Research Topics

Development of a state-of-the-art single molecule force-clamp spectrometer

The laboratory is typically populated by an eclectic mixture of molecular biologists, physiologists, and physicists. We develop all the protein engineering, software and instrumentation necessary for our measurements. One aspect of our work that is under continued improvement effort is the construction of an ever faster and more accurate AFM spectrometer (figure). We have developed a simple automatic force-clamp spectrometer that can accurately set the pulling force with a sub-millisecond time constant and very low drift and can be left running automatically for long periods of time. Direct readout of single protein lengths has an effortless resolution of 1 nm. With digital filtering it reaches 0.1 nm. We are starting to use HaloTag technology to covalently attach single proteins to both the pulling cantilever and the substrate. Our aim is to extend the observation time of a single protein to hours/days.

The new machine is ideal to probe the conformational dynamics of single proteins. This instrument was shipped to Spain and demonstrated at the experimental workshop that we taught in Bilbao(September of 2010). This spectrometer is now being manufactured by the company Luigs and Neumann of Rattingen/Germany and will be available soon by September of 2012. We have already tested a prototype which generated the most amazing force-clamp recordings ever. This instrument will become a standard tool in protein biochemistry laboratories around the world.

Paleoenzymology

It is possible to travel back in time at the molecular level by reconstructing proteins from extinct organisms. In collaboration with Dr. Eric gaucher from Georgia Tech, Dr. Jose Manuel Sanchez-Ruiz from the university of Granada and Dr. Arne Holmgren from the Karolinska Insitute, we have reconstructed, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx) dating back between ~.4 and ~4 billion years (Gyr). The reconstructed enzymes were up to 32 °C more stable than modern enzymes,and the oldest showed markedly higher activity than extant ones at pH 5. We probed the mechanisms of reduction of these enzymes using single-molecule force spectroscopy and found that ancient Trxs use chemical mechanisms of reduction similar to those of modern enzymes while adapting over 4 Gyr to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth. We are using a similar approach to attempt the resuscitation of other enzymes and key protein structures dating back to the Last Universal Common Ancestor (LUCA), such as those involved in motility and vesicle fusion.

Single protein folding.

The development of single molecule force-clamp spectroscopy introduces a novel way to probe the dynamics of proteins by measuring their length and mechanical stability during each stage of folding. We use the the force-quench technique to first unfold and extend a protein and then we quench the force allowing the protein to collapse and then fold. This technique easily separates three distinct states during folding under force: the extended conformation,a weakly stable collapsed conformation corresponding to a "molten globule" and the native mechanically stable conformation. The collapse trajectory of an extended protein to its collapsed molten globule state is complex and governed by the recently discovered entropic barrier that forms when force is applied to a molecule. Once the molten globule state is reached, the protein needs to remain undisturbed for a surprisingly long time (seconds) to fully regain its native mechanically stable conformation. Mechanical stability is an excellent proxy for the distinct stages of folding. Our studies show that the physics of a folding protein can be accurately studied using force-clamp spectroscopy. Once the Physics of a folding protein is known, it should be possible to unify all available experimental observations, including bulk and single molecules. Current projects include an effort to identify the mechanisms by which homo-polypeptide expansions lead to misfolding of the host, triggering neuro-degeneration. Other projects are aimed at studying the folding pathways of the mechano-sensing protein talin (in collaboration with Dr. Mike Sheetz), various proteins of the extra-cellular matrix and of the giant muscle protein titin.

Redox reactions studied in single proteins

We have developed a simple assay to monitor the reduction, oxidation and isomerization of disulfide bonds in single proteins. Disulfide bond reactions are crucial to the function of one third of all proteins, yet these reactions remain poorly understood. We use proteins with engineered disulfide bonds to arrest the mechanical extension that occurs during mechanical unfolding (figure). Reduction of the disulfide bond after unfolding results into a further extension that is easily detectable with force spectroscopy. The reverse reaction (oxidative folding) is also easily studied by applying the force-quench protocol in the presence of a chaperone such as Protein Disulfide Isomerase (PDI). In collaboration with Dr. Brent Stockwell (Columbia) and Dr. Arne Holmgren (Karolinska) we are studying the role of PDI and Glutaredoxin in oxidative protein folding. We are also studying the regioselectivity and isomerization of disulfide bond reactions when there are two or more disulfide bonds within the same protein.