Focus on biological motility: from cellular morphogenesis to DNA repair and recombination
Motility (the capability to perform active movement) forms an essential part of all life processes. The driving force for motility is produced by molecular motors (also called mechanoenzymes), which can convert chemical energy into mechanical work with high efficiency. Part of our research is centered on myosins, a superfamily of molecular motors that use the energy stored in ATP produce directed movement along actin filaments. We also investigate the mechanism of action of DNA helicases, enzymes that use the energy of ATP hydrolysis for translocation along and unwinding of the strands of double-stranded DNA.
We aim to
- initiate new lines of interdisciplinary research on the relationship between biochemical kinetics and the effect of mechanical force on biological macromolecules, and
- explore the diversity of molecular motor mechanisms present in living organisms, and elucidation of the function of new types of biological motility in physiological/developmental processes and disease.
We use a variety of biochemical and biophysical techniques to elucidate the mechanism of action of motor enzymes and their inhibitors.
We employ microscope-based motility and single molecule mechanical assays to correlate the biochemical properties of motor enzymes with their motile and mechanical parameters.
We also investigate the in vivo role of motor enzyme action and its inhibition in model organisms including E. coli, C. elegans, zebrafish and human cell lines.
Load-dependent enzymatic and motile mechanism of non-muscle myosin 2
The dependence of the biochemical parameters of macromolecules on mechanical load is an essential but poorly understood feature of living systems. Since molecular motors almost always exert their functions under load, the investigation of this feature is essential in understanding a wide range of physiological and pathological processes.
Non-muscle myosin 2 isoforms are ubiquitously expressed in animal tissues and they perform essential cellular functions such as cytokinesis and cortical tension maintenance. In recent years we have elucidated the enzymatic and motile mechanism of various non-muscle myosin 2 isoforms in the absence of mechanical load. Because of its highly favorable biochemical properties and the knowledge accumulated, the non-muscle myosin 2 system is ideal for the investigation of load-dependent properties.
We and other groups have devised methods by which the load-dependence of certain enzymatic steps of motor proteins can be quantitatively investigated in solution conditions. We are also investigating these effects on the level of single molecules.
Mechanism of inhibition of myosin 2 by blebbistatin and other inhibitors
Recently we have determined the general mechanism of inhibition of myosin 2 by blebbistatin, a novel potent inhibitor of myosin 2 enzymatic activity and motility. We are currently monitoring the effect of the inhibitor on the structural transitions of actomyosin during the working cycle. In these studies we make use of site-specific fluorescent reporters located in the myosin 2 catalytic domain. We are also investigating the effects of inhibition of myosin 2 function on the development of model organisms.
Mechanism of DNA processing by helicases serving DNA repair and recombination
During various biological processes, unwinding of double-stranded nucleic acids is necessary to access and manipulate their information content. Helicases are molecular motors that use chemical energy liberated in nucleotide (mostly ATP) hydrolysis to power the unwinding reaction. All living organisms use a variety of helicases, which are essential for nucleic acid replication, recombination, transcription, and repair. Thus, helicases are prime therapeutic targets in cancer and viral diseases.
Although information is rapidly growing about the diversity of helicase structures and functions, precise knowledge of their mechanisms is lagging behind. The central aspect of helicase mechanisms is how the steps of the ATP hydrolysis process (ATP binding, hydrolysis, product release and associated structural changes) are coupled to translocation along and separation of DNA strands, how this coupling leads to an energetically efficient unwinding mechanism, and how helicases attain specificity to target nucleic acid structures. To understand these phenomena, we investigate the molecular events leading to helicase activity using fluorescence spectroscopic, transient enzyme kinetic and single-molecule approaches.
Mechanisms of action of non-motor enzymes
Besides our studies on molecular motors, we are also investigating the mechanism of action of other enzymes, including ones working in nucleotide metabolism.