Motor proteins, or mechanoenzymes, convert metabolic energy into force, generating movement in all living organisms. The largest class of such proteins derives energy from the hydrolysis of ATP, and includes the myosin, dynein, and kinesin superfamilies. Despite over a century of study and the arsenal of biochemical and biophysical approaches that have been tried, the molecular basis by which motor proteins work remains obscure. Today, the mechanism of force production by proteins is one of the great outstanding problems in biology, with obvious implications in understanding the basis of motor-related disease. The advent of in vitro motility assays has, at last, allowed mechanoenzymes to be studied in comparative isolation, using purified components interacting in defined experimental geometries. Such systems hold great promise because they facilitate genetic, biochemical, physical and molecular biological manipulations not possible in complex cellular systems. Recent advances, described here, show that physiology is feasible at the level of individual molecules, in an assay using kinesin motors moving along microtubules. The kinesin-microtubule system is particularly amenable to study, because (1) movement is produced by single motors, (2) motion is slow enough for measurement, (3) microtubules can be seen in the light microscope, (4) both recombinant kinesin (and kinesin-like) proteins and protein fragments, expressed in either bacterial or eukaryotic vectors, move in vitro, and (5) methods now exist to produce controlled forces and measure displacements on a molecular scale. The long-term goal of this research is to develop a molecular model for motor protein function, based on detailed physical knowledge combined with biochemical/biostructural information.
Specific aims i nclude measurement of the speeds, forces, displacements, cycle timing, ATP coupling, and other properties of native kinesin, recombinant kinesin (and engineered constructs), and the minus end-directed motor ncd. For this purpose, advanced instrumentation based on optical trapping ('optical tweezers') and optical interferometry will be built and used to track motion at the subnanometer level. Closely-related apparatus will also be used to compile data on the micromechanical properties of kinesin/microtubules, and to accomplish a new kind of imaging, 'optical force microscopy,' that may provide insights into the molecular structures that underlie motility.
| Milic, Bojan; Andreasson, Johan O L; Hogan, Daniel W et al. (2017) Intraflagellar transport velocity is governed by the number of active KIF17 and KIF3AB motors and their motility properties under load. Proc Natl Acad Sci U S A 114:E6830-E6838 |
| Andreasson, Johan O L; Shastry, Shankar; Hancock, William O et al. (2015) The Mechanochemical Cycle of Mammalian Kinesin-2 KIF3A/B under Load. Curr Biol 25:1166-75 |
| Andreasson, Johan O L; Milic, Bojan; Chen, Geng-Yuan et al. (2015) Examining kinesin processivity within a general gating framework. Elife 4: |
| Milic, Bojan; Andreasson, Johan O L; Hancock, William O et al. (2014) Kinesin processivity is gated by phosphate release. Proc Natl Acad Sci U S A 111:14136-40 |
| Clancy, Bason E; Behnke-Parks, William M; Andreasson, Johan O L et al. (2011) A universal pathway for kinesin stepping. Nat Struct Mol Biol 18:1020-7 |
| Gutiérrez-Medina, Braulio; Andreasson, Johan O L; Greenleaf, William J et al. (2010) An optical apparatus for rotation and trapping. Methods Enzymol 475:377-404 |
| Gutiérrez-Medina, Braulio; Fehr, Adrian N; Block, Steven M (2009) Direct measurements of kinesin torsional properties reveal flexible domains and occasional stalk reversals during stepping. Proc Natl Acad Sci U S A 106:17007-12 |
| Guydosh, Nicholas R; Block, Steven M (2009) Direct observation of the binding state of the kinesin head to the microtubule. Nature 461:125-8 |
| Valentine, Megan T; Block, Steven M (2009) Force and premature binding of ADP can regulate the processivity of individual Eg5 dimers. Biophys J 97:1671-7 |
| Fehr, Adrian N; Gutiérrez-Medina, Braulio; Asbury, Charles L et al. (2009) On the origin of kinesin limping. Biophys J 97:1663-70 |
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