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Protein engineering and NMR studies of calmodulin

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Abstract

The calcium regulatory protein calmodulin (CaM) plays a role as an on-off switch in the activation of many enzymes and proteins. CaM has a dumbbell shaped structure with two folded domains, which are connected by a flexible linker in solution. The calmodulin-binding domains of the target proteins are contained in 20 residue long amino acid sequences, that share no obvious amino acid sequence homology. In this contribution, we discuss the features of CaM, which allow it to be rather promiscous, and bind effectively to all these distinct domains. In particular, we describe the role of the methionine-rich hydrophobic surfaces of the protein in providing a malleable and sticky surface for binding many hydrophobic peptides. The enzyme activation properties of various Met→Leu mutants of CaM are discussed. In addition, the role of the flexible linker region that connects the two domains is also analyzed. Finally, we describe various NMR and spectroscopic experiments that aid in determining the CaM-bound structures of synthetic peptides containing various CaM-binding domains. All structures analyzed to date are α-helical when bound to CaM, and they interact with CaM only through amino acid sidechains. This form of protein-protein interaction is rather unique, and may contribute to CaM's capacity to bind effectively to such a wide range of distinct partners.

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Abbreviations

CaM:

calmodulin

CD:

circular dichroism

cNOS:

constitutive Nitric Oxide Synthase

FTIR:

Fourier Transform Infrared Spectroscopy

iNOS:

inducible Nitric Oxide Synthase

MLCK:

Myosin Light Chain Kinase

NMR:

Nuclear Magnetic Resonance

nOe:

nuclear Overhauser effect

NOESY:

two dimensional Nuclear Overhauser Effect Spectroscopy

PDE:

cyclic nucleotide phosphodiesterase

SeMet:

selenomethionine

TFE:

trifluoroethanol

TOCSY:

two dimensional Total Correlation Spectroscopy

trnOe:

transferred nuclear Overhauser effect

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  1. Mingjie Zhang

    Present address: Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong

Authors and Affiliations

  1. Department of Biological Sciences, The University of Calgary, T2N 1N4, Calgary, Canada

    Hans J. Vogel & Mingjie Zhang

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Vogel, H.J., Zhang, M. Protein engineering and NMR studies of calmodulin. Mol Cell Biochem 149, 3–15 (1995). https://doi.org/10.1007/BF01076558

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