and the switch region of cTnI.