Research Interests

Our laboratory is involved in the development of novel methods of single-molecule manipulation and detection (such as optical tweezers and single-molecule fluorescence microscopy) and their application to study the behavior of DNA-binding molecular motors and the mechanical unfolding of globular proteins and RNAs. In addition, we use Scanning Force Microscopy (SFM) to investigate the structure of chromatin and the global structure of protein-nucleic acid complexes relevant to the molecular mechanisms of control of transcription in prokaryotes.

Current Projects

We are studying the structural basis of protein-DNA interactions and their relevance in the processes of control of gene expression. In prokaryotes, and especially in eukaryotes, replication and transcription regulation involve the interaction of many specialized protein factors at regulator locations on the sequence to ensure correct sequence recognition, initiation, processivity, fidelity, and kinetic control. We wish to understand the multiple structural, spatial, and functional relationships among these regulatory factors. We are using direct visualization method such as the SFM to image various protein-DNA complexes involved in transcription initiation and elongation and various processes of recombination in prokaryotes.

Our laboratory is also working actively in the development of methods of single-molecule manipulation, including the use of SFM cantilevers, optical tweezers, and magnetic tweezers to investigate the mechanical properties of macromolecules. Recently, for example, we used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule-like intermediate observed in bulk studies. This intermediate displays unusual mechanical compliance and unfolds at substantially lower forces than the native state. In a narrow range of forces, the molecule hops between the unfolded and intermediate states in real time. Occasionally, hopping was observed to stop as the molecule crossed the folding barrier directly from the intermediate, demonstrating that the intermediate is on-pathway. These studies allow us to map the energy landscape of RNase H, which represents the most complete description of the folding pathway of the protein. We plan to investigate how external conditions in the medium, i.e. temperature, denaturant concentration, etc., or point-directed mutations, affect the shape of the potential energy function. Similar studies are being carried out in our laboratory with RNA molecules capable of attaining secondary and tertiary structures.

In the case of RNA, we have found conditions under which it is possible to unfold the molecules at equilibrium. In this case, it is possible to extract directly both the thermodynamics and kinetics of unfolding. Novel statistical mechanical methods are also being implemented to extract thermodynamics information from non-equilibrium data when the unfolding process does not occur reversibly.

Finally, we are also studying DNA-binding molecular motors (nucleic acid translocases such as RNA polymerase, DNA polymerase, etc.) using optical tweezers to investigate the dynamics of these molecules and their mechanochemical conversion during translocation, as well as the effect of external force load and nucleotide tri-phosphate concentration on their power and force generation. A molecular motor of special interest is the bacteriophage phi 29 connector, which is responsible, together with its associated ATPase (gp16) for the packaging of the viral DNA inside the capsid during bacteriophage assembly. Our single-molecule studies have revealed that this is powerful motor, capable of generating forces as high as 57 pN. We are also characterizing now the mechanochemical properties of this motor. Moreover, we have shown that translocation is coincident with the release of phosphate along the chemical cycle of the motor. We are currently investigating how the several ATPases of the motor coordinate their action during packaging.

More recently, we have been characterizing the mechanism of translocation of FtsK, an E. coli translocase using optical tweezers, magnetic tweezers, and direct visualization methods. FtsK is a membrane-bound and septum-localized E. coli translocase that coordinates cell division with chromosome segregation. We directly observed the movement of purified FtsK, an Escherichia coli translocase, on single DNA molecules. The protein moves at 5 kilobases per second and against forces up to 60 piconewtons, and locally reverses direction without dissociation. On three natural substrates, independent of its initial binding position, FtsK efficiently translocates over long distances to the terminal region of the E. coli chromosome, as it does in vivo. Our results imply that FtsK is a bidirectional motor that changes direction in response to short, asymmetric directing DNA sequences. Moreover, single-molecule observations together with an informatics analysis strongly suggest a particular octamer as the most likely FtsK Recognition Sequence or FRS. Direct testing of this sequence confirms its assignment. Finally, we have discovered the FtsK domain responsible for recognizing and reading the FRS. In parallel, we are developing both microscopic (chemical ratchet-type) and phenomenological models of molecular motors, which will be tested experimentally. We believe that single-molecule experiments can provide a unique look into the molecular mechanisms responsible for the mechano-chemical conversion process in these protein machines.