Research Interests

Allosteric communication in membrane protein complexes linked to conformational transitions on multiple timescales

- Development of novel mechanistic models of molecular machines behind protein translocation

Motivation:

Allosterically modulated molecular machines mediate many of the key processes in all forms of life. The Sec translocon, a membrane bound protein complex, is the principal route for the efficient transport of heterogeneous polypeptides across or into lipid bilayers. The bacterial translocon consists of two main parts, a cytosolic ATPase SecA and membrane channel SecYEG. Based on our group’s recent findings we propose that the Sec translocon is a novel type of stochastically coupled hybrid molecular machine, where the processive SecA steers the energy landscape of a stochastic SecYEG channel allosterically dependent on nucleotide state. We present a number of research projects to map the full allosteric network regulating different stages of protein translocation in the Sec complex using a combination of mutagenesis, single-molecule and in silico methods.

Publications:

• Allen, W. J., et al (2016) Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation, Elife. 5.
• Fessl, T., et al (2018) Dynamic action of the Sec machinery during initiation, protein translocation and termination, Elife. 7.
• Corey, R. A., et al (2019) ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery, Elife. 8. 
• Fessl, T., et al (2020) Dynamics of Membrane Proteins Monitored by Single-Molecule Fluorescence Across Multiple Timescales, Methods in Molecular Biology

Studies of G protein signaling at the single molecule level

Motivation:

The G protein signaling cascade is a major pathway responsible for cellular communication with the external environment. It is present in all eukaryotes from yeast to humans. G proteins transduce signals from a variety of chemical and physical stimuli including hormones, odorants, neurotransmitters, and light. Up to 50% of all modern prescription drugs target this signaling cascade. However, many aspects of G protein signaling remain unclear. Our goal is to gain insights into non-conventional properties of G protein signaling in mammalian and fungal cells. Particularly, we are interested in precoupling between G proteins and G protein-coupled receptors (GPCRs), dimerization and heteromerization of GPCSs, and development of signaling activity sensors (1-5). In our research we use advanced imaging techniques, including single-molecule imaging, combined with molecular biology, cell biology methods and advanced data processing. We strive to utilize cutting-edge techniques and multidisciplinary approaches to do excellent science.

Figure 1.  Outstanding questions in G protein signaling research

Figure 2. A snapshot of real time dual channel single-molecule imaging of interactions between G proteins and G protein-coupled receptors in a live cell

Publications:

1. A. Bondar, J. Lazar, Optical sensors of heterotrimeric G protein signaling. FEBS J 288, 2570-2584 (2021).
2. A. Bondar, O. Rybakova, J. Melcr, J. Dohnalek, P. Khoroshyy, O. Tichacek, S. Timr, P. Miclea, A. Sakhi, V. Markova, J. Lazar  Quantitative Linear Dichroism Imaging of Molecular Processes in Living Cells Made Simple by Open Software Tools Communications Biology 4, 189 (2021).
3. A. Bondar, W. Jang, E. Sviridova, N. A. Lambert, Components of the Gs signaling cascade exhibit distinct changes in mobility and membrane domain localization upon beta2-adrenergic receptor activation. Traffic 21, 324-332 (2020).
4. A. Bondar, J. Lazar, The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors. J Biol Chem 292, 9690-9698 (2017).
5. A. Bondar, J. Lazar, Dissociated GalphaGTP and Gbetagamma protein subunits are the major activated form of heterotrimeric Gi/o proteins. J Biol Chem 289, 1271-1281 (2014).

Lab Members