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Investigating the Mechanism of an ABC Transporter (BmrA) Involved in Antibiotic Resistance Using Time-Resolved Approaches
0:00:05 Introduction: The speaker, an PhD student, is studying drug resistance, specifically focusing on ABC transporters and their role in antibiotic resistance.
0:00:21 Context: Antibiotic Resistance: Antibiotic resistance is a major health concern, with projections of increasing deaths. The presentation focuses on proteins that actively expel drugs, reducing their intracellular concentration.
0:00:59 BmrA, an ABC Transporter: BmrA, from Bacillus subtilis, is an ABC transporter that confers resistance to antibiotics by decreasing their intracellular concentration. It's a multidrug resistance protein, capable of transporting various ligands.
0:01:46 Alternating Access Mechanism: BmrA functions via an alternating access mechanism.
0:01:53 Inward-Facing: The drug binds inside the transmembrane domain. ATP binds to the nucleotide-binding domain, causing dimerization.
0:02:10 Outward-Facing: Conformational changes lead to an outward-facing state, releasing the drug outside the cell.
0:02:25 Return to Start: ATP hydrolysis resets the transporter for a new cycle.
0:02:39 Scientific Question: The main research goal is to understand the different states of this mechanism and how various ligands are transported by BmrA.
0:02:48 Methodology Two approaches are applied: steady state conditions and time resolve conditions.
0:03:08 Specific Transition Studied: The presentation focuses on the transition from the inward-facing to the outward-facing conformation.
0:03:19 Experimental Trick: An inactive mutant for ATPase activity is used to isolate the forward transition.
Biochemical part: The presenter used tryptophane fluorescence to monitor the process.
0:05:00 Tryptophan Fluorescence: Conformational changes are monitored over time using tryptophan fluorescence. Ligand binding causes quenching, and ATP addition increases fluorescence as the tryptophan environment becomes more hydrophobic.
0:05:37 Controls: Controls (buffer, ADP instead of ATP) confirm the specificity of the fluorescence changes.
0:06:19 Modulation: The transition can be modulated by temperature (higher temperature, faster transition) and ATP concentration.
0:06:47 Stopped-Flow Machine: A stopped-flow machine is used (in collaboration) to obtain information on the initial fast kinetics.
0:07:03 Wild-Type vs. Mutant: The transition is faster in the wild type than in the ATPase-inactive mutant. Different mutations (glutamine or alanine) impact conformational changes rather than ATP binding.
0:07:39 Kinetic Analysis: Kinetic rate constants are determined, leading to a proposed new kinetic model.
Structural part. The presenter used cryo-EM to monitor the conformational changes.
0:07:51 Cryo-EM Time-Resolved Experiments: Optimal conditions for time-resolved cryo-EM are determined (mutant, 300 µM ATP, 21°C) to slow down the transition and observe all intermediate states.
0:08:17 Cryo-EM Procedure: BmrA is frozen at key time points, and the particles are analyzed over time.
40 seconds: Majority of particles are outward-facing.
End: 100% outward-facing.
0:09:29 Data Correlation: There's a perfect match between the fluorescence traces and the cryo-EM data, showing the shift in populations over time.
0:09:48 Transient Intermediates: Transient intermediates are observed, such as the nucleotide-binding domains getting closer in response to ATP binding.
0:10:21 Future Perspective: The goal is to understand these local deformations and elucidate the mechanism in detail, in parallel with biochemical experiments.