Development and Characterization of Calmodulin-Based Copolymeric Hydrogels.

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.

Spontaneous Calcium-Independent Dimerization of the Isolated First Domain of Neural Cadherin.

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 ( Kd = 130 µM; τ = 2 days at 4 °C). Our results support the idea that NCAD1 is missing critical contacts that facilitate the rapid exchange of the ßA-strand. Furthermore, the monomer and dimer have equivalent and exceptionally high intrinsic stability for a 99-residue Ig-like domain with no internal disulfides ( Tm = 77 °C; Δ H = 85 kcal/mol). Ultimately, a complete analysis of synapse dynamics requires characterization of the kinetics and equilibria of N-cadherin. The studies reported here take a reductionist approach to understanding the essential biophysics of an atypical Ig-like domain that is the site of the adhesive interface of N-cadherin.

Basic residue at position 14 is not required for fast assembly and disassembly kinetics in neural cadherin.

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.

Impact of pH on the structure and function of neural cadherin.

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.

X-interface is not the explanation for the slow disassembly of N-cadherin dimers in the apo state.

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.

Stability studies of extracellular domain two of neural-cadherin.

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.

Calcium-induced strain in the monomer promotes dimerization in neural cadherin.

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 ßA 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 ßA 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 ßA 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.

Prolines in ßA-sheet of neural cadherin act as a switch to control the dynamics of the equilibrium between monomer and dimer.

Neural cadherins dimerize through the formation of calcium-dependent strand-crossover structures. Dimerization of cadherins leads to cell-cell adhesion in multicellular organisms. Strand-crossover dimer forms exclusively between the first N-terminal extracellular modules (EC1) of the adhesive partners via swapping of their ßA-sheets and docking of tryptophan-2 in the hydrophobic pocket. In the apo-state wild-type cadherin is predominantly monomer, which indicates that the dimerization is energetically unfavorable in the absence of calcium. Addition of calcium favors dimer formation by creating strain in the monomer and lowering the energetic barrier between monomer and dimer. Dynamics of the monomer-dimer equilibrium is vital for plasticity of synapses. Prolines recurrently occur in proteins that form strand-crossover dimer and are believed to be the source of the strain in the monomer. N-cadherins have two proline residues in the ßA-sheet. We focused our studies on the role of these two prolines in calcium-dependent dimerization. Spectroscopic, electrophoretic, and chromatopgraphic studies showed that mutations of both prolines to alanines increased the dimerization affinity by ~20-fold and relieved the requirement of calcium in dimerization. The P5A and P6A mutant formed very stable dimers that required denaturation of protein to disassemble in the apo conditions. In summary, the proline residues act as a switch to control the dynamics of the equilibrium between monomer and dimer which is crucial for the plasticity of synapses.

Dimeric states of neural- and epithelial-cadherins are distinguished by the rate of disassembly.

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.

Sequential binding of calcium leads to dimerization in neural 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.

Effect of linker segments on the stability of epithelial cadherin Domain 2.

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 beta-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.

Calcium-dependent stability studies of domains 1 and 2 of epithelial cadherin.

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.

Ligand-linked stability of mutants of the C-domain of calmodulin.

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.

Thermodynamic stability of domain 2 of epithelial cadherin.

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.

Concentration dependence of variability in growth rates of microtubules.

Growth and shortening of microtubules in the course of their polymerization and depolymerization have previously been observed to occur at variable rates. To gain insight into the meaning of this prominent variability, we studied the way in which its magnitude depends on the growth rate of experimentally observed and computer-simulated microtubules. The dynamic properties of plus-ended microtubules nucleated by pieces of Chlamydomonas flagellar axonemes were observed in real time by video-enhanced differential interference contrast light microscopy at differing tubulin concentrations. By means of a Monte Carlo algorithm, populations of microtubules were simulated that had similar growth and dynamic properties to the experimentally observed microtubules. By comparison of the experimentally observed and computer-simulated populations of microtubules, we found that 1) individual microtubules displayed an intrinsic variability that did not change as the rate of growth for a population increased, and 2) the variability was approximately fivefold greater than predicted by a simple model of subunit addition and loss. The model used to simulate microtubule growth has no provision for incorporation of lattice defects of any type, nor sophisticated geometry of the growing end. Thus, these as well as uncontrolled experimental variables were eliminated as causes for the prominent variability.