title: Thermodynamic effects of noncoded and coded methionine substitutions in calmodulin.
abstract: The methionine residues in the calcium (Ca2+) regulatory protein calmodulin (CaM) are structurally and functionally important. They are buried within the N- and C-domains of apo-CaM but become solvent-exposed in Ca2+-CaM, where they interact with numerous target proteins. Previous structural studies have shown that methionine substitutions to the noncoded amino acids selenomethionine, ethionine, or norleucine, or mutation to leucine do not impact the main chain structure of CaM. Here we used differential scanning calorimetry to show that these substitutions enhance the stability of both domains, with the largest increase in melting temperature (19-26 degrees C) achieved with leucine or norleucine in the apo-C-domain. Nuclear magnetic resonance spectroscopy experiments also revealed the loss of a slow conformational exchange process in the Leu-substituted apo-C-domain. In addition, isothermal titration calorimetry experiments revealed considerable changes in the enthalpy and entropy of target binding to apo-CaM and Ca2+-CaM, but the free energy of binding was largely unaffected due to enthalpy-entropy compensation. Collectively, these results demonstrate that noncoded and coded methionine substitutions can be accommodated in CaM because of the structural plasticity of the protein. However, adjustments in side-chain packing and dynamics lead to significant differences in protein stability and the thermodynamics of target binding.
authors.affiliation: Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Aaron P
authors.initials: AP
authors.lastname: Yamniuk
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Ishida
authors.forename: Dustin
authors.initials: D
authors.lastname: Lippert
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Vogel
mesh_terms: D019943:Amino Acid Substitution; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D002151:Calorimetry; D007930:Leucine / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000235:genetics; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000737:chemistry; D020125:Mutation, Missense; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D055550:Protein Stability; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014026:Nicotiana / Q000737:chemistry
chemical_list: D002147:Calmodulin; D008715:Methionine; D009219:Myosin-Light-Chain Kinase; D054639:Mitogen-Activated Protein Kinase Phosphatases; D007930:Leucine