Engineered solubility tag for solution NMR of proteins

		BioNMR > Educational resources > Journal club
Engineered solubility tag for solution NMR of proteins

Webservers

NMR processing:

MDD

NMR assignment:

Backbone:

Side-chains:

NOEs:

Structure from NMR restraints:

Ab initio:

Fragment-based:

Rosetta-NMR (Robetta)

Template-based:

GeNMR

I-TASSER

Refinement:

Amber

Structure from chemical shifts:

Fragment-based:

Homology-based:

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:

NMR model quality:

NOEs, other restraints:

Chemical shifts:

RDCs:

Pseudocontact shifts:

Protein geomtery:

SAVES2 or SAVES4

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:

Isotope labeling:

Solid-state NMR:

Thread Tools

Search this Thread

Rate Thread

Display Modes

09-20-2013, 02:39 PM

nmrlearner

Senior Member

Join Date: Jan 2005

Posts: 23,777

Points: 193,617, Level: 100

Level up: 0%, 0 Points needed

Activity: 50.7%

Last Achievements

Award-Showcase

NMR Credits: 0

NMR Points: 193,617

Downloads: 0

Uploads: 0

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 nuclear magnetic resonance (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. © 2013 The Protein Society

More...

Did you find this post helpful?

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
[NMR paper] Engineered solubility tag for solution NMR of proteins. Engineered solubility tag for solution NMR of proteins. Engineered solubility tag for solution NMR of proteins. Protein Sci. 2013 Aug 21; Authors: Ruschak AM, Rose JD, Coughlin MP, Religa TL 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...	nmrlearner	Journal club	0	08-23-2013 01:07 AM
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...	nmrlearner	Journal club	0	08-21-2013 06:36 AM
[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 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

« Previous Thread | Next 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