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.

Periplasmic domain of the sensor-kinase BvgS reveals a new paradigm for the Venus flytrap mechanism

“…Similarly, no potential agonists were identified among the components of the growth medium or the metabolites produced by B. pertussis (15). We then used the thermal shift assay (TSA) (16), a technique that has been suc- cessfully applied to PBP-ligand interaction studies (17). The ligands were tested with the single-domain proteins (Table 1).…”

Characterization of DalS, an ATP-binding Cassette Transporter for d-Alanine, and Its Role in Pathogenesis in Salmonella enterica

“…Fluorescence Thermal Shift Assay-The fluorescence thermal shift assay was performed essentially as described previously (15). Amino acids were purchased from Sigma unless otherwise indicated.…”

“…Software calculated the first derivative values (Ϫd/dt) from raw fluorescence data to determine T m . A ⌬T m of Ͼ 2°C upon addition of amino acid was considered positive binding as described (15).…”

“…To identify the substrate(s) of DalS, we purified DalS and used this protein in a fluorescence thermal shift (FTS) assay previously developed to identify the amino acid specificity of solute-binding proteins in the bacterial ABC transport family (15). Amino acids were added to DalS-6HIS in the presence of SyPro Orange and fluorescence measurements were acquired during thermal shift.…”

Structural and functional characterization of the Staphylococcus aureus virulence factor and vaccine candidate FhuD2

“…DSF reveals protein melting temperature (T m ) via a chemical probe that displays enhanced fluorescence upon binding to hydrophobic regions of the protein core exposed upon thermally-induced denaturation [35]. Moreover, DSF has been used previously to demonstrate the specificity of several amino-acid-binding SBPs, since ligand-binding usually induces changes in T m [36]. The T m of FhuD2 increased by over 14 • C in the presence of ferrichrome, from 51.6 + − 0.1 • C to 66.1 + − 0.4 • C ( Figure 1).…”