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Cadherins are the transmembrane component in adherens junctions, structures that link the actin cytoskeletons in adjacent cells within 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 region. Given the complexity of the cell adhesion microenvironment, we are investigating whether other divalent cations might affect calcium-dependent dimerization of neural (N) cadherin. The studies reported herein characterize the impact of binding physiological magnesium(ii) or neurotoxic nickel(ii) on calcium-dependent N-cadherin function. Physiological levels of magnesium have only a small effect on the calcium-binding affinity and calcium-induced dimerization of N-cadherin. However, a tenfold lower concentration of nickel decreases the apparent calcium-binding affinity and calcium-induced dimerization of N-cadherin. Competitive binding studies indicate that the apparent dissociation constants for nickel and magnesium are 0.2 mM and 2.5 mM, respectively. These Kd values are consistent with concentrations observed for a range of divalent cations in the extracellular space. Results from these studies indicate that calcium-induced dimerization by N-cadherin is attenuated by natural and non-physiological divalent cations in the extracellular microenvironment.

Recently, there has been growing interest in harnessing genetically engineered polymers to develop responsive biomaterials, such as hydrogels. Unlike their synthetic counterparts, genetically engineered polymers are produced without the use of toxic reagents and can easily be programmed to incorporate desirable hydrogel properties, including bioactivity, biodegradability, and monodispersity. Herein, we report the development of a copolymeric hydrogel that is based on the calcium-dependent protein, calmodulin (CaM). For our system, CaM and M13, a CaM-binding peptide, were incorporated into genetically engineered polymers with intervening linkers containing cleavable sequences. Spectroscopic and multiple-particle tracking (MPT) studies demonstrate that these polymers self-assemble through calcium-stabilized, noncovalent crosslinking to form a soft viscoelastic material. MPT further revealed that gelation is concentration-dependent. Collagenase digests show that the protein polymers are selectively degraded through specific cleavage. The modularity and stimuli-responsiveness of this system suggest its potential as a flexible scaffold for biomedical applications.

Cadherins are calcium-dependent, transmembrane adhesion molecules that assemble through direct noncovalent association of their N-terminal extracellular modular domains. As the transmembrane component of adherens junctions, they indirectly link adherent cells' actin cytoskeletons. Here, we investigate the most distal extracellular domain of neural cadherin (N-cadherin), a protein required at excitatory synapses, the site of long-term potentiation. This domain is the site of the adhesive interface, and it forms a dimer spontaneously without binding calcium, a surprising finding given that calcium binding is required for proper physiological function. A critical tryptophan at position 2, W2, provides a spectroscopic probe for the "closed" monomer and strand-swapped dimer. Spectroscopic studies show that W2 remains docked in the two forms but has a different apparent interaction with the hydrophobic pocket. Size-exclusion chromatography was used to measure the levels of the monomer and dimer over time to study the kinetics and equilibria of the unexpected spontaneous dimer formation ( K

In spite of their structural similarities, epithelial (E-) and neural (N-) cadherin are expressed at different types of synapses and differ significantly in their dimerization kinetics. Recent studies proposed a transient intermediate in E-cadherin as the key requirement for rapid disassembly kinetics of the adhesive dimer. This E-cadherin intermediate comprises four intermolecular ionic and H-bonding interactions between adhesive partners. These interactions are not preserved in N-cadherin except for a basic residue at the 14th position, which could stabilize the intermediate through either H-bonding or ionic interactions with the partner protomer. To investigate the origin of the rapid dimerization kinetics of N-cadherin in the presence of calcium, studies reported here systematically test the role of ionic and H-bonding interactions in dimerization kinetics using R14S, R14A, and R14E mutants of N-cadherin. Analytical size-exclusion chromatographic and bead aggregation studies showed two primary results. First, N-cadherin/R14S and N-cadherin/R14A mutants showed fast assembly and disassembly kinetics in the calcium-saturated state similar to that of wild-type N-cadherin. These results indicate that the fast disassembly of the calcium-saturated dimer of N-cadherin does not require a basic residue at the 14th position. Second, the dimerization kinetics of N-cadherin/R14E were slow in the calcium-saturated state, indicating that negative charge destabilizes the intermediate state. Taken together, these results indicate that the basic residue at the 14th position does not promote rapid dimerization kinetics but that an acidic amino acid in that position significantly impairs dimerization kinetics.

In spite of structural similarities Epithelial- (E-) and Neural- (N-) cadherins are expressed at two types of synapses and differ significantly in dimer disassembly kinetics. Recent studies suggested that the formation of an X-dimer intermediate in E-cadherin is the key requirement for rapid disassembly of the adhesive dimer (Harrison et al., Nat Struct Mol Biol 2010;17:348–357 and Hong et al., J Cell Biol 2011;192:1073–1083). The X-interface in E-cadherin involves three noncovalent interactions, none of which is conserved in N-cadherin. Dimer disassembly is slow at low calcium concentration in N-cadherin, which may be due to the differences in the X-interface residues. To investigate the origin of the slow disassembly kinetics we introduced three point mutations into N-cadherin to provide the opportunity for the formation of X-interface interactions. Spectroscopic studies showed that the triple mutation did not affect the stability or the calcium-binding affinity of the X-enabled N-cadherin mutant. Analytical size exclusion chromatography was used to assay for the effect of the mutation on the rate of dimer disassembly. Contrary to our expectation, the disassembly of dimers of the X-enabled N-cadherin mutant was as slow as seen for wild-type N-cadherin in the apo-state. Thus, the differences in the X-interface residues are not the origin of slow disassembly kinetics of N-cadherin in the apo-state.

Cadherins are cell adhesion proteins that are important for tissue formation and integrity. Cell-cell adhesion occurs through the formation of the strand-crossover dimer between identical cadherins on the surface of neighboring cells. The strand-crossover dimer forms exclusively between their EC1 domains via swapping of the βA sheet by undocking the conserved tryptophan 2, W2, from its own hydrophobic pocket and docking it into the hydrophobic pocket of its adhesive partner. An interesting aspect of the system is the fact that critical noncovalent interactions in the monomer re-form in the dimer. Thus, as these noncovalent interactions are conserved, what drives the formation of dimer? Moreover, why is dimer formation calcium-dependent? Thus, to probe the structural and energetic effects of calcium on the noncovalent interactions that are necessary for dimer formation, we performed spectroscopic, stability, and assembly studies of wild-type and two mutants, W2A and E89A, of neural (N-) cadherin. We find that while the ionic interaction involving E89 has a minimal effect on the general stability of the closed conformation of the βA sheet, the hydrophobic interaction involving W2 is the source of the calcium requirement for adhesive dimer formation. The binding of calcium creates strain in the W2-hydrophbic pocket interaction through direct connection of E11 at the C-terminus of the βA sheet to calcium. To overcome this unfavorable condition in the monomer, N-cadherin forms a dimer. Taken together, our data provide a thermodynamic basis for the calcium dependence of strand-crossover dimer formation in N-cadherin.

Neural cadherin (N-cadherin) is a calcium-dependent homophilic cell-adhesive molecule and critical for synaptogenesis and synapse maintenance. The extracellular region plays an important role in cadherin-mediated cell adhesion and has five tandemly repeated ectodomains (EC1-EC5) with three calcium-binding sites situated between each of these domains. Adhesive dimer formation is significantly dependent on binding of calcium such that mutations in the calcium-binding sites adversely affect cell adhesion. To investigate the relative significance of the calcium-binding sites at the EC1-EC2 interface in calcium-induced dimerization, we mutated three important amino acids, D134, D136, and D103, in NCAD12, a construct containing EC1 and EC2. Spectroscopic and chromatographic experiments showed that all three mutations affected calcium binding and dimerization. Mutation of D134, a bidentate chelator in site 3, severely impaired the binding of calcium to all three sites. These findings confirm that binding to site 3 is required for binding to occur at site 2 and site 1. Interestingly, while the D103A mutation diminished only the affinity for calcium, it completely eliminated dimerization. Equilibrium dialysis experiments showed a stoichiometry of 3 at 2 mM calcium for D103A, but no dimerization was apparent even at 10 mM calcium. These results indicate that calcium binding alone is not sufficient for dimerization but requires cooperativity between calcium-binding sites. In summary, our findings confirm that the calcium-binding sites are occupied sequentially in the order of site 3, then site 2 and site 1, and that cooperativity between site 2 and site 1 is essential for formation of adhesive dimers by N-cadherin.

Epithelial- and neural-cadherins are specifically localized at synapses in neurons which can change the shape and contact surface on a time scale of seconds to months. We have focused our studies on the role of the extracellular domains of cadherins in the dynamics of synapses. The kinetics of dimer disassembly of the first two extracellular domains of E- and N-cadherin, ECAD12 and NCAD12, were studied with analytical size exclusion chromatography and sedimentation velocity. NCAD12 forms three different dimers that are distinguished by assembly conditions and kinetics of dissociation. ECAD12 dimer disassembles rapidly regardless of the calcium concentration, whereas the disassembly of NCAD12 dimers was strongly dependent on calcium concentration. In addition to the apo- and saturated-dimeric forms of NCAD12, there is a third dimeric form that is a slow exchange dimer. This third dimeric form for NCAD12, formed by decalcification of the calcium-saturated dimer, was kinetically trapped in apo-conditions and did not disassemble over a period of months. Sedimentation velocity experiments showed that this dimer, upon addition of calcium, had similar weighted averages as a calcium-saturated dimer. These studies provide evidence that the kinetics of dimer disassembly of the extracellular domains may be a major contributor to the morphological dynamics of synapses in vivo.

Epithelial cadherin is a transmembrane protein that is essential in calcium-dependent cell–cell recognition and adhesion. It contains five independently folded globular domains in its extracellular region. Each domain has a seven-strand β-sheet immunoglobulin fold. Short seven-residue peptide segments connect the globular domains and provide oxygens to chelate calcium ions at the interface between the domains (Nagar et al., Nature 1995;380:360–364). Recently, stability studies of ECAD2 (Prasad et al., Biochemistry 2004;43:8055–8066) were undertaken with the motivation that Domain 2 is a representative domain for this family of proteins. The definition of a domain boundary is somewhat arbitrary; hence, it was important to examine the effect of the adjoining linker regions that connect Domain 2 to the adjacent domains. Present studies employ temperature–denaturation and proteolytic susceptibility to provide insight into the impact of these linkers on Domain 2. The significant findings of our present study are threefold. First, the linker segments destabilize the core domain in the absence of calcium. Second, the destabilization due to addition of the linker segments can be partially reversed by the addition of calcium. Third, sodium chloride stabilizes all constructs. This result implies that electrostatic repulsion is a contributor to destabilization of the core domain by addition of the linkers. Thus, the context of Domain 2 within the whole molecule affects its thermodynamic characteristics. Proteins 2006. © 2005 Wiley-Liss, Inc.

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

Neural (N-) cadherin is a transmembrane protein within adherens junctions that mediates cell-cell adhesion. It has 5 modular extracellular domains (EC1-EC5) that bind 3 calcium ions between each of the modules. Calcium binding is required for dimerization. N-Cadherin is involved in diverse processes including tissue morphogenesis, excitatory synapse formation and dynamics, and metastasis of cancer. During neurotransmission and tumorigenesis, fluctuations in extracellular pH occur, causing tissue acidosis with associated physiological consequences. Studies reported here aim to determine the effect of pH on the dimerization properties of a truncated construct of N-cadherin containing EC1-EC2. Since N-cadherin is an anionic protein, we hypothesized that acidification of solution would cause an increase in stability of the apo protein, a decrease in the calcium-binding affinity, and a concomitant decrease in the formation of adhesive dimer. The stability of the apo monomer was increased and the calcium-binding affinity was decreased at reduced pH, consistent with our hypothesis. Surprisingly, analytical SEC studies showed an increase in calcium-induced dimerization as solution pH decreased from 7.4 to 5.0. Salt-dependent dimerization studies indicated that electrostatic repulsion attenuates dimerization affinity. These results point to a possible electrostatic mechanism for moderating dimerization affinity of the Type I cadherin family. Extrapolating these results to cell adhesion in vivo leads to the assertion that decreased pH promotes adhesion by N-cadherin, thereby stabilizing synaptic junctions.

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.

Calcium-dependent conformational states of calmodulin (CaM) were probed by thrombin to determine quantitative differences in the susceptibility of two bonds: Arg37-Ser38 (R37-S38, near site I in the N-terminal domain) and Arg106-His107 (R106-H107, near site III in the C-terminal domain). Quantitative thrombin footprinting of a discontinuous equilibrium calcium titration of wild-type calmodulin showed that the R37-S38 bond of the apoprotein was cleaved at a barely detectable level while the R106-H107 bond was maximally susceptible. Calcium binding to sites III and IV monotonically protected R106-H107 from proteolysis; concomitantly, the susceptibility of R37-S38 increased. However, calcium binding to sites I and II protected R37-S38 from cleavage, yielding a peaked biphasic profile composed of equal and opposite transitions. Both bonds were fully protected when calmodulin was saturated with calcium. Susceptibility profiles resolved from the fractional abundance of primary cleavage products (peptides 1-37, 38-148, 1-106, 107-148) were interpreted as directly reflecting calcium-induced conformational changes in whole calmodulin; free energies of calcium binding and cooperativity were estimated. Secondary cleavage was never observed; both R37 and R106 were sites of thrombinolysis in whole calmodulin only. In studies of E140Q-CaM (having a mutation in site IV), the susceptibility of R37-S38 decreased monotonically. Thus, the biphasic character of cleavage of R37 in helix B was not intrinsic to that domain but depended on propagation of effects of calcium-induced changes in the C-terminal domain. The observed patterns of susceptibility indicated that partially saturated wild-type calmodulin adopts at least one intermediate conformation whose structure is determined by calcium-mediated interactions between the domains.

Calmodulin binds up to four calcium ions cooperatively in response to cellular signaling events. To understand the functional energetics of calcium activation of calmodulin, it is important to monitor individual Ca(2+)-binding sites and other positions at partial degrees of saturation. This study is the first use of 1-D proton NMR to monitor the equilibrium Ca(2+)-binding properties of calmodulin. Protein concentrations required for NMR experiments (approximately 1 mM) are approximately 1000-fold greater than the Kd values for calcium binding to calmodulin, preventing a direct continuous equilibrium titration of calmodulin. Thus, dialysates of calmodulin in buffers of experimentally determined [Ca2+]free were prepared to conduct discontinuous equilibrium titrations at both 92 and 152 mM KCl. For the C-terminal domain, the normalized area of the delta-protons of Y138 defined calcium binding isotherms. For N-terminal domain resonances (F16C delta H, T26C alpha H, D64C alpha H, and F65C delta H), the calcium-dependent change in chemical shift defined isotherms. These are the first residue-specific studies to monitor the energetics of Ca2+ binding to the N-terminal domain in wild-type holo calmodulin. Calcium binding to both domains appeared cooperative and binding affinity decreased in higher KCl. Isotherms resolved from the side chain resonances of F16 and F65 had a lower median ligand activity and a slightly higher degree of cooperativity than isotherms resolved from the backbone resonances of D64 and T26. Salt-dependent changes in apparent intradomain cooperativity differed for the domains: at higher salt, delta Gc increased for the C-terminal domain while remaining constant or decreasing for the N-terminal domain.

Calmodulin is the primary eukaryotic intracellular calcium receptor. Cooperative calcium binding to two sites in each of two domains drives large conformational changes that enable it to activate target proteins. An understanding of the molecular mechanism of cooperativity requires determination of the conformational states populated by calmodulin, the intrinsic free energies of binding calcium to four sites, and the nature and degree of intradomain and interdomain interactions. To monitor residue-specific conformational changes within calmodulin as calcium binds, we have developed a new quantitative proteolytic footprinting method using endoproteinase GluC (EndoGluC). Under conditions of very limited proteolysis, cleavage occurred at only five of the sixteen positions possible in calmodulin. The relative abundance of fragments indicated that calcium induced changes in the susceptibility of individual peptide bonds. Quantitative susceptibility profiles were resolved for two positions: E31-L32, in site I in the N-terminal domain, and E87-A88, preceding site III in the C-terminal domain. In apocalmodulin, E87-A88 was susceptible to EndoGluC; calcium binding to sites III and IV caused monotonic protection from proteolysis. The response of E31-L32 was biphasic. In apocalmodulin, it was resistant to cleavage. Susceptibility was induced by calcium binding to sites III and IV, followed by protection induced by calcium binding to sites I and II. This indicated that calmodulin must adopt at least three distinguishable conformations and suggested that the two domains interact. Model-dependent equilibrium constants were resolved from the EndoGluC susceptibility profiles for E31 and E87; they indicated cooperative binding within each domain. Approaches taken to validate this proteolytic footprinting method are described.

Neural- (NCAD) and epithelial- (ECAD) cadherin are calcium-dependent cell-adhesive molecules, and are localized at excitatory and inhibitory synapses respectively. They play an important role in synaptogenesis, synapse maintenance and plasticity. The extracellular region plays a critical role in cadherin-mediated cell adhesion, and has five tandemly repeated ectodomains (EC1-EC5). Calcium binding is required for dimer formation between first two N-terminal domains (EC1-EC2). Despite similarity in the primary structure, the extracellular domains of NCAD and ECAD have different intrinsic stability, dimerization affinity and kinetics of disassembly. To investigate the origin of these differences, we are characterizing the modular domains individually. Here, we report studies of NCAD2, EC2 of NCAD. This domain is important for calcium binding and is the physical linkage between the dimerization interface in EC1 and the membrane proximal modular domains. Thermal-denaturation studies show that NCAD2 is less stable than ECAD2 and less influenced by the adjoining 7-residue, N- and C-terminal linker segments. In addition the NCAD2 constructs are less influenced by added salt. This difference is likely due to variation in the overall number and distribution of charges on these anionic proteins. Our studies indicate that despite their sequence similarity and apparently passive role in adhesive dimer formation, EC2 of E- and N-cadherins are distinctly different and may contribute to the differences in energetics and kinetics of dimerization.

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.

Epithelial cadherin is important in establishing and maintaining cell to cell interactions in epithelial cells, thereby playing an important role during morphogenesis. The epithelial cadherin molecules have three main regions: the N-terminal extracellular region, the transmembrane region that spans the cell membrane once, and the C-terminal cytoplasmic region that communicates with the cytoskeletal actin filaments through catenins. We report studies of the calcium-dependent stability of extracellular domains 1 and 2 of epithelial cadherin as a two-domain construct (MECAD12). Circular dichroism (CD) spectra of MECAD12 indicated a typical beta-sheet conformation in all solution conditions. Thermal- and denaturant-induced unfolding was monitored by CD. Distinct calcium stabilization was observed as a shift in T(m) from 40 (apo) to 65 degrees C (10 mM Ca2+). Spectroscopic experiments agreed well with calorimetric (DSC). In the absence of calcium, the unfolding transition was shallow (deltaH(m) = 40 kcal/mol) but not obviously three state. Model-dependent analysis indicated that a second transition could be assigned to the unfolding of domain 2. A calcium-binding constant was derived from the calcium-dependent shift in temperature denaturation profiles. The Kd that was obtained (55 microM) was consistent with literature values. Thus, the modular domains of epithelial cadherin exhibit context-dependent behavior in both the apo and calcium-bound states. This cooperativity between the modules is consistent with the physiological role of epithelial cadherin in signal transduction through cell-adhesive contacts.

There is a necessary energetic linkage between ligand binding and stability in biological molecules. The critical glutamate in Site 4 was mutated to create two mutants of the C-domain of calmodulin yielding E140D and E140Q. These proteins were stably folded in the absence of calcium, but had dramatically impaired binding of calcium. We determined the stability of the mutant proteins in the absence and presence of calcium using urea-induced unfolding monitored by circular dichroism (CD) spectroscopy. These calcium-dependent unfolding curves were fit to models that allowed for linkage of stability to binding of a single calcium ion to the native and unfolded states. Simultaneous analysis of the unfolding profiles for each mutant yielded estimates for calcium-binding constants that were consistent with results from direct titrations monitored by fluorescence. Binding to the unfolded state was not an important energetic contributor to the ligand-linked stability of these mutants.

Cadherin is a cell adhesion molecule that participates in ordered calcium-dependent self-association interactions both between molecules on the same cell surface (cis-interactions) and on neighboring cell surfaces (trans-interactions). Cadherin is a transmembrane protein that has 3-7 independently folded beta-barrel extracellular domains. Both types of self-association interactions are mediated through the most N-terminal domain (Domain 1). Although the structural nature of the trans-interactions is clear, the nature of the cis-interactions is ambiguous despite several high-resolution structural studies. From earlier studies, it is understood that for the trans-interactions to happen, cis-interactions are mandatory. Hence, our first steps are to study the energetic driving forces for the cis-interactions. We have simplified the approach by first examining participating extracellular domains individually. We report here our initial experiments into the stability of Domain 2 of E-cadherin (ECAD2). ECAD2 appears monomeric, according to results from mass spectrometry and sedimentation equilibrium studies. We report denaturation data from differential scanning calorimetric experiments, and temperature and denaturant-induced unfolding experiments monitored by circular dichroism. These studies give a unified picture of the energetics of ECAD2-folding and stability, for which DeltaG degrees is 6.6 kcal/mol, T(m) is 54 degrees C, DeltaH(m) is 90 kcal/mol, and DeltaC(p) is 1300 cal/Kmol. These parameters are independent of calcium up to 5 mM, indicating that ECAD2 does not bind calcium at physiological calcium levels.

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.

To dissect the chemical basis for interactions controlling regulatory properties of macromolecular assemblies, it is essential to explore experimentally the linkage between ligand binding, conformational change, and subunit assembly. There are many advantages to using techniques that will probe the occupancy of individual binding sites or monitor conformational responses of individual residues, as described here. Proteolytic footprinting titrations may be used to infer binding free energies for ligands interacting with multiple sites or domains and to detect otherwise unrecognized “silent” interdomain interactions. Microgram quantities of pure protein are required, which is low relative to the hundreds of milligrams needed for comparable discontinuous equilibrium titrations monitored by NMR.21 By running comparative studies with several proteases, it is easy to determine whether resulting titration curves are consistent, independent of the protease used and therefore representative of the structural response of the protein to ligand binding or other differences in solution conditions (pH, salt, temperature). The results from multiple techniques (e.g., NMR, fluorescence, and footprinting) applied to aliquots from the same discontinuous titration may be compared easily to test for consistency. Classic methods for determining thermodynamic and kinetic properties of calcium binding to calmodulin include filter binding and equilibrium or flow dialysis (employing the isotope 45Ca), spectroscopic studies of stopped-flow fluorescence, calorimetry, and direct ion titrations. A cautionary note is that many different sets of microscopic data would be consistent with a single set of macroscopic constants determined by classic methods. This was well illustrated in Fig. 9. Thus, while it is important to compare results with those obtained by classic binding methods, they are, by definition, incapable of resolving the microscopic constants of interest. Thus, there is only one “direction” for comparison. Quantitative proteolytic footprinting titrations applied to studying calmodulin provided the first direct quantitative estimate of negative interactions between domains. Although studies of site-knockout mutants had suggested interactions between domains, this approach gave the first evidence for the pathway of anticooperative interactions between domains by showing that helix B responds structurally to calcium binding to sites III and IV in the C-domain. Despite two decades of study of calmodulin and the application of limited proteolysis studies to the apo and fully saturated forms, this finding emerged only when titration studies were undertaken as described. This highlights the general observation that while the behavior of the intermediate states in a cooperative switch are the key elements of the transition mechanism, they are the most difficult to observe. The unexpected finding that the isolated domains are nearly equivalent in their calcium-binding properties (Fig. 23 B) leaves us with many of the questions we had at the start: How does the sum of two nearly equivalent domains result in a molecule that switches sequentially rather than simultaneously? But it underscores why it is not yet possible to understand similar proteins by sequence gazing alone.

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.