Abstract/Details

Development of a steric trap method to study water soluble and membrane protein folding


2010 2010

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Abstract (summary)

The unique environment of the membrane protein has caused a severe discrepancy between the level of importance of membrane proteins and the level of our current understanding of them. To rectify this disparity we have developed a novel method, The Steric Trap Method, which couples protein unfolding to a measurable binding event. The simultaneous binding of two monovalent streptavidin (mSA) molecules to a dually, biotinylated target protein is inhibited when the protein is folded due to steric hindrance created by the bulky bound molecules and can occur only when an unfolded state is sampled under the condition of dynamic equilibrium. Therefore, the binding affinity of mSA for the second biotin site on the target protein is correlated to the unfolding energy of the protein. This allows the investigation of folding energetics in the native environment of lipid bilayers without the harsh denaturing conditions utilized by existing methods. It also eliminates the need for dilution, a disadvantage of existing methods, essential for probing high affinity interactions.

We have successfully applied the steric trap method to study both soluble protein folding for the model enzyme, dihydrofolate reducatase (DHFR), and membrane protein folding for glycophorin A (GpA) and diacylglycerol kinase (DGK). We have used the steric trap method to study protein folding and stability, protein-protein interaction energetics, and unfolding kinetics. We can investigate the effects of different mutations, detergents, lipids, or any other change to the folding conditions. Lastly, we can also irreversibly (on the order of several days) trap the unfolded state to enable its characterization.

Indexing (details)


Subject
Biochemistry
Classification
0487: Biochemistry
Identifier / keyword
Pure sciences; Dihydrofolate reductase; Membrane protein; Monovalent streptavidin; Protein folding; Steric trap
Title
Development of a steric trap method to study water soluble and membrane protein folding
Author
Blois, Tracy Mitchell
Number of pages
129
Publication year
2010
Degree date
2010
School code
0031
Source
DAI-B 71/10, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9781124225029
Advisor
Bowie, James U.
University/institution
University of California, Los Angeles
University location
United States -- California
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3424209
ProQuest document ID
753281816
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/753281816
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