Wayne Frasch

Single-Molecule Studies of the FoF1 ATP Synthase Molecular Motors.

We developed a novel technology that can measure the rotational position of single molecules of rotary motors with unprecedented precision, time resolution, and signal-to-noise. Using this approach, my lab is gaining new insight into the mechanism of the FOF1 ATP synthase, which is responsible for >80% of the ATP in almost every living organism. This complex is composed of two rotary motors. The FO motor uses a transmembrane proton gradient to power CW rotation, which forces F1 to synthesize ATP. Conversely, F1 can hydrolyze ATP to power CCW rotation.

 The F1 ATPase Molecular Motor. Using our single-molecule assay, we resolved the angular velocity of F­1-ATPase rotation as a function of rotary position and found that the power stroke undergoes a series of accelerations and decelerations that we divided into two 60° phases. We recently showed that the energy that drives Phase-2 results from the binding energy of ATP to the empty catalytic site. Elevated ADP concentrations not only increase dwells at 35° consistent with competitive product inhibition but also decrease angular velocity during Phase-2. We have now also identified similarities and differences in the F1 power stroke angular velocity profiles with that from thermophilic bacteria, an A1-ATPase from an archae bacterium, and most notably the F1 from a mycobacterium. The latter is notable because structural differences that may contribute to the changes in the power stroke provide possible new drug targets for tuberculosis.

• Martin, J., Hornung, T., Ishmukhametov, R., Spetzler, D., and Frasch, W. D. (2018) “Elastic Coupling Power Stroke Mechanism of the F1-ATPase”, Proc. Natl. Acad. Sci. USA, 115, 5750-5755
• Ragunathan, R., Sielaff, J., Sundararaman, L., Biukovic, G., Sony, M., Manimekalai, S., Singh, D., Kundu, S., Wohland, T., Frasch, W.D., Dick, T., and Grüber, G. (2017) “The Uniqueness of Subunit a of Mycobacterial F-ATP synthases: An evolutionary Variant for Niche Adaptation”, J. Biol. Chem. 292, 11262-11279.
• Sielaff, H., Martin, J., Grüber, G., and Frasch, W. D. (2016) “Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors” J. Biol. Chem. 291, 25351-25363.
• Martin, J., Ishmukhametov, R., Hornung, T., Ahmad, Z., and Frasch, W. D. (2014) “Anatomy of F1-ATPase Powered Rotation” Proc. Natl. Acad. Sci. USA 111, 3715-3720.
• Hornung, T., Martin, J., Ishmukhametov, R., Spetzler, D., and Frasch, W. D. (2011) “Microsecond Resolution of Single Molecule Rotation Catalyzed by Molecular Motors”, Methods in Molecular Biology 778, 273-289
• Spetzler, D., Ishmukhametov, R., Day, L. J., Hornung, T., Martin, J., and Frasch, W. D. (2009) “Single Molecule Measurements of F1-ATPase Reveal an Interdependence between the Power Stroke and the Dwell Duration”, Biochemistry 49, 7979-7985.
• Hornung, T., Ishmukhametov, R., Spetzler, D., Martin, J., and Frasch, W. D. (2008) “Determination of Torque Generation from the Power Stroke of Escherichia coli F1-ATPase.” Biochim. Biophys. Acta- Bioenergetics 1777, 579-582.
• York, J., Spetzler, D., Hornung, T., Ishmukhametov, R., Martin, J., and Frasch, W.D. (2007) “Abundance of Escherichia coli F1-ATPase Molecules Observed to Rotate via Single-Molecule Microscopy with Gold Nanorod Probes”, J. Bioenergetics and Biomembranes, 39, 435-439.
• Spetzler, D., York, J., Lowry, D., Daniel, D., Fromme, R. and Frasch, W. D. (2006) “Microsecond Time Resolution of Single Molecule F1-ATPase Rotation”, Biochemistry 45, 3117-3124. Accelerated publication designated a Hot Article by the ACS based on top-10 down loads from the journal.
• Boltz, K. and Frasch, W.D. (2006) “Hydrogen Bonds between the alpha and beta Subunits of the F1-ATPase Allow Communication between the Catalytic site and the Interface of the beta-Catch Loop and the gamma Subunit.” Biochemistry 45, 11190-11199.
• Boltz, K. W. and Frasch, W. D. (2005) “Interactions of gammaT273 and gammaE275 with the beta Subunit PSAV Segment that Links the gamma-Subunit to the Catalytic Site Walker Homology B Aspartate are Important to the Function of Escherichia coli F1Fo ATP Synthase”, Biochemistry 44, 9497-9506.
• Lowry, D. and Frasch, W. D. (2005) “Interactions between betaD372 and gamma-Subunit N-terminus residues gamma-K9 and gamma-S12 are Important for ATP Synthase Activity Catalyzed by the E. coli FoF1 ATP Synthase” Biochemistry 44, 7275-7281.
• Greene, M. D. and Frasch, W. D. (2003) “Interactions between gammaR268, gammaQ269 and the beta Subunit Catch-Loop of E. Coli F1-ATPase are Critical for Catalytic Activity, J. Biol. Chem. 278, 51594-51598.

The Fo Molecular Motor. Using our nanorod method, we have been able to resolve the rotational stepping of single c-subunits of the Fo c-ring and observed that Fo subunit-a can push the c-ring in the ATP synthesis direction against the force of F1 ATPase-dependent rotation. These results are providing important new insight into the molecular mechanism of the Fo motor. In FoF1, the Fo motor uses a transmembrane proton gradient as an energy source to drive rotation in the ATP synthesis direction against the force generated by the F­1-ATPase.

Single molecule studies of this motor have been limited due to the technical difficulties inherent in studying a membrane embedded protein complex. Detergent solubilized FoF1 is unstable resulting in subunit dissociation and rapid loss of activity. We overcame this problem by incorporating purified FoF­1 into lipid nanodiscs that have the physical properties of a biological membrane but are small enough for single molecule work. The use of gold nanorods to monitor rotation is the only approach capable of resolving the ~100 ms rotational stepping of Fo. Most recently, we reported that the c-subunit stepping increases inversely with pH. Since, using lipid bilayer nanodiscs, the half-channels on each side of the membrane that supply protons to the c-ring are exposed to the same pH, these results show that the proton input channel for ATP synthesis is more easily protonated than is the ATPase channel. This provides new insight concerning how the FO motor can maintain a high ratio of ATP/ADP-Pi under steady state conditions.

• Yanagisawa, S. and Frasch, W. D. (2017) “Protonation Dependent Stepped Rotation of the F-type ATP synthase c-Ring Observed by Single-Molecule Measurements”, J. Biol. Chem., 292, 17093-17100.
• Martin, J., Hudson, J., Hornung, T., and Frasch, W.D. (2015) “Fo-driven Power Stroke Rotation Occurs against the Force of F1ATPase-dependent rotation in the FoF1 ATP synthase”, J. Biol. Chem., 290, 10717-10728.
• Spetzler, D., Ishmukhametov, R., Hornung, T., Martin, J., York, J., Jin-Day, L., and Frasch, W. D. (2012) “Energy Transduction by the Two Molecular Motors of the F1Fo ATP Synthase” Advances in Photosynthesis and Respiration 34, Dordrecht, The Netherlands, 561-590.
• Frasch, W. D. and Chapsky, L. (2012) “Polarization-Enhanced Detector with Gold Nanorods for Detecting Nanoscale Rotation and Method Therefore” U.S. Patent 8,207,323
• Frasch, W. D. and Chapsky, L. (2011) “Polarization-Enhanced Detector with Gold Nanorods for Detecting Nanoscale Rotational Motion and method therefor” U.S. Patent 8,003,316
• Ishmukhametov, R., Hornung, T., Spetzler, D., and Frasch, W. D. (2010), “Direct Observation of stepped proteolipid ring rotation in E. coli FoF1-ATP synthase”, EMBO J 29, 3911-3923.

Nanodevices for Molecular Detection and Computation. We adapted the single-molecule assay of F1-ATPase rotation using gold nanorods for use in molecular detection of DNA sequences, proteins, and metabolites that are biomarkers for cancer and infectious diseases. I realized that the F1 and the nanorod can be engineered such that assembly of the F1-ATPase motor with a nanorod is only possible by the binding of a specific target molecule. The target molecule can a protein biomarker for cancer, or the Stx2 toxin from E. coli O157:H7. Through the creation of these nanodevices, we have been able to achieve limits of detection for proteins that are 5 orders of magnitude more sensitive than commercially available approaches. This is possible because F1-ATPae-dependent nanorod rotation is only possible when the nanodevice has correctly assembled via the target molecule. We have made nanodevices to detect target metabolites, and for DNA profiling that avoids the need for signal amplification with PCR, which can introduce artifacts. I invented the ligation-exonuclease reaction (LXR) as part of our DNA detection. As a secondary spin-off of this technology, we adapted LXR for use in DNA computing where we solved the largest, most complex mathematical problem at that time using DNA. To increase the speed and accuracy of the computations with DNA, I invented omega probe-mediated qRT-PCR nanodevices. We are developing these nanodevices for use in molecular computations with direct biomedical applications.

• Xiong, F. and Frasch, W. D. (2017) “Telomere Measuring Nanodevice and Method Therefor,” U.S. Patent Disclosure
• Frasch, W. D. (2013)  “Detection of Target Metabolites,” U.S. Patent Nationalized PCT application13/808,567
• Frasch, W. D., Spetzler, D., and York, J. (2013) “High Speed, High Fidelity, High Sensitivity Nucleic Acid Detection”, U.S. Patent 8,530,199.
• Frasch, W. D., Spetzler, D., and York, J., Xiong, F. (2013) “Methods for Generating a Distribution of Optimal Solutions to Nondeterministic Polynomial Optimization Problems” U.S. Patent 8,126,649.
• Frasch, W. D. and Chapsky, L. (2012) “Polarization-Enhanced Detector with Gold Nanorods for Detecting Nanoscale Rotational Motion and method therefor” U.S. Patent 8,192,936
• Frasch, W. D., Spetzler, D., and York, J., Xiong, F. (2012) “Methods for Generating a Distribution of Optimal Solutions to Nondeterministic Polynomial Optimization Problems” U.S. Patent 8,126,649
• Frasch, W. D., Spetzler, D., and York, J. (2012) “High Speed, High Fidelity, High Sensitivity Nucleic Acid Detection”, U.S. Patent 8,084,206
• Frasch, W. D. and He, Liyan (2011) “Single Molecule Detection using Molecular Motors”, U.S. Patent 8,076,079
• Xiong, F. and Frasch, W. D. (2010) “Padlock Probe-Mediated qRT-PCR for DNA Computing Answer Determination”, Natural Computing 10, 947-959
• Xiong, F., Spetzler, D., and Frasch, W. D. (2009) “Solving the Fully-Connected 15-City TSP using Probabilistic DNA Computing”, Integr. Biol., 1, 275-280
• York, J., Spetzler, D., Xiong, F., and Frasch, W. D. (2008) “Single Molecule Detection of DNA via Sequence-Specific Links between F1-ATPase Motors and Gold Nanorod Sensors”, Lab. Chip 8, 415-419. Among top-10 LOC articles accessed on-line. Highlighted in Chemical Biology, a Royal Society of Chemistry news magazine that provides a snapshot of the latest, most exciting, chemical biology developments.
• Spetzler, D., Xiong, F., and Frasch, W. D. (2008) “Heuristic solution to a 10-City Traveling Salesman Problem Using Probabilistic DNA Computing”, LNCS 4848, 152-160.
• Spetzler, D., York, J., Dobbin, C., Martin, J., Xiong, F., Ishmukhametov, R., Day, L., Yu, J., Kang, H., Porter, K., Hornung, T., and Frasch, W.D. (2007) “Recent Developments of Biomolecular Motors as On-Chip Devices using Single Molecule Techniques”, Lab. Chip 7, 1633-1643. Among top 10 most accessed LOC articles in 2007.
• Spetzler, D., Xiong, F., and Frasch, W.D. (2007) “Probabilistic DNA Computing Solution to a Fully Connected 10-City Asymmetric Traveling Salesman Problem” Proc. DNA 13, 9-18.
• Xiong, F., Spetzler, and Frasch, W. D. (2007) “Elimination of Secondary Structures for DNA Computing”, Proc. DNA 13, 241-249.
• Chapsky, L., Frasch, W. D., Chou, C., Zenhausern, F., and Goronkin, H. (2006) “Single-Molecule Detection of Biological Warfare Agents Using the F1-ATPase Biomolecular Motor”, U.S. Patent 6,989,235