CONFORMATIONAL CHANGES IN THE SUBSTRATE-BINDING DOMAIN OF GlnPQ BY SINGLE-MOLECULE FRET

• Main Author: Agustin Husada, Florence
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/34598
• Institution: Institut Teknologi Bandung

ABC (ATP-binding cassette) transporters are vital to any living system and involved in the translocation of a wide variety of substrates. They are involved in many crucial processes, including nutrient uptake, lipid trafficking, drug and antibiotic excretion, cell volume regulation and many others. The core component of the ABC transporters is formed by the transmembrane domains (TMDs), together with the nucleotide binding domains (NBDs) and the substrate binding domains (SBDs), they constitute the actual translocator. The presence of SBDs allows the system to bind a specific substrate for the subsequent translocation process. The ABC transporter of interest in this work is the glutamine transporter (GlnPQ) from Lactococcus lactis IL1403 that has two substrate-binding domains linked in tandem. Dimeric GlnPQ is essential for the uptake of glutamine/glutamic acid into the cell. In particular, we investigated the SBDs of this transporter. SBDs are made by two rigid ?/? subdomains connected by a flexible hinge. Upon substrate binding, the two rigid subdomains come together via a conformational change driven by the flexible hinge. The aim of this project is to identify the conformational changes occurring on the SBDs of GlnPQ in the presence and absence of substrates. Single molecule Förster-resonance energy-transfer was used as a tool to monitor conformational changes. For this purpose, a number of double cysteine mutants were engineered in the two different strategic sites of GlnPQ?s SBD genes. Proteins were labeled on these cysteines with donor (Cy3B) and acceptor (Atto647N) fluorophores, then distance changes during the conformational transitions will be monitored using alternating laser excitation (ALEX). In these experiments, a low FRET population (open-unliganded state) was observed in the soluble protein, while upon substrate addition this population was converted into high a FRET population (close-liganded state). These results demonstrate that we can probe the two different conformational states at the single molecule level. The constructs of the mutation in SBD1, SBD2, and SBD1+2 tandem were successfully generated and expressed. The SBDs protein were also successfully purified and labelled with Cy3B (donor) and ATTO647N (acceptor) at strategic positions. As indicated for the mutant SBD2 S451C/T369C, the static crystal structures suggest that the distance of the fluorophore was 4.9 nm (low FRET = 0.5159) for the open and 4.0 nm for the closed conformation (high FRET = 0.6869). Our results showed only a single peak in the absence of the ligand as well as for saturating concentrations of glutamine. Close to the KD-value of ~1.0 µ M, clear populations of both species can be observed. Similar results were found for different labelling positions in GlnPQ-SBP2 (mutant H319C/T392C), and GlnPQ-SBP1 (mutant Q87C/T159C) indicating a similar binding mechanism independent of the labelling position. There is no cooperativity between SBD1 and SBD2 upon substrate binding.