skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.

Abstract

We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity and to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.

Authors:
; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
990158
Report Number(s):
ANL/BIO/JA-62493
Journal ID: ISSN 0006-2960; BICHAW; TRN: US201020%%181
DOE Contract Number:  
DE-AC02-06CH11357
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochem.; Journal Volume: 47; Journal Issue: 52 ; 12/8/2008
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; AMINO ACIDS; FUNCTIONALS; PLATES; PROTEINS; SCREENS; SPECIFICITY; TARGETS; VALIDATION

Citation Formats

Giulliani, S. E., Frank, A. E., Collart, F. R., and Biosciences Division. Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.. United States: N. p., 2008. Web. doi:10.1021/bi801648r.
Giulliani, S. E., Frank, A. E., Collart, F. R., & Biosciences Division. Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.. United States. doi:10.1021/bi801648r.
Giulliani, S. E., Frank, A. E., Collart, F. R., and Biosciences Division. Mon . "Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.". United States. doi:10.1021/bi801648r.
@article{osti_990158,
title = {Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.},
author = {Giulliani, S. E. and Frank, A. E. and Collart, F. R. and Biosciences Division},
abstractNote = {We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity and to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.},
doi = {10.1021/bi801648r},
journal = {Biochem.},
number = 52 ; 12/8/2008,
volume = 47,
place = {United States},
year = {Mon Dec 08 00:00:00 EST 2008},
month = {Mon Dec 08 00:00:00 EST 2008}
}