415 South Street
Waltham, MA 02453-2728
1990 Searle Scholar
The general interest of our laboratory is macromolecular motion. Movement is essential for the biological function of enzymes and nucleic acids. A major focus of our research is motor enzymes. These remarkable molecular machines catalyze a chemical reaction, capture the free energy released by the reaction, and use this energy to perform biologically useful mechanical work. Motor enzymes play essential roles in diverse biological processes ranging from muscle contraction to neuronal development to mitosis to gene transcription. To learn how the enzymes work, we study them in vitro using molecular cloning, enzymology, protein chemistry, and biophysical chemistry techniques. The laboratory has also pioneered methods for visualizing nanometer-scale movements and individual chemical reaction events in single enzyme molecules. Such methods reveal the crucial dynamic features of enzyme mechanisms that are missing from the information produced by static techniques like x-ray crystallography. In addition, single molecule methods can examine reaction mechanism steps that are difficult or impossible to study by conventional biochemical techniques, which are limited to analyzing the population-average properties of large molecular ensembles.
The motor enzyme kinesin binds to subcellular organelles and then transports the organelles through the cytoplasm by pulling them along microtubules. Kinesin or its homologs drive chromosome movement in meiosis and mitosis, axonal transport, and formation of the endoplasmic reticulum. Like other motor enzymes, a kinesin molecule is a chemically-powered molecular engine: it catalyzes the hydrolysis of ATP and uses the energy from this reaction to move the enzyme from site to site on the microtubule lattice. We want to learn how this engine works by elucidating the steps in the movement mechanism and determining how these steps are coupled to the reactions of ATP hydrolysis. A major direction of our work is to prepare kinesin derivatives using directed mutagenesis techniques. Once this is done, we analyze the structural and functional properties of the derivatives. These studies have demonstrated the importance of particular enzyme structures for efficient processive movement along the microtubule, and we are now investigating how these structures function. One way we study function is to tag single enzyme molecules with microscopic polystyrene beads and then directly observe movement along microtubules by light microscopy. Digital image processing techniques allow us to track bead positions with nanometer-scale precision, allowing us to directly observe the kinetics of kinesin-microtubule interactions and the pattern of single kinesin molecule movements on the microtubule lattice.
Enzymes that move processively along DNA -- DNA polymerases, RNA polymerases, and DNA helicases are some examples -- are also motor enzymes. Our laboratory has developed the first methods for directly observing the movement of single enzyme molecules along DNA and for measuring the associated forces. This approach has allowed us to investigate important mechanistic features of these enzymes that could not previously be studied. For example, our studies on the E. coli RNA polymerase have revealed the kinetic behavior of the enzyme at factor-independent transcriptional terminators. We have also demonstrated that this enzyme is the most powerful molecular motor yet characterized and is by itself capable of overcoming mechanical obstacles to transcription that exist in living cells. The overall goal of our studies in this area is to explore the translocation and regulation mechanisms of DNA motor enzymes and, by doing so, to more completely define the molecular bases for DNA replication, transcription and recombination.
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