Rapid NMR-scale purification of 15N,13C isotope-labeled recombinant human STIM1 coiled coil fragments.
Protein Expr Purif. 2018 Feb 01;146:45-50
Authors: Rathner P, Stadlbauer M, Romanin C, Fahrner M, Derler I, Müller N
Abstract
We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapid purification procedure for two 15N,13C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded 15N,13C isotope-labeled cytosolic coiled coil fragments followed by detergent screening provide for initial NMR characterization of these domains. The longer 30.5 kDa fragment represents the largest STIM1 wild type fragment that has been recombinantly prepared and characterized in solution without need for mutation or refolding.
PMID: 29414068 [PubMed - as supplied by publisher]
[NMR paper] Probing coiled-coil assembly by paramagnetic NMR spectroscopy.
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http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.rsc.org-images-entities-char_z_RSClogo.gif Related Articles Probing coiled-coil assembly by paramagnetic NMR spectroscopy.
Org Biomol Chem. 2015 Jan 28;13(4):1159-68
Authors: Zheng T, Boyle A, Robson Marsden H, Valdink D, Martelli G, Raap J, Kros A
Abstract
Here a new method to determine the oligomeric state and orientation of coiled-coil peptide motifs is described. Peptides K and E, which...
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Abstract
Alzheimer's disease (AD) is a progressive neurodegenerative disease...
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Protein Expr Purif. 2013 Aug 1;
Authors: Bhate MP, Wylie BJ, Thompson A, Tian L, Nimigean C, McDermott AE
Abstract
We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled...
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Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein ?i1
Expression, purification and preliminary NMR characterization of isotopically labeled wild-type human heterotrimeric G protein ?i1
August 2012
Publication year: 2012
Source:Protein Expression and Purification, Volume 84, Issue 2</br>
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Molecular-level investigation of proteins is increasingly important to researchers trying to understand the mechanisms of signal transmission. Heterotrimeric G proteins control the activation of many critical signal transmission cascades and are also implicated in numerous diseases. As part of a longer-term investigation of...
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Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.
Expression and purification of (15)N- and (13)C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.
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[NMR paper] Rapid and accurate structure determination of coiled-coil domains using NMR dipolar couplings: application to cGMP-dependent protein kinase Ialpha.
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Authors: Schnell JR, Zhou GP, Zweckstetter M, Rigby AC, Chou JJ
Coiled-coil motifs play essential roles in protein assembly and molecular recognition, and are therefore the targets of many ongoing...
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Nat Struct Biol. 1998 Aug;5(8):687-91
Authors: Dames SA, Kammerer RA, Wiltscheck R, Engel J, Alexandrescu AT
The solution structure of the oligomerization domain of cartilage matrix protein (also known as matrilin-1) has been determined by heteronuclear NMR spectroscopy. The domain folds into a parallel, disulfide-linked, three-stranded, alpha-helical coiled coil, spanning five heptad repeats in the amino acid sequence....
Rapid NMR-scale purification of 15N,13C isotope-labeled recombinant human STIM1 coiled coil fragments.
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