title: The energetics and dynamics of molecular recognition by calmodulin.
abstract: Amide hydrogen exchange has been used to examine the structural dynamics and energetics of the interaction of a peptide corresponding to the calmodulin binding domain of smooth muscle myosin light chain kinase with calcium-saturated calmodulin. Heteronuclear NMR 15N-1H correlation techniques were used to quantitate amide proton exchange rates of both 15N-labeled and unlabeled amide protons of the smMLCK peptide complexed to calmodulin. Hydrogen exchange slowing factors were determined for 18 of the 19 amide hydrogens and found to span 6 orders of magnitude. The first six residues of the bound peptide were found to have slowing factors near 1 and are considered not to be hydrogen bonded, consistent with the previously reported model for the structure of the peptide. The pattern of hydrogen exchange of hydrogen-bonded amide hydrogens is indicative of end-fraying behavior characteristic of helix-coil transitions. The effective statistical mechanical parameters revealed by the end fraying are consistent with exchange from a highly solvated state. However, the slowing factors of the first hydrogen-bonded amide hydrogens are large, indicating the requirement for a reorganization of the calmodulin-peptide complex before the helix-coil transitions leading to exchange can occur. Taken together, these observations suggest that the collapsed complex reorganizes with an associated free energy change of 5.5 kcal/mol to a more open state where the helical peptide is highly solvated and undergoes helix-coil transitions leading to exchange. The free energy difference between the most and least stable intrahelical amide hydrogen bonds of the bound peptide is estimated to be approximately 2.5 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana, Champaign 61801.
authors.forename: M R
authors.initials: MR
authors.lastname: Ehrhardt
authors.forename: J L
authors.initials: JL
authors.lastname: Urbauer
authors.forename: A J
authors.initials: AJ
authors.lastname: Wand
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D004734:Energy Metabolism; D004926:Escherichia coli / Q000235:genetics; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D009587:Nitrogen Isotopes; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013816:Thermodynamics
chemical_list: D002147:Calmodulin; D009587:Nitrogen Isotopes; D010455:Peptides; C065738:MLCK peptide; D006859:Hydrogen