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Protein Sci. 2017 Nov;26(11):2240-2248. doi: 10.1002/pro.3282. Epub 2017 Sep 27.

NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma-tubulin complex.

Author information

1
Institut de Pharmacologie et Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, Toulouse, France.
2
Centre de Biologie du Développement, Université de Toulouse, CNRS, UPS, Toulouse, France.

Abstract

Mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ-tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha-helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N-terminus (residues 1-250) of GCP3 (γ-tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.

KEYWORDS:

GCP3; MOZART1; NMR; oligomeric state; γ-tubulin

PMID:
28851027
PMCID:
PMC5654863
DOI:
10.1002/pro.3282
[Indexed for MEDLINE]
Free PMC Article

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