Fifty years ago, biochemists described cells as small vessels that contain a complex mixture of chemical species undergoing reactions through diffusion and random collision. This description was satisfactory inasmuch as the intricate pathways of metabolism and, later, the basic mechanisms of gene regulation and signal transduction were still being unraveled. Gradually, and in part as a result of the parallel growth in the structural understanding of the molecular components of the cell, together with the development of single molecule manipulation methods, scientists have become increasingly aware that the cell resembles more a mechanical factory in which many of the processes are performed by specialized machinery whose behavior is essentially mechanical in nature. Biochemical processes as diverse as the elastic response of DNA, protein-induced DNA bending, chromosomal segregation, replication, transcription, translation, protein translocation across membranes, catalyzed protein and nucleic acid folding and unfolding, and even the ubiquitous processes of induced-fit molecular recognition, are all examples in which forces and torques develop in molecules as they move along their reaction coordinates. They are thus, amenable of study by direct manipulation methods. Methods of single-molecule manipulation (such as optical tweezers or atomic force micrsocopy) are being used today to: a) measure directly the forces holding molecular structures together, b) determine the stresses and strains generated in the course of chemical and biochemical reactions, c) exert external forces or torques to alter the extent and fate of these reactions, and d) reveal the rules that govern the interconversion of mechanical and chemical forces in these processes. This area of research can be rightly called mechanochemistry.
Single molecule methods are ideal to investigate the dynamics of complex reactions for, unlike their bulk counterparts, they make it possible to follow the trajectories of the individual molecules as they undergo their reactions in real-time, thus avoiding the ensemble average over the uncorrelated signals from a molecular population.
We use single molecule manipulation methods to:
- Determine mechanical properties of macromolecules;>
- Characterize the forces that maintain and hold together the structure of proteins and RNA (folding studies);
- Characterize the mechanochemical energy conversion of nucleic-acid-binding molecular motors;
- Develop the theoretical framework for the analysis and the interpretation of single molecules data including how to extract equilibrium data from non-equilibrium measurements.
We are studying the structural basis of protein-DNA interactions and their relevance in the processes of control of gene expression using single molecule manipulation methods. In prokaryotes, and specially in eukaryotes, replication and transcription regulation involve the interaction of many specialized protein factors at regulator locations on the sequence to insure correct sequence recognition, initiation, processivity, fidelity, and kinetic control. We wish to understand the multiple structural, spatial, and functional relationships among these regulatory factors.
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 interme- diate 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 folded state of the protein.
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.
Cecconi, C., Shank, E.A., Bustamante, C., Marqusee, S., “Direct observation of the three-state of a single protein molecule,” Science, 309, 2057-2060, (2005).
Bustamante, C., Liphardt, J., Ritort, F., “The nonequilibrium thermodynamics of small systems,” Physics Today 58, 43-48 (2005).
Levy, O., Ptacin, J.L., Pease, P.J., Gore, J., Eisen, M.B., Bustamante, C., and Cozzarelli, N.R., “Identification of oligonucleotide sequences that direct the movement of the Escherichiacoli FtsK translocase,” PNAS, 102, 17618 – 17623 (2005).
Dumont, S., Cheng, W., Serebrov, V., Beran, R.K., Tinoco, I Jr., Pyle, A.M., and Bustamante, C., “RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP,” Nature 439, 105-108 (2006).
Gore, J., Bryant, Z., Stone, M.D., Nollmann, M., Cozzarelli, N.R., and Bustamante, C., “Mechanochemical analysis of DNA gyrase using rotor bead tracking,” Nature 439, 100-104 (2006).
Moffitt J.R., Chemla Y.R., Izhaky D., Bustamante C., “Differential detection of dual traps improves the spatial resolution of optical tweezers,” PNAS 103 (24), 9006-11 (2006).
Gore J., Bryant, Z., Nöllmann M., Le M.U., Cozzarelli N.R., Bustamante C., “DNA overwinds when stretched,” Nature 442, 836-839 (2006).