The Protein Biochemistry Lab at UWindsor
  • Research
    • Research Interest
    • Facilities
    • Publications
  • Team
    • Lab and members
    • Future trainees
    • Collaborators
  • Teaching
  • Media & Blog
    • Blog
    • Media Coverage
  • Resources
    • Accumulated Wisdom
    • Codes
    • Fundings
    • Links
    • Windsor
  • Contact

The fusion protein approach to protein complex crystallization

13/11/2019

0 Comments

 
In our recent Nature Structural & Molecular Biology paper (Nov. 2019) on the interaction of the Myc oncoprotein with TATA-box binding transcription factor TBP, in collaboration with the Penn (Toronto, Canada) and Sunnerhagen (Linköping, Sweden) labs, we used a fusion protein approach to make a tripartite fusion protein that finally yielded a complex structure, which formed the cornerstone of the discovery described in the paper.

We used three copies of the GGGS motif for each linker between the target proteins. These linkers are so flexible that they did not show up in the electron density map and shall not impose any structural restraints. Several lines of evidence suggested the observed crystal structure of the fusion protein is physiologically relevant and not an artifact:
  1. A version of the tripartite fusion construct with only one copy of the GGGS motif resulted in a crystal structure with a nearly identical conformation of the three parts.
  2. Mutagenesis and protein interaction studies using NMR and SPR confirmed the interfacial residues observed in the crystal structure are involved in the interactions in solution.
  3. These interfacial residues are further confirmed to be important in in vivo cellular biology studies.

The reason we adopted this fusion protein approach is multi-faceted:
  1. The interaction between Myc and TBP is relatively week at a single-digit micromolar affinity.
  2. TBP forms a dimer that precludes a straight-forward reassembly of the recombinant ternary complex.
  3. The length of the TAF1-TAND1 peptide is 32 a.a. and that of Myc is 30 a.a., which could be costly to synthesize for crystallization work, especially when multiple constructs of the peptides are to be tried to find a well-diffracting crystal. A genetically-encoded peptide sequence gave us much flexibility in trying out different constructs.
Tips:
The fusion protein approach is versatile and has been used previously in other works, for example, in the structure of an Arf6 GTPase and ArfGAP(ASAP3) complex, where the affinity of the complex is low. However, this approach must be combined with other validation methods to confirm the interfaces observed in the structure are physiologically relevant. One such case of an artifact caused by the fusion approach is our unpublished structure (released in the public domain) of the fusion protein of Arf1 and ArfGAP1 (PDB:3O47), in which the interface is neither consistent with that of the Arf6/ASAP3 complex nor with an early report of the complex structure on Cell (1999).

The choice of linker is also worth considering. In the PDB:3O47 structure, we used a natural intrinsically disordered peptide (IDP) region of ArfGAP1 to serve as the linker to obtain the structure.
Picture
0 Comments

Avoiding Plagiarism and SafeAssign at UWindsor

1/11/2019

0 Comments

 
Plagiarism, including self-plagiarism, is a serious offence in academics. UWindsor Leddy library has a list of tips on avoiding plagiarism.

​SafeAssign is a plagiarism checking service available for UWindsor. However, it is slightly tricky to access the service. It is available in the Blackboard Learn system. Details can be found at the Wiki-page of UWindsor Blackboard Learn. "Direct submit" in the BB Sandbox course, is a quick way to check one's writing to avoid unintended plagiarism. It is not necessary to be a course assignment to use this service.
0 Comments

    Twitter
    ​@tongalumina

    Tong

    That's me.

    Archives

    December 2020
    November 2019
    October 2019

    Categories

    All
    Website
    Wisdom
    Writing

    RSS Feed

Powered by Create your own unique website with customizable templates.
  • Research
    • Research Interest
    • Facilities
    • Publications
  • Team
    • Lab and members
    • Future trainees
    • Collaborators
  • Teaching
  • Media & Blog
    • Blog
    • Media Coverage
  • Resources
    • Accumulated Wisdom
    • Codes
    • Fundings
    • Links
    • Windsor
  • Contact