NMR studies of the methionine methyl groups in calmodulin

Abstract

Calmodulin (CaM) is a ubiquitous Ca2+‐binding protein that can regulate a wide variety of cellular events. The protein contains 9 Met out of a total of 148 amino acid residues. The binding of Ca2+ to CaM induces conformational changes and exposes two Met‐rich hydrophobic surfaces which provide the main protein‐protein contact areas when CaM interacts with its target enzymes. Two‐dimensional (1H,13C)‐heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy was used to study selectively 13C‐isotope labelled Met methyl groups in apo‐CaM, Ca2+‐CaM and a complex of CaM with the CaM‐binding domain of skeletal muscle Myosin Light Chain Kinase (MLCK). The resonance assignment of the Met methyl groups in these three functionally different states were obtained by site‐directed mutagenesis (Met→Leu). Chemical shift changes indicate that the methyl groups of the Met residues are in different environments in apo‐, calcium‐, and MLCK‐bound‐CaM. The T 1 relaxation rates of the individual Met methyl carbons in the three forms of CaM indicate that those in Ca2+‐CaM have the highest mobility. Our results also suggest that the methyl groups of the unbranched Met sidechains in general are more flexible than those of aliphatic amino acid residues such as Leu and Ile.

Substitution of the methionine residues of calmodulin with the unnatural amino acid analogs ethionine and norleucine: Biochemical and spectroscopic studies

“…The outcome of our studies provides further evidence for the importance of the polarizability of the sulfur atoms and the optimal side-chain length and flexibility of Met residues in CaM. This paper extends on our earlier studies with Met r Leu mutants~Zhang et al, 1994b; Siivari et al, 1995;Edwards et al, 1998!, selenomethionine substituted CaM~Zhang & Vogel, 1994aYuan et al, 1998! and difluoromethionyl …”

Energetics of Target Peptide Binding by Calmodulin Reveals Different Modes of Binding

“…There is a relatively small negative entropy associated with the loss of translational and rotational degrees of freedom upon complex formation (87)(88)(89)(90). The immobilization of the side chains (20,21) and the loss of side chain entropy will also have a small and negative contribution (91,92). The major contribution to the entropy of binding will, however, be defined by the large positive entropy of dehydration of the binding interface (41).…”

“…However, in a recent calorimetric study, it was shown that binding of a CaM target peptide from smooth muscle myosin light chain kinase (smMLCK) is driven by enthalpic not entropic factors (19). It could be that the increase in solvent entropy is offset by other phenomena, such as the loss of mobility of some amino acid side chains (20,21) or the loss of backbone entropy in the target peptide as it forms an ␣-helix in the complex (22). Enthalpic effects must also play a key role, and these could include van der Waal's interactions among the hydrophobic residues in the complex, as well as salt bridges and hydrogen bonds between acidic residues on CaM and basic residues on the target peptide.…”

Calmodulin in Complex with Proteins and Small Molecule Ligands: Operating with the Element of Surprise; Implications for Structure-Based Drug Design

“…CaM contains Met side chains in unusually high abundance, accounting for nearly half of the hydrophobic surface of each patch [11]. These methionines were shown to become solvent exposed in the open conformation of CaM [35,36], and are essential for CaM's ability to accommodate such different ligands [37,38] (see Fig. (3)).…”

“…(3)). Met side chains are highly pliable [36,39], and they are ideal for creating favorable van der Waals interactions due to the polarizability of sulfur and are at the same time fairly compatible with the surrounding aqueous medium [38,40].…”