Difference between revisions of "Team:Toronto/Notebook-w03-thu"

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Revision as of 05:53, 19 October 2016

console.js:26

Week 3: June 2 2016

Thursday, 6/2
Members present: Kat, Karim, Tam, Bohdan, Andrea, Hamed
Lab Stuff: (research, plasmid design, protocol design, troubleshooting)
*Our RRFs were finally approved, and we were able to obtain two keys from Susie*
GolS Structure
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2.6. 3D modeling of GolS proteins
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3D structure of GolS proteins was predicted by 3DLigandSite server (http://www.sbg.bio.ic.ac.uk/3dligandsite/) (Wass et al., 2010) and analyzed by using Swiss Pdb Viewer 4.1.0. 3D model quality was checked by Rampage Ramachandran plot analysis (http://mordred.bioc.cam.ac.uk/∼rapper/rampage.php) (Lovell et al., 2003).
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3D model of GolS found to resemble our model
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Rosetta as a possible modelling program option
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GolS Dimerization and metal-binding
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Metal binding involves Ser77 (of other monomer), Cys112, Cys120
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Dimerization involves S77-X2-V-K
Rosetta: https://www.rosett
acommons.org/
We figured that since GolS is not a well studied protein so not many predicted 3D structurers were established. We look into a couple of articles on how they monitor GolS ligand docking and dimerization. There is an article in which GolS binding to DNA was monitored using program called Rosetta 3.1. Unfortunately the program is written under Python language so therefore, the wet lab will ask for assistance from the computational lab.
Input to I-TASSER structure of cleaved His-tag (Ser & Gly versions) GolS:
Job ID: S277159 - GolS cleaved His (Gly) - Kat
Job ID: S277165 - GolS cleaved His (Ser) - Cathy
Results for GolS cleaved His (Gly):
GolS appeared to have a higher similarity to CueR after the cleavage of linker His-tag.
There is a suggestion that the modified GolS cleaved with His-tag (Gly) may appear to behave more like CueR in terms of structure wise. The difference in terms of confidence for the wt GolS is 0.08 and the cleaved wt GolS is 0.09, very small.
GolS cleaved His-tag (Gly).gif
thumbnail
There is a little shift with the pocket binding and C-terminus that would suggest for a little change in the binding of the GolS to metal ions. DNA domain remains the same.
GolS.gif
thumbnail
Besides that, we conclude cleavable linker His-tag maybe affect the protein dimerization significantly once cleavage is done.
GolS cleavable linker His-tag.gif
thumbnail
On the other hand, the uncleaved linker His-tag affects the structure drastically that it results in a huge shift and bent in the structure of the protein.
In conclusion, it possesses some certain risks that the protein may not be able to refold back after the cleavage and chaperones may not assist refolding for the protein efficiently enough. Therefore, we believe His-tag presents some certain risks to the protein loss of metal binding affinity. However, we still assemble a plasmid with the cleavable linker and His-tag parallel to the experiment.
Purification Procedures
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Alternative purification procedures from 1987, 1989
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uses heparin sepharose affinity column
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2-mercaptoethanol used in procedure (for disulphide bridges) does not appear to affect stability of the protein.
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His-tag purifications used in previous experiments (MerR, ZntR) do not appear to impact function
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However, GolS is ~14 residues longer
Administrative: (shipment orders, inventory)
Attained Lockers in the Walbeur Building basement # 646-651
Locker #650 will be designated as the IGEM locker (needs to have a lock supplied)
Email Updates: (Correspondance with proffessors, shipping companies, etc)
Got in touch with Anthoula Vlahakis in regard to accessing MedStore account along with last years order reciepts.
Emailed Susie regarding transporting materials from Steipe's lab into 403 in the morning along with requesting for permission for the use of consumables: ... from room 303
Meeting Notes:
ON MONDAY: Ask if the the pheumatic press downstairs is a "french press"
References:
http://ac.els-cdn.com/S1476927115300773/1-s2.0-S1476927115300773-main.pdf?_tid=102556c8-28d2-11e6-ae05-00000aacb361&acdnat=1464879516_b2f75238850ae006540d58f443f845b5
-possible procedure for finding ligand binding domains
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2007.05590.x/epdf
-Figure 1b: aas involved in binding in black, (77)S-X2-V-K involved in dimerization
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711316/
-Figure 4
-3D structure of GolS
-Rosetta 3.1 used
http://www.ganino.com/games/Science/Science%201986-1988/root/data/Science_1986-1988/pdf/1987_v235_n4785/p4785_0211.pdf
-Figure 2
-old school procedure
-contains denaturant
http://pubs.acs.org.myaccess.library.utoronto.ca/doi/pdf/10.1021/bi00431a053
-ctrl+f purification
-modified version of above 1987 procedure
-followed up with DNA and Hg binding assays, implying that despite denaturant involved in purification procedure, protein is still functional
http://pubs.acs.org/doi/full/10.1021/bi9817562
-his-tags do not impact MerR
http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1999.01466.x/full
-his-tags do not impact ZntR