User:Jeff Quinn

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Contact Info

Jeff Quinn

Jeffrey Quinn, MIT 2010, Course 20

• (916)765-9607
• jquinn[at]mit.edu

Module 3 Research Proposition Information

Project Overview

• By inserting the sequence of one protein into the sequence of another, the functionality of the protein can be lost; however, the new chimeric protein can assume new characteristics. For example, by inserting the sequence for an allosteric substrate-binding protein into the sequence of a metabolic enzyme, it is possible that the two proteins together can behave in a new way: as a switch. When substrate binds to the chimera, it can induce a conformational change in the enzyme that either activates or inactivates its enzymatic behavior, effectively creating a novel control mechanism for modulating the enzyme's activity with an input molecule (the substrate).
• Based on the experiment described by Guntas and Ostermeier, we plan to develop our own "molecular switch" protein in the same way that they did, by creating an allosteric enzyme that "couples effector levels (input) to enzyme activity (output)."
• Enzymes to work with (IDEAS):
  • hemoglobin
  • EGFP (easy to moniter!)
  • pyruvate decarboxylase (alcohol fermentation?)
  • insulin
• Substrate-Binding proteins to work with (IDEAS):
  • calmodulin
  • oxygen sensor?

Background Information

• End-to-End Fusion vs. Domain Insertion:
  • end-to-end fusion takes two proteins and fuses them together, often with a linkage, such that the each protein sequence (domain) is intact and separate. this nearly always guarantees that all parts of the chimeric protein will function as their native constituents. Examples of this include fluorescently-tagged proteins or Pericam, which end-to-end fuses calmodulin, M13, and a circularly-permuted EYFP.
  • Domain insertion, however, involves taking the sequence from one protein (insert domain) and inserting it into the middle of another (host? better word?) protein, such that the host protein's conformation and functionality are greatly disrupted. The advantage of this is that this new chimeric protein can adopt new behavior that reflects both the insert protein's and the host protein's behaviors, but only when the respective domains "cooperate" with eachother. For example, Guntas and Ostermeier took an enzyme that hydrolyzes penicillin-like antibiotics and inserted a protein that binds to maltose, a sugar molecule. After a library* of chimera proteins was produced, two such proteins showed new behavior: the antibiotic hydrolyzing enzyme would only break down antibiotics if the maltose-binding protein was bound to maltose. In effect, this creates a molecular switch such that the enzymatic activity can be controlled (turned off or on) by maltose.
• Domain Insertion Libraries are produced by randomly inserting the insert protein DNA domain into the host DNA sequence to generate numerous plasmids with the insert in various positions within the host sequence. The plasmids can then be expressed and individually tested for a) substrate binding ability and b) enzymatic activity.

Research Problem and Goals

• Develop a protein via domain insertion that can act as a molecular switch, in order to modulate its activity.
• Depending on what proteins we select for this, the goals will vary.
• We can also do some studies in order to determine how and why some random insertions work and why others don't. Does it have to do with linkages between the domains? Or the position at which the insert is inserted? Or how does the size of the insert domain affect overal functionality of the final chimeric protein? If the insert is pared down to the minimum to do its job, such that the insert domain is small, does that cause less disruption and therefore better library generations? We could possibly explore one or two of these questions as well...

Methods

• TBD based on our experiment
• However, it would likely follow what Guntas and Ostermeier have previously done.