Relating the structural changes to enzymatic reactions and mechanical steps of the energy transduction mechanism by mapping the real-time domain motions of the motor proteins and ribosomal elongation factors.
Motor proteins and GTP-binding proteins (G-proteins) share many structural and functional attributes. Molecular motors myosin, dynein and kinesin are prototype biological energy transducers that can be understood at a particularly fine level of detail. The obvious functional output (force and motion) allow the reaction sequence to be probed by single molecule biophysical, chemical and structural studies. A cyclic interaction between actin and myosin transforms free energy of splitting ATP into motion and mechanical work. Modified forms of this mechanism power other cell biological motions such as targeted vesicle transport and cell division. We are using novel biophysical techniques, including nanometer tracking of single fluorescent molecules, FRET sensors, bifunctional fluorescent probes and infrared optical traps (laser tweezers) to map the real-time domain motions of the motor proteins.
Although the ribosome has been studied extensively since the unraveling of the genetic code, how it accomplishes the enormous fidelity of messenger RNA translation into amino acid sequences during protein biosynthesis is not understood. The ribosome is a motor translocating along the mRNA exactly 3 bases per elongation cycle. Energy from splitting GTP by G-protein elongation factors (EFs) is transformed into translational accuracy and maintenance of the reading frame. Codon-anticodon base pairing between mRNA and tRNA ‘reads’ the code, but EF-Tu ‘proofreads’ it. EF-G is the motor catalyzing translocation of tRNAs and mRNA. Powerful techniques developed for studies on motor proteins, including single molecule fluorescence and optical traps, may be applied to understand the structural biology, energetics, and function of EFs in their working environment.
The microscopes were designed and built by the laboratory of Dr. Yale E. Goldman using modified Nikon TE-2000 and TE-I microscopes and Photometrics Cascade II and Evolve EMCCD cameras.
Prism-type and objective-type total internal reflection fluorescence microscopes equipped with optics on the excitation and emission pathways for detecting changes in orientation and rotational mobility of single fluorescently molecules and for applying pN-level mechanical forces. The instruments are capable of tracking the 3-dimensional position of fluorescent molecules with nanometer-level precision in vitro and in live cells. Excitation and emission wavelengths are optimized for macromolecules fluorescently-labeled with probes in the visible wavelength range.