Abstract NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally labeled MecA protein conjugate is ~40%. The resultant HSQC spectrum obtained from this domain-labeled conjugate demonstrates successful application of sortase A for segmental labeling of multi-domain proteins for solution NMR study.
Content Type Journal Article
DOI 10.1007/s10858-010-9464-2
Authors
Mary Anne Refaei, Department of Chemistry, University of Cincinnati, Cincinnati, 45221-0172 USA
Al Combs, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, 45267-0524 USA
Douglas J. Kojetin, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, 45267-0524 USA
John Cavanagh, Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA
Carol Caperelli, College of Pharmacy, University of Cincinnati, Cincinnati, 45267-0004 USA
Mark Rance, Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, 45267-0524 USA
Jennifer Sapitro, Department of Chemistry, University of Cincinnati, Cincinnati, 45221-0172 USA
Pearl Tsang, Department of Chemistry, University of Cincinnati, Cincinnati, 45221-0172 USA
Multiplet-filtered and gradient-selected zero-quantum TROSY experiments for 13C1H3 methyl groups in proteins
Multiplet-filtered and gradient-selected zero-quantum TROSY experiments for 13C1H3 methyl groups in proteins
Abstract Multiplet-filtered and gradient-selected heteronuclear zero-quantum coherence (gsHZQC) TROSY experiments are described for measuring 1Hâ??13C correlations for 13CH3 methyl groups in proteins. These experiments provide improved suppression of undesirable, broad outer components of the heteronuclear zero-quantum multiplet in medium-sized proteins, or in flexible sites of larger proteins, compared to previously described HZQC sequences (Tugarinov et al. in J Am Chem Soc...
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09-17-2011 10:20 AM
Narrowing the conformational space sampled by two-domain proteins with paramagnetic probes in both domains
Narrowing the conformational space sampled by two-domain proteins with paramagnetic probes in both domains
Abstract Calmodulin is a two-domain protein which in solution can adopt a variety of conformations upon reorientation of its domains. The maximum occurrence (MO) of a set of calmodulin conformations that are representative of the overall conformational space possibly sampled by the protein, has been calculated from the paramagnetism-based restraints. These restraints were measured after inclusion of a lanthanide binding tag in the C-terminal domain to supplement the data obtained...
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08-13-2011 02:47 AM
A segmental labeling strategy for unambiguous determination of domainâ??domain interactions of large multi-domain proteins
A segmental labeling strategy for unambiguous determination of domainâ??domain interactions of large multi-domain proteins
Abstract NMR structural determination of large multi-domain proteins is a challenging task due to significant spectral overlap with a particular difficulty in unambiguous identification of domainâ??domain interactions. Segmental labeling is a NMR strategy that allows for isotopically labeling one domain and leaves the other domain unlabeled. This significantly simplifies spectral overlaps and allows for quick identification of domainâ??domain interaction. Here, a...
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07-08-2011 07:01 PM
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.
J Biomol NMR. 2010 Dec 29;
Authors: Refaei MA, Combs A, Kojetin DJ, Cavanagh J, Caperelli C, Rance M, Sapitro J, Tsang P
NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to...
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12-29-2010 04:04 PM
[NMR paper] Evaluation of parameters critical to observing proteins inside living Escherichia col
Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy.
Related Articles Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy.
J Am Chem Soc. 2001 Sep 19;123(37):8895-901
Authors: Serber Z, Ledwidge R, Miller SM, Dötsch V
Our recently developed in-cell NMR procedure now enables one to observe protein conformations inside living cells. Optimization of the technique demonstrates that distinguishing the signals produced by a...
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11-19-2010 08:44 PM
[NMR paper] Chemical ligation of folded recombinant proteins: segmental isotopic labeling of doma
Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.
Related Articles Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.
Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):388-93
Authors: Xu R, Ayers B, Cowburn D, Muir TW
A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology...
Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy
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