THE PROPERTIES OF ENERGY-COUPLING FACTOR TRANSPORTER COMPLEX WITH THE NANOBODIES

• Main Author: Ramdhan Anshari, Zaid
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/41391
• Institution: Institut Teknologi Bandung

ATP-binding cassette (The ABC) proteins serve many functions, including the transport of nutrients into the cell, transport of compounds across organellar membranes, the secretion of proteins, antigen (peptide) presentation, cell volume regulation. regul ation of protein synthesis, detoxifaction and antibiotic resistance. Energy-coupling factor (ECF) transporter arc a group of ATP-binding cassette transporters responsible for micronutrients transport in prokaryotes. ECF transporters consist of two proteins in cystoplasm and two membrane proteins in lipid bilayer. The hypothetical mechanism of ECF transporter was defined. To obtain the complete mechanism of ECF transporter we need to define the structure of ECF transporter in many states. To determine the complete mechanism of ECF transporter, we decided to usc crystallography approach. The preparation of high-quality crystal s of membrane proteins is a major bottleneck for structure determination by using X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as a promising tool to increase the crystallization probability of a selected target protein. Crystallization chaperones is an auxiliary protein, such as fragments of monoclonal antibodies. Here, nanobodies were used as crystal lization chaperones, which had already been derived from immunized llamas and selected by phage display. The diversity of the nanobodies i s high, with sequence simi l arity screening revealing 21 different families based on the target recognition si te. Here, we used 7 different families of nanobodies to further characterization. Nanobodies was produced in E. coli WK6 and induced by Isopropyl P-D-1-thiogalactopyranoside (IPTG). This protein was purified by the nickel affinity chromatography (IMAC). Then, the el ution buffer of nanobodies was changed with desalting column. The concentration of purified nanobodies was obtained -1-2 mg in I L of culn1re. ECFtransporterwas produced in E. coli MCI061 and induced by L-(+)-Arabinosc. Membrane vesicles ofECF transporter was prepared by cell disrupter and collected with ultracentrifugation. This protein was solubilized in n-dodecyl-P-0-maltoside (DDM). The solubilized ECF transporter was purified by the !M AC and analytical gel filtration (AGF). The characterization of 7 different nanobodies regarding their interaction with the ECF transporter was done using analytical gel filtration (AGF), SDS-PAGE analysis, and transpo11activity. Based on AGF. six nanobodics were found binding to the ECF transporter, specifica lly in the ECF module domain. One nanobodies, Nb 62, could not bind to ECF transporter ECF PanT in this experiment condition. Nb 86 showed 95% inhibition on transport activity and bound to the target with affinity in the nanomolar range. In addition, Nb have profound effect on the crystallization of ECF transporter. The crystal of EcfFoiT2-Nb86 was obtained in 0.05 M Mg(CH3COOh 4H20, 0.1 M MES pH 6.5 and 26o (v/v) PEG 400 condition for 5 days incubation at 8 °C. The co-crysta lli zation of ECF Pdx was then screened with three different nanobodies, for instance Nb 81, Nb 89, and Nb 91. ECF Pdx with Nb 91 gave promising crystal s in 0.08 M Mg(CH,C00)2 4H20, 0.1 M Na-citrate pH 6 and 14% (v/v) PEG 5000 MME condition for 35 days incubation at 8 °C. Together with previous data, we show that nanobodies arc a versatile and valuable tool in structural biology by widening the crysta lli zation space to get novel structures of ECF transporters.