Research

Below we highlight the main research areas of our research group, encompassing different areas of single molecule force spectroscopy.

1. Capturing the individual folding trajectories in a single protein
In sharp contrast with the prevailing 2-state view of protein folding, using single molecule force-clamp spectroscopy we discovered that proteins visit different dynamic conformations before attaining the functional and native state. In particular, a generic collapsed conformation is a necessary precursor to the native state, and may hold the key to explain how proteins fold. The different and elusive conformations might be vital to understand chaperone-assisted protein folding and also protein misfolding and aggregation.

We are mostly interested in studying the mechanical resistance of those proteins that are in vivo continuously exposed to mechanical forces. Understanding the mechanisms of tissue elasticity requires comprehending how these proteins continuously and reversibly extend and recoil physiologically under the presence of a mechanical force. Paradigmatic examples of these proteins are muscle proteins, and in particular those present in cardiomyocytes, such as titin or myosin binding protein C.We are interested in studying the molecular mechanisms underpinning the reversible extension/collapse/folding dynamic cycle in these proteins. Finally, along the same lines, we are extending the single molecule mechanical studies to intercellular proteins that link the plasma membrane with the nuclear envelope as a putative way to swiftly transmit mechanical stimulus from the extracellular matrix to the cell nucleus.

fig3new

Force quench folding trajectory of an individual ubiquitin polyprotein, exhibiting the whole conformational dynamics marked by the presence of the ensemble of mechanically labile molten globule state and the mechanically resistant native state of the protein.

2. Chemistry under force
A chemical reaction is classically activated by temperature, light or electricity. Force is an alternative, albeit much less explored, way to promote a chemical reaction. Using a combination of force clamp spectroscopy with molecular engineereing techniques we demonstrated that the SN2 reduction of an individual protein embedded disulfide bond is a force-activated process  along a smooth energy landscape. We plan to experimentally reconstruct the full free-energy landscape of a chemical reaction under force. These novel experiments have opened a totally new way of inquiry in the chemistry field.

3.    The nanomechanics of lipid membranes

FC schamtic presentationimagebilayer 3D

(Left). Schmatics of a stacked supported lipid bilayer being imaged and/or indented by an AFM probe. (Right). 3D plot of an AFM image on a stacked supported lipid bilayer.

Many cellular processes, such as endo- and exocytosis or cytokinesis, demand fast rearrangements of cell membranes to accommodate the dramatic change in cell shape. Such physical restructuring implies that the cell is exposed to high mechanical forces. Most of these studies have focused on the role of the actin cortex, however, the role of the lipid bilayer as a way to withstand mechanical forces has remained elusive. Since 2005, we study the mechanical properties of individual supported lipid bilayers using force spectroscopy AFM. We discovered that lipid bilayers withstand significant amount of force, and that the mechanical stability is intimately related to both the environment (temperature, ionic strength) and the chemical composition of the lipids forming the supported lipid bilayer.  We are now applying a constant force to a series of supported stacked lipid bilayers to study, for the first time, the kinetics of rupture and reformation (healing) at the single bilayer level. We are also extending our in-vitro experiments to the in-vivo study of the mechanical properties of live cells.

FC DPhPC 13-03-05 03

Force clamp indenting trajectory on a DPhPC lipid multibilayer.

Funding

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