Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.
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.
Giuliani
Sarah E
SE
Biosciences Division, Argonne National Laboratory, Lemont, Illinois 60439, USA.
Frank
Ashley M
AM
Collart
Frank R
FR
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000596:Amino Acids; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D005453:Fluorescence; D008024:Ligands; D008722:Methods; D015336:Molecular Probe Techniques; D011485:Protein Binding; D011489:Protein Denaturation; D013696:Temperature
D018528:ATP-Binding Cassette Transporters; D000596:Amino Acids; D002352:Carrier Proteins; D008024:Ligands