Document Detail


Differential scanning fluorimetry measurement of protein stability changes upon binding to glycosaminoglycans: a screening test for binding specificity.
MedLine Citation:
PMID:  20353159     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The interaction between glycosaminoglycans (GAGs) and proteins is important for the regulation of protein transport and activity. Here we present a novel method for the measurement of protein-GAG interactions suitable for high-throughput screening, able to discriminate between the interactions of a protein with GAGs of different structures. Binding of proteins to the GAG heparin, a proxy for sulfated regions of extracellular heparan sulfate, was found to enhance the stability of three test proteins, fibroblast growth factors (FGFs)-1, -2, and -18. Chemically modified heparins and heparin oligosaccharides of different lengths stabilized the three FGFs to different extents, depending on the pattern of sugar binding specificity. The method is based on a differential scanning fluorescence approach. It uses a Sypro Orange dye, which binds to exposed core residues of a denatured protein and results in an increased fluorescence signal. It is convenient, requiring low micromolar amounts of protein and ligand compared to other interaction assays, employing only a real-time polymerase chain reaction (PCR) instrument.
Authors:
Katarzyna A Uniewicz; Alessandro Ori; Ruoyan Xu; Yassir Ahmed; Mark C Wilkinson; David G Fernig; Edwin A Yates
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical chemistry     Volume:  82     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2010 May 
Date Detail:
Created Date:  2010-04-30     Completed Date:  2010-08-18     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3796-802     Citation Subset:  IM    
Affiliation:
School of Biological Sciences, University of Liverpool, Liverpool, L69 7ZB, United Kingdom.
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MeSH Terms
Descriptor/Qualifier:
Binding Sites
Calorimetry, Differential Scanning
Fibroblast Growth Factor 1 / chemistry,  metabolism
Fibroblast Growth Factor 2 / chemistry,  metabolism
Fluorometry*
Glycosaminoglycans / chemistry*,  metabolism*
Polymerase Chain Reaction
Protein Binding
Protein Stability
Chemical
Reg. No./Substance:
0/Glycosaminoglycans; 103107-01-3/Fibroblast Growth Factor 2; 104781-85-3/Fibroblast Growth Factor 1

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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