Related ArticlesImproved initial yields in C-terminal sequence analysis by thiohydantoin chemistry using purified diphenylphosphoryl isothiocyanate: NMR evidence for a reaction intermediate in the coupling reaction.
Anal Biochem. 2002 Aug 15;307(2):202-11
Authors: Graham K, Shively JE
A thiohydantoin method for C-terminal sequence analysis of proteins on Zitex membranes involves derivatization of the free alpha-carboxyl group with diphenylphosphoryl isothiocyanate (DPPITC) plus treatment with pyridine to form a peptidylthiohydantoin derivative, cleavage of the thiohydantoin (TH) amino acid from the protein with potassium trimethylsilanolate, and identification of the released TH-amino acid by online reversed-phase HPLC. This automated chemistry, which was adapted to the Hewlett-Packard G 1009A sequencer, has been shown to identify two or three cycles on a wide variety of proteins, but suffers from low initial yields and instability of the DPPITC reagent. We report here an improved method for synthesis and purification of DPPITC. With this procedure the DPPITC reagent is a clear liquid that is stable at room temperature under vacuum for more than 9 months or for more than 24 months as a 1.0M solution in benzene at -20 degrees C. Using the purified reagent we were able to more than double the initial yield (from 30.7 to 72.4%) of TH-amino acid from a test protein and substantially decrease sequencer background. Examination of the reaction between DPPITC and the carboxylate of model N-terminally protected dipeptides with 31P NMR provides spectroscopic evidence for a postulated intermediate formed between the DPPITC and the peptide carboxylate. The reaction intermediate provides new insight into the coupling mechanism.
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
Abstract An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract,...
nmrlearner
Journal club
0
10-05-2011 08:57 PM
Use of optimized 1D TOCSY NMR for improved quantitation and metabolomic analysis of biofluids
Use of optimized 1D TOCSY NMR for improved quantitation and metabolomic analysis of biofluids
Abstract One dimensional selective TOCSY experiments have been shown to be advantageous in providing improved data inputs for principle component analysis (PCA) (Sandusky and Raftery 2005a, b). Better subpopulation cluster resolution in the observed scores plots results from the ability to isolate metabolite signals of interest via the TOCSY based filtering approach. This report reexamines the quantitative aspects of this approach, first by optimizing the 1D TOCSY experiment as it relates to...
nmrlearner
Journal club
0
03-13-2011 05:24 AM
[NMR paper] NMR solution structure and membrane interaction of the N-terminal sequence (1-30) of
NMR solution structure and membrane interaction of the N-terminal sequence (1-30) of the bovine prion protein.
Related Articles NMR solution structure and membrane interaction of the N-terminal sequence (1-30) of the bovine prion protein.
Biochemistry. 2004 Nov 30;43(47):14940-7
Authors: Biverståhl H, Andersson A, Gräslund A, Mäler L
The structure and membrane interaction of the N-terminal sequence (1-30) of the bovine prion protein (bPrPp) has been investigated by NMR spectroscopy in phospholipid membrane mimetic systems. CD spectroscopy...
nmrlearner
Journal club
0
11-24-2010 10:03 PM
[NMR paper] Expression and initial structural insights from solid-state NMR of the M2 proton chan
Expression and initial structural insights from solid-state NMR of the M2 proton channel from influenza A virus.
Related Articles Expression and initial structural insights from solid-state NMR of the M2 proton channel from influenza A virus.
Biochemistry. 2002 Sep 17;41(37):11294-300
Authors: Tian C, Tobler K, Lamb RA, Pinto LH, Cross TA
The M2 protein from influenza A virus has been expressed, purified, and reconstituted into DMPC/DMPG liposomes. SDS-PAGE analysis of reconstituted M2 protein in DMPC/DMPG liposomes demonstrates a stable...
nmrlearner
Journal club
0
11-24-2010 08:58 PM
[NMR paper] Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal doma
Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.nrc-cnrc.gc.ca-cisti-journals-rp-gifs-PubMed_logo_e.gif Related Articles Bacterial overexpression, isotope enrichment, and NMR analysis of the N-terminal domain of human apolipoprotein E.
Biochem Cell Biol. 1997;75(1):45-53
Authors: Fisher CA, Wang J, Francis GA, Sykes BD, Kay CM, Ryan RO
The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human...
nmrlearner
Journal club
0
08-22-2010 03:31 PM
[BMNRC community] Protein sequence analysis websites
Protein sequence analysis websites
ProtParam tool
Go to BMNRC community to find more info about this topic.
nmrlearner
News from other NMR forums
0
08-22-2010 01:23 AM
[NMR paper] Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger o
Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger of the HIV-1 nucleocapsid protein: NMR structure of the complex with the Psi-site analog, dACGCC.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.pubmedcentral.nih.gov-corehtml-pmc-pmcgifs-pubmed-pmc.gif Related Articles Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger of the HIV-1 nucleocapsid protein: NMR...
nmrlearner
Journal club
0
08-21-2010 11:53 PM
[NMR paper] Nucleotide sequence and analysis of NMR, a negative-acting regulatory gene in the nit
Nucleotide sequence and analysis of NMR, a negative-acting regulatory gene in the nitrogen circuit of Neurospora crassa.
Related Articles Nucleotide sequence and analysis of NMR, a negative-acting regulatory gene in the nitrogen circuit of Neurospora crassa.
Mol Gen Genet. 1990 Jun;222(1):120-8
Authors: Young JL, Jarai G, Fu YH, Marzluf GA
In Neurospora the expression of a set of unlinked structural genes, which allows utilization of various nitrogen-containing compounds, is controlled by the positive-acting nit-2 gene and the negative-acting...
Improved initial yields in C-terminal sequence analysis by thiohydantoin chemistry us
Linear Mode
