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Cadherins are transmembrane proteins responsible for calcium dependent cell-cell adhesion. Classical cadherins have a common structure with five extracellular domains, connected by a 7-residue sequence linker region. Cadherins mediate adhesion via Adherens Junctions by forming a strand-swapped structure between identical protomers from apposing cells. The adhesive interface has been characterized structurally, and biophysical studies have been used to elucidate the forces that stabilize the strand-swapped structure. Upon calcium binding, the swapping of the N-terminal strands between protomers allows for the symmetrical docking of a tryptophan residue required for adhesion. Fundamental questions still remain regarding the striking difference in the calcium-dependent kinetics of dimerization between N- and E-cadherin. This study compares the first extracellular domain of N-cadherin (NCAD1) and E-cadherin (ECAD1). With NCAD1, the binding pocket for calcium is not complete, so dimerization was not expected. However, we observed a dimeric form that was not in exchange with monomer. This dimer was reversibly converted to monomer by heating the protein. Noticeably, NCAD1 was found to unfold at an uncharacteristically high temperature. Results for NCAD1 construct were similar to results for domain 1 in studies of the two-domain construct, NCAD12. Similar studies of ECAD1 show that it is significantly lower stability. In summary, our results indicate that the difference in kinetics of dimerization may be due to a difference in the intrinsic stability of domain 1.GAANN P200A120046

Developing new quantitative methods for studying the interactions of cell surface proteins is imperative to advance understanding of the molecular determinants of cell adhesion. Our interest in the dimerization kinetics of cadherin, a calcium-dependent cell-cell adhesion molecule, has motivated us to develop a quantitative bead aggregation assay to study the effects of calcium concentration on the kinetics of association. Although bead aggregation has been used for some time to study protein-protein interactions, the data are usually only interpreted qualitatively. Here we establish a quantitative method for studying the aggregation of beads that are coated with neural-cadherin. Data indicated exponential growth in aggregate size as a function of time. Time constants of association ranged from 18 to 3 minutes over a 100 fold increase in calcium concentration. We used this method to study the slow association kinetics of a mutant cadherin (NCAD1/R14E). These studies yielded a time constant of 28 minutes, which indicates that the association rate of mutant is slower than wild type (9 minutes). Experimental factors were controlled to ensure precision in the data including protein binding to beads, incubation time in the presence of calcium, mixing of the beads prior to placing on the slide and the residence time on the slide before data were acquired. This method is the first report of experimental determination of the kinetics of the association of proteins tethered to a surface, and is broadly applicable to study the kinetics of protein-protein interactions.

The two most obvious differences between alkyl-bonded silica phases and polystyrene-divinylbenzene resins as reversed-phase chromatographic supports are the aromaticity and the lack of hydrogen bonding ability in the polymeric resin. The effect of these differences on the selectivity for a set of small solutes was studied through the use of a solvatochromic comparison method. For retention on a polymeric phase for mobile phases with the modifiers methanol and acetonitrile, the linear solvation energy relationship indicated an increased dependence on the polarizability/dipolarity of solutes. For the modifier tetrahydrofuran, retention on the polystyrene-divinylbenzen resin was indistinguishable from that on the alkyl-bonded silica phase. The hydrogen bonding ability of a solute was found to play a greater role in retention on alkyl-bonded silica than on the polymeric resin for all three modifiers. Since the mobile phase compositions were chosen such that the Hildebrand solubility parameters were equal, the dependence of retention on molar volume was found to be the same for all mobile phase-stationary phase combinations examined.

Cadherins are the primary transmembrane component in adherens junctions, structures that link the actin cytoskeleton in adjacent cells within tissue. Adherens junctions occur in most solid tissues including neurological synapses, epithelium and endothelium. Cell-cell adhesion by cadherins requires the binding of calcium ions to specific sites in the extracellular domain. Requirement of calcium binding beckons the question of whether other divalent cations might substitute for or compete with calcium for site occupancy. The studies reported herein characterize the biophysics of nickel binding to neural cadherin. Based on results from thermal-denaturation studies, it was clear that neural cadherin was stabilized by the presence of nickel in solution. Subsequent experiments tested whether the presence of constant low levels of nickel affected calcium binding affinity and calcium-induced dimerization. Interestingly, there was a significant effect upon the apparent calcium binding affinity and on adhesive dimer formation as a function of nickel concentration, suggesting that detrimental competition for site occupancy occurs between these divalent cations.

Cadherins are calcium dependent homophilic cell adhesive protein molecules that are critical for morphogenesis, synaptogenesis and synapse maintenance. Cadherins comprise an extracellular region, a single transmembrane region and a cytoplasmic region. The extracellular region has five tandemly repeated ectodomains (EC1-EC5), with three calcium binding sites situated between each of these domains. Cell-cell adhesion is mediated by the dimerization of cadherins presented on neighboring cell surfaces. We are focusing our studies on two members of type I classical cadherins, Neural (N) - and Epithelial (E)-cadherins. In spite of high sequence similarity between E- and N-cadherin, they have distinct physiological localization implying differences in their adhesive properties. Here, we compare the spectral characteristics, stability, calcium binding and assembly properties of the first two domains of N- and E- cadherins. Spectroscopic studies of these proteins were predictable and indicated typical β-sheet conformation with only partial exposure of tryptophans. Although both proteins are stabilized by calcium, apo-ECAD12 is less stable than apo-NCAD12. Direct calcium titrations that found the proteins bind calcium with equally high affinity (−6.2 kcal/mol). There was a striking difference between these proteins in terms of the kinetics of disassembly. Analytical size exclusion chromatography experiments showed that disassembly of ECAD12 dimers is rapid and disassembly of NCAD12 dimers is slow regardless of the calcium concentration. We observe this striking difference with constructs containing only the first 220 residues of a 700 residue protein. Thus, not only is this an interesting protein folding-function question, this remarkable difference in these cadherins may explain their segregation into different physiological niches.

The retention behavior of a series of non-polar solutes on two polystyrene-divinylbenzene high-performance liquid chromatography supports was studied. The solvent strength of several organic solvents was measured over a range of solvent compositions. Poor peak symmetry was observed for some solvents, and this appears to be correlated with the ability of the solvent to swell the polymer. Addition of a solvent that cna swell the polymer to another solvent which cannot usually improves peak shape. The observed improvement in peak symmetry also correlates with the ability of the ternary mixture to decrease the void volume of the column.