Abstract Here we present a novel NMR method for the structure determination of calcium-calmodulin (Ca2+-CaM)-peptide complexes from a limited set of experimental restraints. A comparison of solved CaM-peptide structures reveals invariability in CaMâ??s backbone conformation and a structural plasticity in CaMâ??s domain orientation enabled by a flexible linker. Knowing this, the collection and analysis of an extensive set of NOESY spectra is redundant. Although RDCs can define CaM domain orientation in the complex, they lack the translational information required to position the domains on the bound peptide and highlight the necessity of intermolecular NOEs. Here we employ a specific isotope labeling strategy in which the role of methionine in CaM-peptide interactions is exploited to collect these critical NOEs. By 1H, 13C-labeling the methyl groups of deuterated methionine against a 2H, 12C background, we can acquire a 13C-edited NOESY characterized by simplified, easily analyzable spectra. Together with measured CaM backbone HN-N RDCs and intrapeptide NOE-based distances, these intermolecular NOEs provide restraints for a low temperature torsion-angle dynamics and simulated annealing protocol used to calculate the complex structure. We have applied our method to a CaM complex previously solved through X-ray crystallography: Ca2+-CaM bound to the CaM kinase I peptide (PDB code: 1MXE). The resulting structure has a backbone RMSD of 1.6 Ã? to that previously published. We have also used this test complex to investigate the importance of homologous model selection on the calculated outcome. In addition to having application for fast complex structure determination, this method can be used to determine the structures of difficult complexes characterized by chemical shift overlap and broad signals for which the traditional method based on the use of fully 13C, 15N-labeled CaM fails.
Content Type Journal Article
Pages 1-11
DOI 10.1007/s10858-011-9495-3
Authors
Jessica L. Gifford, Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada
Hiroaki Ishida, Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada
Hans J. Vogel, Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada
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Structure determination of protein?RNA complexes in solution provides unique insights into factors that are involved in protein/RNA recognition. Here, we review the methodology used in our laboratory to overcome the challenges of protein?RNA structure determination by nuclear magnetic resonance (NMR). We use as two examples complexes recently...
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[NMR paper] Structure determination of protein complexes by NMR.
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This chapter describes nuclear magnetic resonance (NMR) methods that can be used to determine the structures of protein complexes. Many of these techniques are also applicable to other systems (e.g., protein-nucleic acid complexes). In the first section, we discuss methodologies for optimizing the sample conditions for...
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Structure Determination of Protein Complexes by NMR
Structure Determination of Protein Complexes by NMR
D. Nietlispach, H.R. Mott, K.M. Stott, P.R. Nielsen, A. Thiru & E.D. Laue
The Department of Biochemistry, University of Cambridge
Introduction
As the structures of more proteins and domains are solved by structural genomics projects, the future of structural biology will be oriented more toward the study of macromolecular complexes. Since so many biological processes are mediated by interactions between proteins, it is important to study them at a molecular level. The study of protein-protein interactions also has applications in a...
Fast methionine-based solution structure determination of calcium-calmodulin complexes
Linear Mode
