BioNMR
NMR aggregator & online community since 2003
BioNMR    
Learn or help to learn NMR - get free NMR books!
 

Go Back   BioNMR > Educational resources > Journal club
Advanced Search



Jobs Groups Conferences Literature Pulse sequences Software forums Programs Sample preps Web resources BioNMR issues


Webservers
NMR processing:
MDD
NMR assignment:
Backbone:
Autoassign
MARS
UNIO Match
PINE
Side-chains:
UNIO ATNOS-Ascan
NOEs:
UNIO ATNOS-Candid
UNIO Candid
ASDP
Structure from NMR restraints:
Ab initio:
GeNMR
Cyana
XPLOR-NIH
ASDP
UNIO ATNOS-Candid
UNIO Candid
Fragment-based:
BMRB CS-Rosetta
Rosetta-NMR (Robetta)
Template-based:
GeNMR
I-TASSER
Refinement:
Amber
Structure from chemical shifts:
Fragment-based:
WeNMR CS-Rosetta
BMRB CS-Rosetta
Homology-based:
CS23D
Simshift
Torsion angles from chemical shifts:
Preditor
TALOS
Promega- Proline
Secondary structure from chemical shifts:
CSI (via RCI server)
TALOS
MICS caps, β-turns
d2D
PECAN
Flexibility from chemical shifts:
RCI
Interactions from chemical shifts:
HADDOCK
Chemical shifts re-referencing:
Shiftcor
UNIO Shiftinspector
LACS
CheckShift
RefDB
NMR model quality:
NOEs, other restraints:
PROSESS
PSVS
RPF scores
iCing
Chemical shifts:
PROSESS
CheShift2
Vasco
iCing
RDCs:
DC
Anisofit
Pseudocontact shifts:
Anisofit
Protein geomtery:
Resolution-by-Proxy
PROSESS
What-If
iCing
PSVS
MolProbity
SAVES2 or SAVES4
Vadar
Prosa
ProQ
MetaMQAPII
PSQS
Eval123D
STAN
Ramachandran Plot
Rampage
ERRAT
Verify_3D
Harmony
Quality Control Check
NMR spectrum prediction:
FANDAS
MestReS
V-NMR
Flexibility from structure:
Backbone S2
Methyl S2
B-factor
Molecular dynamics:
Gromacs
Amber
Antechamber
Chemical shifts prediction:
From structure:
Shiftx2
Sparta+
Camshift
CH3shift- Methyl
ArShift- Aromatic
ShiftS
Proshift
PPM
CheShift-2- Cα
From sequence:
Shifty
Camcoil
Poulsen_rc_CS
Disordered proteins:
MAXOCC
Format conversion & validation:
CCPN
From NMR-STAR 3.1
Validate NMR-STAR 3.1
NMR sample preparation:
Protein disorder:
DisMeta
Protein solubility:
camLILA
ccSOL
Camfold
camGroEL
Zyggregator
Isotope labeling:
UPLABEL
Solid-state NMR:
sedNMR


Reply
Thread Tools Search this Thread Rate Thread Display Modes
  #1  
Unread 09-13-2020, 09:18 AM
nmrlearner's Avatar
Senior Member
 
Join Date: Jan 2005
Posts: 23,135
Points: 193,617, Level: 100
Points: 193,617, Level: 100 Points: 193,617, Level: 100 Points: 193,617, Level: 100
Level up: 0%, 0 Points needed
Level up: 0% Level up: 0% Level up: 0%
Activity: 50.7%
Activity: 50.7% Activity: 50.7% Activity: 50.7%
Last Achievements
Award-Showcase
NMR Credits: 0
NMR Points: 193,617
Downloads: 0
Uploads: 0
Default Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.

Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.

Related Articles Sparse isotope labeling for nuclear magnetic resonance (NMR) of glycoproteins using 13C-glucose.

Glycobiology. 2020 Sep 08;:

Authors: Rogals MJ, Yang JY, Williams RV, Moremen KW, Amster IJ, Prestegard JH

Abstract
Preparation of samples for nuclear magnetic resonance (NMR) characterization of larger proteins requires enrichment with less abundant, NMR-active, isotopes such as 13C and 15N. This is routine for proteins that can be expressed in bacterial culture where low-cost isotopically enriched metabolic substrates can be used. However, it can be expensive for glycosylated proteins expressed in mammalian culture where more costly isotopically enriched amino acids are usually used. We describe a simple, relatively inexpensive procedure in which standard commercial media is supplemented with 13C-enriched glucose to achieve labeling of all glycans plus all alanines of the N-terminal domain of the highly glycosylated protein, CEACAM1. We demonstrate an ability to detect partially occupied N-glycan sites, sites less susceptible to processing by an endoglycosidase, and some unexpected truncation of the amino acid sequence. The labeling of both the protein (through alanines) and the glycans in a single culture requiring no additional technical expertise past standard mammalian expression requirements is anticipated to have several applications, including structural and functional screening of the many glycosylated proteins important to human health.


PMID: 32902634 [PubMed - as supplied by publisher]



More...
Reply With Quote


Did you find this post helpful? Yes | No

Reply
Similar Threads
Thread Thread Starter Forum Replies Last Post
[NMR paper] Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems.
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems. Related Articles Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems. J Biomol NMR. 2018 Feb 28;: Authors: Yanaka S, Yagi H, Yogo R, Yagi-Utsumi M, Kato K Abstract Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing...
nmrlearner Journal club 0 03-02-2018 03:20 PM
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems
Stable isotope labeling approaches for NMR characterization of glycoproteins using eukaryotic expression systems Abstract Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope labeling in recombinant glycoproteins have been developed recently using a variety of eukaryotic production vehicles,...
nmrlearner Journal club 0 02-28-2018 03:32 PM
NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13 C-methyl alanine
NMR characterization of HtpG, the E. coli Hsp90, using sparse labeling with 13 C-methyl alanine Abstract A strategy for acquiring structural information from sparsely isotopically labeled large proteins is illustrated with an application to the E. coli heat-shock protein, HtpG (high temperature protein G), a 145Â*kDa dimer. It uses 13C-alanine methyl labeling in a perdeuterated background to take advantage of the sensitivity and resolution of Methyl-TROSY spectra, as well as the backbone-centered structural information from 1Hâ??13C residual dipolar...
nmrlearner Journal club 0 06-27-2017 03:27 AM
[NMR paper] Sparse labeling of proteins: Structural characterization from long range constraints
Sparse labeling of proteins: Structural characterization from long range constraints Publication date: April 2014 Source:Journal of Magnetic Resonance, Volume 241</br> Author(s): James H. Prestegard , David A. Agard , Kelley W. Moremen , Laura A. Lavery , Laura C. Morris , Kari Pederson</br> Structural characterization of biologically important proteins faces many challenges associated with degradation of resolution as molecular size increases and loss of resolution improving tools such as perdeuteration when non-bacterial hosts must be used for expression. In...
nmrlearner Journal club 0 03-22-2014 01:28 AM
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins
Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins Abstract In the last 15 years substantial advances have been made to place isotope labels in native and glycosylated proteins for NMR studies and structure determination. Key developments include segmental isotope labeling using Native Chemical Ligation, Expressed Protein Ligation and Protein Trans-Splicing. These advances are pushing the size limit of NMR spectroscopy further making larger proteins accessible for this technique. It is just emerging that segmental isotope...
nmrlearner Journal club 0 01-09-2011 12:46 PM
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a m
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system. Related Articles Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system. Prog Nucl Magn Reson Spectrosc. 2010 May;56(4):346-59 Authors: Kato K, Yamaguchi Y, Arata Y
nmrlearner Journal club 0 10-19-2010 04:51 PM
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a m
Stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G as a model system Publication year: 2010 Source: Progress in Nuclear Magnetic Resonance Spectroscopy, In Press, Accepted Manuscript, Available online 19 March 2010</br> Koichi, Kato , Yoshiki, Yamaguchi , Yoji, Arata</br> More...
nmrlearner Journal club 0 08-16-2010 03:50 AM
Enhanced production and isotope enrichment of recombinant glycoproteins produced in c
Abstract NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant...
nmrlearner Journal club 0 08-14-2010 04:19 AM


Thread Tools Search this Thread
Search this Thread:

Advanced Search
Display Modes Rate This Thread
Rate This Thread:

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is On
Trackbacks are Off
Pingbacks are Off
Refbacks are Off



BioNMR advertisements to pay for website hosting and domain registration. Nobody does it for us.



Powered by vBulletin® Version 3.7.3
Copyright ©2000 - 2024, Jelsoft Enterprises Ltd.
Copyright, BioNMR.com, 2003-2013
Search Engine Friendly URLs by vBSEO 3.6.0

All times are GMT. The time now is 06:03 AM.


Map