Linked Life Data

19689113

Source:http://linkedlifedata.com/resource/pubmed/id/19689113

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
18
pubmed:dateCreated
2009-9-14
pubmed:abstractText
The GM2 activator protein (GM2AP) is an 18 kDa nonenzymatic accessory protein involved in the degradation of neuronal gangliosides. Genetic mutations of GM2AP can disrupt ganglioside catabolism and lead to deadly lysosomal storage disorders. Crystallography of wild-type GM2AP reveals 4 disulfide bonds and multiple conformations of a flexible loop region that is thought to be involved in lipid binding. To extend the crystallography results, a cysteine construct (L126C) was expressed and modified with 4-maleimide TEMPO for electron paramagnetic resonance (EPR) studies. However, because a ninth cysteine has been added by site-directed mutagenesis and the protein was expressed in E. coli in the form of inclusion bodies, the protein could misfold during expression. To verify correct protein folding and labeling, a sequential multiple-protease digestion, nano-liquid chromatograph (LC) electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry assay was developed. High-magnetic field and robust automatic gain control results in subppm mass accuracy for location of the spin-labeled cysteine and verification of proper connectivity of the four disulfide bonds. The sequential multiple protease digestion strategy and ultrahigh mass accuracy provided by FTICR MS allow for rapid and unequivocal assignment of relevant peptides and provide a simple pipeline for analyzing other GM2AP constructs.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
1520-6882
pubmed:author
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
81
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7611-7
pubmed:dateRevised
2011-9-26
pubmed:meshHeading
pubmed-meshheading:19689113-Amino Acid Sequence, pubmed-meshheading:19689113-Amino Acid Substitution, pubmed-meshheading:19689113-Crystallography, X-Ray, pubmed-meshheading:19689113-Cyclic N-Oxides, pubmed-meshheading:19689113-Disulfides, pubmed-meshheading:19689113-Electron Spin Resonance Spectroscopy, pubmed-meshheading:19689113-Fourier Analysis, pubmed-meshheading:19689113-G(M2) Activator Protein, pubmed-meshheading:19689113-Maleimides, pubmed-meshheading:19689113-Molecular Sequence Data, pubmed-meshheading:19689113-Mutagenesis, Site-Directed, pubmed-meshheading:19689113-Peptides, pubmed-meshheading:19689113-Protein Folding, pubmed-meshheading:19689113-Recombinant Proteins, pubmed-meshheading:19689113-Spectrometry, Mass, Electrospray Ionization, pubmed-meshheading:19689113-Spin Labels, pubmed-meshheading:19689113-Trypsin
pubmed:year
2009
pubmed:articleTitle
Sequential proteolysis and high-field FTICR MS to determine disulfide connectivity and 4-maleimide TEMPO spin-label location in L126C GM2 activator protein.
pubmed:affiliation
Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural