Titin is the largest protein in the human proteome. It is responsible for the passive elasticity of cardiac and skeletal muscle. Titin is the paradigmatic example of a protein that works under mechanical load. We have been studying titin for many years now. We pioneered the concept that Ig domains in titin unfold and refold during titin activity. In our recent article in Cell, we described a new form of molecular memory that allows titin to set its elasticity according to the redox state of myocytes. This new mechanism is based on the posttranslational modification of cryptic residues that become exposed after mechanical unfolding of Ig domains. Similar mechanisms may account for the fact that stretching increases elasticity, for instance after a yoga session. We have also determined that disulfide formation and isomerization are possible in many Ig domains in titin, which provides another checkpoint to control the elasticity of titin. Very clearly, titin is a mechanical computer that outputs the optimized elasticity after integrating multitude of intra and extracellular signals. We are just beginning to understand how titin really works. We are currently implementing tools that can simulate the elastic behavior of titin, and how posttranslational modifications or disease-linked mutations affect the elastic output of titin, our amazing molecular computer.
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. The most recent development is the application of Magnetic Tweezers (MT) to pull from single polyproteins. This is the result of a collaboration with the group of Jie Yan at the Mechanbiology Institute (Singapore). Using MT, we have been able to examine a single protein for over 15 hours, and we believe this is only the beginning! For the first time, we have resolved collapse trajectories from single protein domains, which we are now actively investigating. These advancements have been made possible thanks to the HaloTag technology, which allows to covalently attach single proteins a glass surface. We are developing a second covalent anchoring technology that will enable end-to-end covalent tethering.
The focus of the lab has been for many years AFM. We have distilled over 15 years of experience in AFM to produce 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. The new machine is ideal to probe the conformational dynamics of single proteins. This spectrometer is now being manufactured by the company Luigs and Neumann of Rattingen/Germany. In November 2013, Luigs and Neumann shipped one of the new AFMs to an experimental workshop in Stellenbosch (South Africa) where students were able to obtain state-of-the-art single-molecule recordings.
Disclosure: Columbia University has licensed intellectual property to Luigs and Neumann Gmbh. In accordance with University policy, Dr. Fernandez is entitled to royalties through this license.
Our next goal is to build a force spectrometer with high-throughput capabilities, to be able to screen for molecules that alter mechanical properties of proteins. This technological advancement is the first step towards Mechano-Pharmacology, a new way of finding drugs that could be used to treat cardiac disease or fight against bacterial infections.
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. Covalent tethering techniques in combination with drift-less Magnetic Tweezers have enabled single recordings that last over 10 hours. It should be feasible to hang on to the same molecule for weeks and monitor thousands of folding and unfolding transitions. Single-molecule techniques have always promised to capture rare events that can lead to misfolding diseases such as Alzheimer's and Parkinson's. Now, for the first time, we have the means to explore such rare and conformations in proteins.
In our experiments, we use the the force-quench technique to first unfold and extend a protein and then allow the protein to collapse and then fold by quenching the force to a lo value. 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 by AFM and MT. We are developing techniques that make possible to extract the free energy of a single protein from our experimental recordings. Thanks to our unique perspective on mechanical unfolding and folding of proteins, we are beginning to understand the mechanical architecture of adhesive pilins from Gram-positive bacteria.
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), as we describe in our 2012 paper in Cell. Now, we are expanding our studies to DsbA, the main catalyst of oxidative folding in bacteria, to investigate oxidative folding of bacterial virulence factors. Using our cutting edge single-molecule techniques, we have also observed that thiol chemistry is altered in mutants of superoxide dismutase that cause familial Amyotrophic Lateral Sclerosis (Lou Gehrig's disease).
Bacteria must withstand tremendous shearing forces when binding to host, such as those induced by coughing, chewing, or urination. Consequently, they have evolved some pretty remarkable adhesive structures to address these mechanical challenges. We have been focused on understanding the mechanics of adhesion in Gram-positive bacteria, with a particular interest in their extracellular protein appendages, notably their pili. Pili are micron-long fibrous structure that can withstand the highest forces known of any globular proteins. Moreover, Gram-positive pili harbor a slew of internal covalent bonds, including disulfide, isopeptide, and thioester bonds, that confer remarkable mechanical stabilities, folding properties, and covalent binding to ligands. How these unique mechanical features conspire to facilitate bacterial adhesion is a fundamental question with implications for the development of novel anti-adhesive drugs.