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Protein Eng. 1998 Oct;11(10):847-53
Authors: McAlister MS, Davis B, Pfuhl M, Driscoll PC
CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and a subset of B-cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine-rich (SRCR) superfamily for which the three-dimensional polypeptide fold is as yet unknown. Glycosylated CD5 domain 1 (CD5d1) has been obtained by expression by secretion from both Chinese hamster ovary (CHO) cells and Pichia pastoris. Recombinant CD5d1 expressed in this manner was shown to be correctly folded by binding to anti-CD5 L17F12/Leu1 monoclonal antibody. Preliminary nuclear magnetic resonance (NMR) spectra obtained for CD5d1 (residues 1-118) had spectral dispersion typical of a folded protein, but otherwise of such poor quality that NMR structural studies were not feasible. The analysis of glycoproteins by NMR is frustrated by sample heterogeneity and poor spectral quality associated with glycan resonance overlap and the potential for increased line-widths due to the large hydrodynamic volume. In order to pursue NMR structural studies of CD5d1 it was necessary to optimize the quality of NMR spectra of CD5d1. A range of constructs of varying length and carbohydrate content were expressed in CHO cells and in P. pastoris. In addition the P. pastoris CD5d1 proved susceptible to N-glycan cleavage with endoglycosidase H. The protein products were characterised using size exclusion chromatography, NMR measurement of translational self-diffusion coefficients and two-dimensional 1H nuclear Overhauser effect spectroscopy experiments. Removal of an eight residue O-glycosylated C-terminal peptide, in particular, resulted in significant improvements in the quality of the CD5d1 NMR data, while retaining native protein structure. Two-dimensional heteronuclear NMR spectroscopy of nitrogen-15 isotope labelled deglycosylated CD5d1 (residues 1-110) prepared from P. pastoris suggests that this protein product is now amenable to solution structure determination.
N-terminal Dbl domain of the RhoGEF, Kalirin
N-terminal Dbl domain of the RhoGEF, Kalirin
N-terminal Dbl domain of the RhoGEF, Kalirin
Content Type Journal Article
Category NMR structure note
Pages 1-8
DOI 10.1007/s10858-012-9605-x
Authors
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Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain.
Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain.
Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain.
Biochimie. 2011 Sep 22;
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Abstract
The insulin-like growth factor binding proteins are a family of six proteins (IGFBP-1 to 6) that bind insulin-like growth factors-I and -II (IGF-I/II) with high affinity. In addition...
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09-30-2011 06:00 AM
Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain.
Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain.
Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain.
Biochimie. 2011 Sep 22;
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Abstract
The insulin-like growth factor binding proteins are a family of six proteins (IGFBP-1 to 6) that bind insulin-like growth factors-I and -II (IGF-I/II) with high affinity. In...
nmrlearner
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09-30-2011 05:59 AM
[NMR paper] Solution NMR structure of the SH3 domain of human nephrocystin and analysis of a muta
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Human nephrocystin is a protein associated with juvenile NPH, an autosomal recessive, inherited kidney disease responsible for chronic renal failure in children. It...
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Interferons are cytokines that play a complex role in the resistance of mammalian hosts to pathogens. IFNgamma (interferon-gamma) is secreted by activated T-cells and...
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[NMR paper] Solution NMR structure of the C-terminal domain of the human protein DEK.
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The chromatin-associated protein DEK was first identified as a fusion protein in patients with a subtype of acute myelogenous leukemia. It has since become associated with diverse human ailments ranging from cancers to autoimmune diseases. Despite much research effort, the biochemical basis for these...
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11-24-2010 09:51 PM
[NMR paper] The N-terminal domain of the human Rad51 protein binds DNA: structure and a DNA bindi
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J Mol Biol. 1999 Jul 9;290(2):495-504
Authors: Aihara H, Ito Y, Kurumizaka H, Yokoyama S, Shibata T
Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination. In higher eukaryotes, Rad51 protein is essential for cell viability. The...
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[NMR paper] NMR studies of U1 snRNA recognition by the N-terminal RNP domain of the human U1A pro
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EMBO J. 1994 Aug 15;13(16):3873-81
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The RNP domain is a very common motif found in hundreds of proteins, including many protein components of the RNA processing machinery. The 70-90...
NMR analysis of the N-terminal SRCR domain of human CD5: engineering of a glycoprotei
Linear Mode
