Giuliani Sarah E, Frank Ashley M, Collart Frank R
Biosciences Division, Argonne National Laboratory, Lemont, Illinois 60439, USA.
Biochemistry. 2008 Dec 30;47(52):13974-84. doi: 10.1021/bi801648r.
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.
我们使用了基于荧光的热迁移（FTS）分析方法来鉴定与细菌ABC转运蛋白家族中溶质结合蛋白相结合的氨基酸。该分析方法通过一组六个具有已知结合特异性的蛋白质进行了验证，并且始终能够将蛋白质与其已知的结合配体对应起来。该分析方法还为验证集中的几种氨基酸结合蛋白鉴定出了额外的候选结合配体。我们将此方法扩展到其他靶点，并证明了FTS分析能够明确鉴定出具有已知特异性的氨基酸结合蛋白的几种同系物的优先结合情况，并对具有未知结合特异性的蛋白进行功能注释。该分析以微孔板形式进行，为验证预期功能或筛选未知功能的蛋白质提供了一种快速方法。ABC型转运蛋白家族广泛存在，可转运多种生物化合物，但目前对配体结合蛋白的注释大多局限于功能的一般性描述。结果表明FTS分析在改善与ABC型转运蛋白相关的结合蛋白功能注释方面的可行性，并表明这种方法也可扩展到其他蛋白质家族。
