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 08-21-2013, 06:36 AM
nmrlearner's Avatar
Senior Member
 
Join Date: Jan 2005
Posts: 23,134
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 Engineered solubility tag for solution NMR of proteins

Engineered solubility tag for solution NMR of proteins

Abstract

The low solubility of many proteins hinders large scale expression and purification as well as biophysical measurements. Here, we devised a general strategy to solubilize a protein by conjugating it at a solvent exposed position to a 6 kDa protein that was re-engineered to be highly soluble. We applied this method to the CARD domain of Apoptosis-associated speck-like protein containing a CARD (ASC), which represents one member of a class of proteins that are notoriously prone to aggregation. Attachment of the tag to a cysteine residue, introduced by site-directed mutagenesis at its self-association interface, improved the solubility of the ASC CARD over 50-fold under physiological conditions. Although it is not possible to use NMR to obtain a high quality 2D correlation spectrum of the wild type domain under physiological conditions, we demonstrate that NMR relaxation parameters of the solubilized variant are sufficiently improved to facilitate virtually any demanding measurement. The method shown here represents a straightforward approach for dramatically increasing protein solubility, enabled by ease of labeling as well as flexibility in tag placement with minimal perturbation to the target.




More...
Reply With Quote


Did you find this post helpful? Yes | No

Reply
Similar Threads
Thread Thread Starter Forum Replies Last Post
Improving protein solubility & long-term stability
Has anybody successfully used the following method? A simple method for improving protein solubility and long-term stability. Golovanov AP, Hautbergue GM, Wilson SA, Lian LY. Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, P. O. Box 88, Manchester M60 1QD, UK. a.golovanov@umist.ac.jp J Am Chem Soc. 2004 Jul 28;126(29):8933-9.
nmrlearner Proteins 4 01-22-2014 07:04 PM
[NMR paper] Structure determination of ?-helical membrane proteins by solution-state NMR: Emphasis on retinal proteins.
Structure determination of ?-helical membrane proteins by solution-state NMR: Emphasis on retinal proteins. Structure determination of ?-helical membrane proteins by solution-state NMR: Emphasis on retinal proteins. Biochim Biophys Acta. 2013 Jul 2; Authors: Gautier A Abstract The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical...
nmrlearner Journal club 0 07-09-2013 02:47 PM
Structure determination of ?–helical membrane proteins by solution-state NMR: Emphasis on retinal proteins
Structure determination of ?–helical membrane proteins by solution-state NMR: Emphasis on retinal proteins Publication date: Available online 2 July 2013 Source:Biochimica et Biophysica Acta (BBA) - Bioenergetics</br> Author(s): Antoine Gautier</br> The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical reactions and mediate protein–protein interactions. Indeed, the...
nmrlearner Journal club 0 07-02-2013 09:44 AM
Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility
Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility Abstract A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS2) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using...
nmrlearner Journal club 0 03-08-2012 08:46 AM
Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies
Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies Abstract Although the rapid progress of NMR technology has significantly expanded the range of NMR-trackable systems, preparation of NMR-suitable samples that are highly soluble and stable remains a bottleneck for studies of many biological systems. The application of solubility-enhancement tags (SETs) has been highly effective in overcoming solubility and sample stability issues and has enabled structural studies of important biological systems previously deemed...
nmrlearner Journal club 0 01-09-2011 12:46 PM
[NMR paper] A straight-forward method of optimising protein solubility for NMR.
A straight-forward method of optimising protein solubility for NMR. Related Articles A straight-forward method of optimising protein solubility for NMR. J Biomol NMR. 2004 Nov;30(3):283-6 Authors: Howe PW Maximising solubility is a key step in applying solution-state NMR to proteins. The 'microbatch' crystallisation screening method can be adapted to screen for protein solubility. In this approach, drops of test solutions are placed under paraffin oil in 96-well screening plates. This requires very small amounts of protein, is easy to set up...
nmrlearner Journal club 0 11-24-2010 10:03 PM
[NMR paper] A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins.
A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins. Related Articles A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins. J Biomol NMR. 2001 May;20(1):11-4 Authors: Zhou P, Lugovskoy AA, Wagner G Protein-fusion constructs have been used with great success for enhancing expression of soluble recombinant protein and as tags for affinity purification. Unfortunately the most popular tags, such as GST and MBP, are large, which hinders direct NMR studies of the fusion proteins. Cleavage of the...
nmrlearner Journal club 0 11-19-2010 08:32 PM
Optimisation of protein solubility for NMR
A straight-forward method of optimising protein solubility for NMR. Howe PW. Analytical Sciences, Syngenta, Jealott's Hill Research Centre, Bracknell, Berkshire, RG42 6EY, UK. http://public.metapress.com/images/covers/publications/102922.jpg J Biomol NMR. 2004 Nov;30(3):283-6 Abstract: Maximising solubility is a key step in applying solution-state NMR to proteins. The 'microbatch' crystallisation screening method can be adapted to screen for protein solubility. In this approach, drops of test solutions are placed under paraffin oil in 96-well screening plates. This requires very...
nmrlearner Proteins 0 08-14-2005 07:53 PM


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 10:24 AM.


Map