Related Experiment Video
Updated: Jun 25, 2026
07:51
Pull-down of Calmodulin-binding Proteins
Published on: January 23, 2012
Thermodynamic effects of noncoded and coded methionine substitutions in calmodulin
Aaron P Yamniuk1, Hiroaki Ishida, Dustin Lippert
1Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
Biophysical Journal
|February 17, 2009
Summary
Methionine substitutions in calmodulin (CaM) enhance protein stability and alter target binding thermodynamics. These changes occur due to adjustments in side-chain packing and dynamics, highlighting CaM's structural plasticity.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Dynamics
Background:
- Methionine residues are crucial for calmodulin's (CaM) structure and function.
- These residues transition from buried in apo-CaM to solvent-exposed in Ca2+-CaM, mediating target interactions.
Purpose of the Study:
- To investigate the structural and thermodynamic consequences of methionine substitutions in CaM.
- To assess the impact of noncoded and coded amino acid substitutions on CaM stability and target binding.
Main Methods:
- Differential scanning calorimetry (DSC) to measure protein stability.
- Nuclear magnetic resonance (NMR) spectroscopy to analyze conformational dynamics.
- Isothermal titration calorimetry (ITC) to determine binding thermodynamics.
Main Results:
- Methionine substitutions (selenomethionine, ethionine, norleucine, leucine) increased CaM domain stability, with leucine/norleucine in the apo-C-domain showing the largest effect (19-26°C increase in melting temperature).
- NMR revealed the loss of a slow conformational exchange process in Leu-substituted apo-C-domain.
- ITC showed significant enthalpy-entropy compensation in target binding to both apo- and Ca2+-CaM, with minimal change in free energy.
Conclusions:
- CaM accommodates noncoded and coded methionine substitutions due to its structural plasticity.
- Side-chain packing and dynamics adjustments upon substitution lead to altered protein stability and target binding thermodynamics.
More Related Videos
Related Concept Videos
01:16Calmodulin-dependent Signaling
Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
01:39Mutations
Overview
01:35Mutations
Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations can be characterized in a number of different ways: whether and how they alter the amino acid sequence of the protein, whether they occur over a small or large area of DNA, and whether they occur in somatic cells or germline cells.
Chromosomal Alterations Are Large-Scale Mutations
While point mutations are changes in a single nucleotide in...
Chromosomal Alterations Are Large-Scale Mutations
While point mutations are changes in a single nucleotide in...
01:58Cooperative Allosteric Transitions
Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
01:58Cooperative Allosteric Transitions
Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
01:58Cooperative Allosteric Transitions
Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...