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Computationally Guided Monomerization of Red Fluorescent Proteins of the Class Anthozoa


Wannier, Timothy Milton (2015) Computationally Guided Monomerization of Red Fluorescent Proteins of the Class Anthozoa. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9571908.


Red fluorescent proteins (RFPs) have attracted significant engineering focus because of the promise of near infrared fluorescent proteins, whose light penetrates biological tissue, and which would allow imaging inside of vertebrate animals. The RFP landscape, which numbers ~200 members, is mostly populated by engineered variants of four native RFPs, leaving the vast majority of native RFP biodiversity untouched. This is largely due to the fact that native RFPs are obligate tetramers, limiting their usefulness as fusion proteins. Monomerization has imposed critical costs on these evolved tetramers, however, as it has invariably led to loss of brightness, and often to many other adverse effects on the fluorescent properties of the derived monomeric variants. Here we have attempted to understand why monomerization has taken such a large toll on Anthozoa class RFPs, and to outline a clear strategy for their monomerization. We begin with a structural study of the far-red fluorescence of AQ143, one of the furthest red emitting RFPs. We then try to separate the problem of stable and bright fluorescence from the design of a soluble monomeric β-barrel surface by engineering a hybrid protein (DsRmCh) with an oligomeric parent that had been previously monomerized, DsRed, and a pre-stabilized monomeric core from mCherry. This allows us to use computational design to successfully design a stable, soluble, fluorescent monomer. Next we took HcRed, which is a previously unmonomerized RFP that has far-red fluorescence (λemission = 633 nm) and attempted to monomerize it making use of lessons learned from DsRmCh. We engineered two monomeric proteins by pre-stabilizing HcRed’s core, then monomerizing in stages, making use of computational design and directed evolution techniques such as error-prone mutagenesis and DNA shuffling. We call these proteins mGinger0.1 (λem = 637 nm / Φ = 0.02) and mGinger0.2 (λem = 631 nm Φ = 0.04). They are the furthest red first generation monomeric RFPs ever developed, are significantly thermostabilized, and add diversity to a small field of far-red monomeric FPs. We anticipate that the techniques we describe will be facilitate future RFP monomerization, and that further core optimization of the mGingers may allow significant improvements in brightness.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:RFP, Protein Engineering
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Molecular Biology and Biochemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Mayo, Stephen L.
Thesis Committee:
  • Pierce, Niles A. (chair)
  • Arnold, Frances Hamilton
  • Bjorkman, Pamela J.
  • Mayo, Stephen L.
Defense Date:19 December 2014
Record Number:CaltechTHESIS:01272015-110638252
Persistent URL:
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URLURL TypeDescription adapted for ch. 2
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8761
Deposited By: Timothy Wannier
Deposited On:04 Jan 2016 19:55
Last Modified:04 Oct 2019 00:07

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