Transmembrane Proteins Studied at the Single Molecule Level – Niels Bohr Institute - University of Copenhagen

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Transmembrane Proteins Studied at the Single Molecule Level

Studies at the Single Molecule (SM) level are a new and expanding field of biophysics providing unique information about bio-molecule function. Trans-membrane (TM) proteins have only been studied at the SM level a few times due to difficulties in analysis and acquisition of SM signals and sample preparation. SM studies allow access to the time-scale and pathway of TM protein movement (conformational dynamics), which is a perquisite for their function, and determine energy barriers intrinsic to these processes. Studies probing the effect of different thermodynamic parameters (T, pH, p), small molecules (drugs, inhibitors, etc.) or mutations, on the conformational dynamics of each individual protein are now feasible. These studies reveal protein heterogeneity in ensemble and the heterogeneity of individual proteins.

Here we develop automated methods to extract and process SM signals. We initially characterize performance of these methods with simulated SM signals. These methods were then employed for analysis of real SM data, obtained using total internal reflection fluorescence (TIRF) microscopy, of the TM Leucine transporter (LeuT). The Tetramethylrhodamine fluorophore (TMR) was covalently linked to LeuT and functioned as a conformation sensitive probe. Experiments were carried out with LeuT-TMR either: 1) solubilized in detergent and immobilized on mono Nitrilo Triacetic Acid (mNTA)-hydrogel, or 2) reconstituted in vesicles and immobilized on Neutravidin modified coverslip surface. We also characterized the photo-physics of TMR at the SM level. Activity of LeuT-TMR was verified through its response to Leucine with ensemble measurements in both a spectrometer and a TIRF microscope. Methods developed in this work were employed to extract information about conformational dynamics of SM LeuT-TMR.

Master Thesis defence by Marijonas Tutkus