Research Synopsis

Membrane proteins are proteins located in biological membranes. They play fundamental roles in some of the most important physiological processes in living organisms, including ion and nutrient transport, signal transduction, intra- and inter- cellular communication, pathogenesis and defense, representing ~30% of all protein-coding genes. Not surprisingly, membrane proteins are linked to many human disorders including cancer, heart disease, diabetes, and autoimmune diseases. Therefore, membrane proteins are highly attractive drug targets accounting for ~50% of existing pharmaceutical drugs. However, despite their significance and abundance, membrane proteins are relatively understudied and challenging to work with owing to protein instability outside the membrane.

The overall goal of our lab is to understand comprehensively the mechanisms by which membrane proteins operate. To do so, we use cutting-edge techniques that allow us to study simultaneously the structure, dynamics, and function of these proteins whilst still in the membrane. We directly visualize, on a single-molecule level, their structures, conformational changes, kinetic activity, and dynamic interactions with other proteins besides themselves. Most particularly, we aspire to discover rare and transient states crucial for the function of these membrane proteins, undiscoverable by average-based structural methods.

How do we do so?

The two major methods used in our lab are high-speed atomic force microscopy (HS-AFM) and single-particle cryo electron microscopy (cryo-EM), complemented by an array of biochemical and biophysical techniques.

High-Speed Atomic Force Microscopy (HS-AFM)

HS-AFM is a cutting-edge technique in protein biophysics, allowing us to acquire “videos” of proteins under near native conditions. HS-AFM uniquely provides us both structural and dynamic information of proteins on a single-molecule level. It does so by quickly measuring the topography of a surface by raster-scanning it with a sharp tip placed at the extremity of a cantilever. This allows direct imaging of the proteins in aqueous solutions, ambient temperature, without labelling or staining procedures, at a lateral resolution of ~1 nm, vertical resolution of ~0.1 nm, and time resolution of ~10 ms. 
We use HS-AFM to directly visualize membrane protein structures, conformational changes, dynamics and kinetics, and protein-protein interactions, and aim to discover rare and transient states undiscoverable by “classic” structural methods. HS-AFM is a powerful method with qualitatively different abilities than other structural biology methods, and therefore provides a new approach to study many unresolved questions concerning membrane proteins.

Cryo Electron Microscopy (Cryo-EM)

Cryo-EM has developed dramatically during the past decade following the “resolution revolution”, becoming a powerful and central tool in the field of structural biology to determine high-resolution structures of membrane proteins. 
Using cryo-EM, membrane protein structures can now be determined much more easily, providing a wealth of atomic-level information regarding the molecular details governing protein function, the conformational states they assume, and their protein-protein complexes. We aim to determine these structures in their native membrane bilayer (i.e., in native nanodiscs or vesicles), thereby visualizing as accurately as possible the structures they assume in a native environment.