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

Hobson, Kenosha F.; Housley, Nicole A.; Pedigo, Susan

doi:10.1016/j.bpc.2004.11.002

Cited by 4 publications

(5 citation statements)

References 57 publications

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“…The discussion of linked equilibria is well established in the literature (45,58). This approach was used to determine the energetics of binding of cytidine 2′-monophosphate to ribonuclease A using calorimetry (59) and calcium binding to mutants of the C-terminal domain of calmodulin from urea denaturation profiles (60). Using this approach, we found a dissociation constant of 22-55 µM.…”

Section: Discussionmentioning

confidence: 98%

Calcium-Dependent Stability Studies of Domains 1 and 2 of Epithelial Cadherin

Prasad

Pedigo

2005

Biochemistry

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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 β-sheet conformation in all solution conditions. Thermal-and denaturantinduced unfolding was monitored by CD. Distinct calcium stabilization was observed as a shift in T m from 40 (apo) to 65 °C (10 mM Ca 2+ ). Spectroscopic experiments agreed well with calorimetric (DSC). In the absence of calcium, the unfolding transition was shallow (∆H 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 K d that was obtained (55 µM) was consistent with literature values. Thus, the modular domains of epithelial cadherin exhibit context-dependent behavior in both the apo and calciumbound states. This cooperativity between the modules is consistent with the physiological role of epithelial cadherin in signal transduction through cell-adhesive contacts.

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Section: Discussionmentioning

confidence: 98%

Calcium-Dependent Stability Studies of Domains 1 and 2 of Epithelial Cadherin

Prasad

Pedigo

2005

Biochemistry

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“…In evolutionary aspects, not surprisingly, the acidic residues on the CaM loop; Asp-93, Asp-95, and Glu-104, are highly conserved among virtually all EF-hand Ca 21 binding motifs (34). Point mutations in these residues of the EF-hand motif basically eliminate the Ca 21 binding ability unless extremely high Ca 21 concentrations are explored (35)(36)(37)(38)(39).…”

Section: Discussionmentioning

confidence: 99%

Protein Grabs a Ligand by Extending Anchor Residues: Molecular Simulation for Ca2+ Binding to Calmodulin Loop

Kobayashi¹,

Takada

2006

Biophysical Journal

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The structural difference in proteins between unbound and bound forms directly suggests the importance of the conformational plasticity of proteins. However, pathways that connect two-end structures and how they are coupled to the binding reaction are not well understood at atomic resolution. Here, we analyzed the free-energy landscape, explicitly taking into account coupling between binding and conformational change by performing atomistic molecular dynamics simulations for Ca2+ binding to a calmodulin loop. Using the AMBER force field with explicit water solvent, we conducted umbrella sampling for the free-energy surface and steered molecular dynamics for the pathway search. We found that, at an early stage of binding, some key residue side chains extend their "arms" to catch Ca2+ and, after catching, they carry the Ca2+ to the center of the binding pocket. This grabbing motion resulted in smooth and stepwise exchange in coordination partners of Ca2+ from water oxygen to atoms in the calmodulin loop. The key residue that first caught the ion was one of the two acidic residues, which are highly conserved. In the pathway simulations, different pathways were observed between binding and dissociation reactions: The former was more diverse than the latter.

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“…To test the stabilizing effect of calcium ions on hydrogel formation, the following experiment was performed. Polymers were mixed (1:1 mole ratio) in microcentrifuge tubes to form hydrogels in the absence of added calcium; contaminating calcium ion concentration is ∼5 μM as determined by atomic absorption spectroscopy. , Calcium was added to one gel sample to a total concentration of 20 mM. The chelator EDTA was added to the other to a total of 5 mM concentration to chelate the contaminating calcium ions.…”

Section: Methodsmentioning

confidence: 99%

“…We will refer to these polymers as calmodulin collagen-like polymers (CCLP3) and peptide collagen-like polymers (PCLP3). CaM was chosen due to its specific, calcium-dependent, high-affinity interaction with M13 and its remarkable resilience to denaturants , and high temperatures . CaM is a well-studied, intracellular calcium signaling protein with two globular domains connected by a flexible central helix .…”

Section: Introductionmentioning

confidence: 99%

Development and Characterization of Calmodulin-Based Copolymeric Hydrogels

2020

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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 multipleparticle 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.

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

Cited by 4 publications

References 57 publications

Calcium-Dependent Stability Studies of Domains 1 and 2 of Epithelial Cadherin

Calcium-Dependent Stability Studies of Domains 1 and 2 of Epithelial Cadherin

Protein Grabs a Ligand by Extending Anchor Residues: Molecular Simulation for Ca2+ Binding to Calmodulin Loop

Development and Characterization of Calmodulin-Based Copolymeric Hydrogels

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