Douglas D. Root, Ph.D., (UCLA)

Contact Information

Phone: 940-565-2683 

FAX: 940-565-4136 


Department of Biological Sciences 

PO Box 305220 

Denton, Texas 76203-5220

Actin Atomic Model

Research Interests: Structural and Functional Molecular Analyses of Actin Binding Proteins, Myosin, Nebulin, Troponin/Tropomyosin, and Dystrophin. My laboratory’s research on actin and actin-binding proteins uses sophisticated microscopic, spectroscopic, and biochemical techniques to investigate the structure and function of these proteins in solution. Myosin is the most familiar of the actin-binding proteins. Mutations in myosin have recently been found to underlie the disease familial hypertrophic cardiomyopathy which is the most common cause of sudden cardiac arrest in young adults. My research investigates the molecular mechanisms of muscle contraction that are thought to be impeded in this condition. Extensive use is made of resonance energy transfer between luminescent probes to determine the precise molecular transitions that occur in myosin and actin during this fundamental biological process. Chemical attachments of the probes to specific sites on actin and myosin are characterized using FPLC, electrophoretic, western blotting, ultracentrifugation, and enzymatic analyses. By measurements of resonance energy transfer between specific sites under different phases in the contractile process, atomic models of these events can be tested.

The absence of the protein dystrophin is known to cause Duchenne muscular dystrophy in humans which causes an early death in about 1/3,500 male children; however, the function of dystrophin is not yet elucidated. To test the hypothesis that dystrophin is an actin-binding protein in muscle cells, we have conjugated resonance energy transfer probes to monoclonal antibodies which bind specifically to the giant muscle protein dystrophin on thin cryostat muscle tissue sections. By labeling both actin and dystrophin in muscle cells with these probes, the association of these two proteins in muscle tissue sections has been established. This result supports current hypotheses that dystrophin plays a role in transduces stress generated by actin and myosin during shortening of the muscle to the extracellular matrix thereby preventing muscle atrophy. Additionally, the new technology developed in this study can be applied to the study of interactions between other proteins in muscle tissue. This technology has the potential for the study or diagnosis of diseases by examining the presence or absence of interactions at molecular resolution even within cells.

Nebulin is a giant protein (600 kDa - 900 kDa) of skeletal muscle which coextends with actin filaments. Nebulin is made up primarily of more than twenty highly conserved sequences of approximately 245 amino acids in size which are called superrepeats. My work has shown that nebulin fragments of approximately one superrepeat in length bind with high affinities to actin and inhibit potently normal actin-myosin functions such as ATPase activity and actin sliding over myosin. This effect can be reversed by the addition of calmodulin in a calcium dependent manner. These results suggest that nebulin might play a regulatory role in mediating actin and myosin interactions. The long length of a single nebulin molecule provides a mechanism by which the primary sequence of nebulin might have modified roles at different positions along the actin filament in addition to regulating the lengths of the filament. Investigations into these intriguing possibilities are underway.

Representative Publications:

Gawalapu, R. K., and Root, D. D. (2006) “Fluorescence labeling and computational analysis of the strut of myosin’s 50 kDa cleft” Arch. Biochem. Biophys. 456, 102-111. (Cover photo for issue)

Hong, F., and Root, D. D. (2006) “Downscaling Functional Bioassays by Single Molecule TechniquesDrug Discovery Today 11, 640-5.

Root, D. D., Yadavalli, V.M., Forbes, J. G., and Wang, K. (2006) “Coiled-coil Nanomechanics and Uncoiling and Unfolding of the Superhelix and Alpha-Helices of MyosinBiophys. J. 90, 2852-2866.

Gundapaneni, D., Xu, J., and Root, D. D. (2005) “High Flexibility of the Actomyosin Crossbridge Resides in Skeletal Muscle Myosin Subfragment-2 as Demonstrated by a New Single Molecule Assay” J. Struct. Biol. 149, 117-126.

Zhang, Z., Jin, J.-P., and Root, D. D. (2004) "Binding of Calcium Ions to an Avian flight Muscle Troponin T," Biochemistry 43, 2645-2655.

Root, D. D., Vaccaro, C., Zhang, Z., and Castro, M. (2004) “Detection of Single Nucleotide Variations by a Hybridization Proximity Assay Based on Molecular Beacons and Luminescence Resonance Energy Transfer.” Biopolymers 75, 60-70.

Root, D. D. (2002) “The Dance of Actin and Myosin: A Review of the Mechanism of Actomyosin Motility.” Cell Biochem. Biophys. 37, 111-139.

Root, D. D. (2002) “A Computational Comparison of the Atomic Models of the Actomyosin Interface” Cell Biochem. Biophys. 37, 97-110.

Root, D. D., Stewart, S., and Xu, J. (2002) “Dynamic Docking of Myosin and Actin Observed with Resonance Energy Transfer.” Biochemistry  41, 1786-1794.

Root, D. D., and Wang, K. (2001) "High-Affinity Actin-Binding Nebulin Fragments Influence the ActoS1 Complex," Biochemistry  40, 1171-1186.

Xu, J., and Root, D. D. (2000) "Conformational Selection During Weak Binding at the Actin and Myosin Interface," Biophys. J. 79, 1498-1510.

Jin, J.-P., and Root, D. D. (2000) "Modulation of Troponin T Molecular Conformation and Flexibility by Metal Ion Binding to the NH2-Terminal Variable Region," Biochemistry 39, 11702-11713.

Teal, H. E., Hu, Z., and Root, D. D. (2000) "Native Purification of Biomolecules with Temperature-Mediated Hydrophobic Modulation Liquid Chromatography," Anal. Biochem. 283, 159-165.

Root, D. D., Shangguan, X., Xu, J., and McAllister, M. (1999) " Determination of Fluorescent Probe Orientations on Biomolecules by Conformational Searching: Algorithm Testing and Applications to the Atomic Model of Myosin," J. Struct. Biol. 127, 22-34. (+ Cover Photo)

Xu, J., and Root, D. D. (1998) "Domain Motion between the Regulatory Light Chain and the Nucleotide Site in Skeletal Muscle Myosin," J. Struct. Biol. 123, 150-161.

Root, D. D. (1997) "In Situ Molecular Association of Dystrophin with Actin Revealed by Sensitized Emission Immuno-Resonance Energy Transfer," Proc. Natl. Acad. Sci., USA, 94, 5685-5690.

Root, D. D., and Wang, K. (1996) "Copper Iodide Staining of Proteins on Solid Phases," In Protein Protocols Handbook (Ed. James Walker), Humana Press, Totowa, NJ, pp. 39-43.

Root, D. D., and Wang, K. (1994) "Calmodulin-Sensitive Interaction of Human Nebulin Fragments With Actin And Myosin," Biochemistry 33, 12581-12591.

Cook, R. K., Root, D. D., Miller, C., Reisler, E., and Rubinstein, P. A. (1992) "Enhanced Stimulation of Myosin Subfragment 1 ATPase Activity by Addition of Negatively Charged Residues to the Yeast Actin N-Terminus." J. Biol. Chem. 268, 2410-2415.

Root, D. D., and Reisler, E. (1992) "Cooperativity of Thiol-Modified Myosin Filaments: ATPase and Motility Assays of Myosin Function," Biophysical Journal. 63, 730-740.