Report
Teaser, summary, work performed and final results
Periodic Reporting for period 3 - SM-IMPORT (Substrate import at work: single-molecule studies of ABC transporters)
Teaser
ABC (ATP Binding Cassette) transporters represent the most abundant and diverse family of transport proteins known that play crucial roles in numerous cellular processes. Despite their importance, all proposed molecular models for transport are based on indirect evidence due...
Summary
ABC (ATP Binding Cassette) transporters represent the most abundant and diverse family of transport proteins known that play crucial roles in numerous cellular processes. Despite their importance, all proposed molecular models for transport are based on indirect evidence due to the inability of classical biophysical and biochemical techniques to directly visualize dynamic structural changes. To solve this problem, the SM-IMPORT team will decipher the molecular mechanisms of transport using novel single-molecule methodology with the ultimate goal to use this knowledge against pathogenic bacteria, for treatment of ABC-related diseases or multi-drug resistance of cancer cells. The team uses single-molecule fluorescence microscopy for the study of conformational states of an ABC model system in vitro, and thus to observe directly how elementary transport steps are coordinated. This will open up a virtually unexplored biophysical research area and provide a detailed understanding of the molecular mechanisms of ABC transporters.
The overall objectives this proposal are: (i) What is the mechanism of substrate binding in ABC transporters? The conformational equilibrium of ABC-associated substrate-binding proteins will be studied to understand the molecular mechanism of binding. (ii) What are relevant conformational states and changes for substrate translocation? The time- and length-scales of conformational changes in transmembrane and nucleotide binding domains as well as interactions with other domains will be characterized. (iii) How are substrate binding, energy utilization and translocation coordinated in ABC transporters? Finally, a complete model of transport will be developed to decipher the coordination of transport events, i.e., how substrate binding and ATP-hydrolysis are coupled and transferred into conformational changes that drive substrate transport.
Work performed
Work during the first 30 months:
(aim 1): Six different SBDs of bacterial ABC importers were analyzed with smFRET in solution and dynamically on the surface to assess their conformational states and allosteric changes in the structure upon ligand binding. The SM-IMPORT team additionally examined a protein with a structurally related fold and found that the binding mechanism of all proteins is an induced fit (except for new protein which uses lock-and-key). Besides these general implications of the ligand binding-mode as a central theme within this action for ABC importers, the studied the additional scientific questions regarding SBDs ABC importers ligand selectivity and structural evolution.
(aim 2): The SM-IMPORT team made an improved homology model of the ABC importer OpuA to allow design of the smFRET experiments in both TMDs and NBDs. A large number of 27 OpuA-TMD cysteine mutants were designed and produced for smFRET analysis; all proteins are available in the host group now. Functional checks excluded only two of the mutants for further biophysical studies. After successful reconstitution of OpuA into lipid nanodiscs, the team further screened the mutants for ATPase activity and their labelling efficiency with fluorescent dyes; mutants with high efficiency and activity were selected for further analysis. Unfortunately, all labelling attempts for TMD mutants failed (even at the detergent state) and pose the fundamental challenge to continue this project with OpuA as a model system. Alternative strategies and model systems are currently screened.
(aim 3): Work on this aim has just been started with the development of two different techniques to monitor conformational states with self-induced fluorophore quenching and PIFE to allow visualization of correlated movement. The research is still ongoing to establish the dynamic range of both techniques.
Final results
As indicated in the publication section, the project team developed novel biophysical assays (PIFE-FRET), photostable self-healing fluorophores and studied protein systems that have related biochemical mechanisms to ABC transporters.