Abstract/Details

Protein engineering and design of thermostable biosensors


2007 2007

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Abstract (summary)

Here I describe the application of rational protein engineering of bacterial periplasmic binding proteins to the design of thermostable biosensors. Periplasmic glucose- and ribose-binding proteins from thermophilic organisms have been identified, cloned and characterized. The structure of periplasmic glucose-binding protein from Thermotoga maritima (tmGBP) has been solved to 1.7 Å resolution by X-ray crystallography. A series of robust glucose biosensors have been constructed by coupling single, environmentally sensitive fluorophores to unique cysteines introduced by site-specific mutagenesis at positions responsive to ligand-induced conformational changes. Among tmGBP-based glucose biosensors, the Y13C•Cy5 conjugate signals strongly and responds to glucose concentrations appropriate for in vivo monitoring of blood glucose levels (1-30 mM). The Y13C•Cy5 conjugate has been immobilized onto microtiter plates in both semi-specific and orientation-specific manners to give reversible responses to glucose. The immobilized protein also retains its response after long term storage at room temperature.

In addition, E. coli ribose-binding protein (ecRBP) has been stabilized by rational protein engineering to enhance its suitability as a scaffold protein for use in computational design. Several approaches have been exploited to improve the thermostability of ecRBP, including the introduction of mutations to decrease the entropy of the unfolded form, the replacement of un-favored polar amino-acids in the protein core with non-polar residues, the engineering of disulfide bonds, and the incorporation of features from thermophilic RBPs. The stabilizing mutations achieved from these approaches were evaluated individually and then combined in a stepwise manner, resulting in a variant with a melting temperature 17.5°C higher than ecRBP, which can also serve as a stable scaffold protein for biosensor design.

Indexing (details)


Subject
Biochemistry
Classification
0487: Biochemistry
Identifier / keyword
Pure sciences; Biosensors; Protein engineering; Thermostability
Title
Protein engineering and design of thermostable biosensors
Author
Tian, Yaji
Number of pages
104
Publication year
2007
Degree date
2007
School code
0066
Source
DAI-B 68/06, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9780549072386
Advisor
Hallinga, Homme W.
University/institution
Duke University
University location
United States -- North Carolina
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3267812
ProQuest document ID
304863520
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/304863520
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