Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and AT... more Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and ATP synthases. The V 1 complex consumes ATP to drive rotation of a central rotor that pumps protons across membranes via the V o complex. Eukaryotic V-ATPases are regulated by reversible disassembly of subunit C, V 1 without C, and V O. ATP hydrolysis is thought to generate an unknown rotary state that initiates regulated disassembly. Dissociated V 1 is inhibited by subunit H that traps it in a specific rotational position. Here, we report the first single-molecule studies with high resolution of time and rotational position of Saccharomyces cerevisiae V 1-ATPase lacking subunits H and C (V 1 ΔHC), which resolves previously elusive dwells and angular velocity changes. Rotation occurred in 120°power strokes separated by dwells comparable to catalytic dwells observed in other rotary ATPases. However, unique V 1 ΔHC rotational features included: 1) faltering power stroke rotation during the first 60°; 2) a dwell often occurring~45°after the catalytic dwell, which did not increase in duration at limiting MgATP; 3) a second dwell,~2-fold longer occurring 112°that increased in duration and occurrence at limiting MgATP; 4) limiting MgATP-dependent decreases in power stroke angular velocity where dwells were not observed. The results presented here are consistent with MgATP binding to the empty catalytic site at 112°and MgADP released at~45°, and provide important new insight concerning the molecular basis for the differences in rotary positions of substrate binding and product release between V-type and F-type ATPases.
A novel method for detecting F 1-ATPase rotation in a manner sufficiently sensitive to achieve ac... more A novel method for detecting F 1-ATPase rotation in a manner sufficiently sensitive to achieve acquisition rates with a time resolution of 2.5 μs (equivalent to 400 000 fps) is reported. This is sufficient for resolving the rate at which the γ-subunit travels from one dwell state to another (transition time). Rotation is detected via a gold nanorod attached to the rotating γ-subunit of an immobilized F 1-ATPase. Variations in scattered light intensity allow precise measurement of changes in the angular position of the rod below the diffraction limit of light. Using this approach, the transition time of Escherichia coli F 1-ATPase γ-subunit rotation was determined to be 7.62 ± 0.15 (standard deviation) rad/ms. The average rate-limiting dwell time between rotation events observed at the saturating substrate concentration was 8.03 ms, comparable to the observed Mg 2+-ATPase k cat of 130 s −1 (7.7 ms). Histograms of scattered light intensity from ATP-dependent nanorod rotation as a function of polarization angle allowed the determination of the nanorod orientation with respect to the axis of rotation and plane of polarization. This information allowed the drag coefficient to be determined, which implied that the instantaneous torque generated by F 1 was 63.3 ± 2.9 pN nm. The high temporal resolution of rotation allowed the measurement of the instantaneous torque of F 1 , resulting in direct implications for its rotational mechanism. The F 1 F o-ATP synthase couples the energy provided by a transmembrane proton gradient to the production of ATP from ADP and phosphate. The intrinsic membrane complex of ab 2 c 10-14 subunits known as F o 1 functions as a proton channel, and the F 1 peripheral membrane complex of α 3 β 3 γδϵ subunits contains one site for ATP synthesis and/or hydrolysis per αβ heterodimer. When F 1 is purified from F o and the membrane, it retains the ability to hydrolyze ATP (1-3). There are three catalytic sites which are coupled such that the binding of the substrate to one site induces conformational changes that result in product
The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, ... more The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F1 (αβ)3 ring stator contains three catalytic sites. Single-molecule F1 rotation studies revealed that ATP hydrolysis at each catalytic site (0°) precedes a power-stroke that rotates subunit-γ 120° with angular velocities that vary with rotational position. Catalytic site conformations vary relative to subunit-γ position (βE, empty; βD, ADP bound; βT, ATP-bound). During a power stroke, βE binds ATP (0°–60°) and βD releases ADP (60°–120°). Årrhenius analysis of the power stroke revealed that elastic energy powers rotation via unwinding the γ-subunit coiled-coil. Energy from ATP binding at 34° closes βE upon subunit-γ to drive rotation to 120° and forcing the subunit-γ to exchange its tether from βE to βD, which changes catalytic site conformations. In F1FO, the membrane-bound FO complex contains a ring of...
Most cellular ATP is made by rotary F 1 F O ATP synthases using proton translocationgenerated clo... more Most cellular ATP is made by rotary F 1 F O ATP synthases using proton translocationgenerated clockwise torque on the F O c-ring rotor, while F 1-ATP hydrolysis can force counterclockwise rotation and proton pumping. The F O torque-generating mechanism remains elusive even though the F O interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F 1 F O structures. Here, single-molecule F 1 F O rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of F O to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through F O operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the Escherichia coli c 10-ring of F 1 F O against the torque of F 1-ATPase-dependent rotation that result from H + transfer events from F O subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c 10-ring occur to sustain F 1 F O ATP synthesis. Editor's evaluation This manuscript describes single molecule spectroscopic work to probe the mechanisms by which protonation/deprotonation steps produce torque between the a-subunit and the C10 ring, which is subsequently conveyed to F1 to couple to ATP synthesis. This is an important bioenergetics question and the approach yields some tantalizing clues as to which protonation steps are involved. In principle, this knowledge could lead to direct experimental or computational tests to resolve the overall mechanism.
ΩqPCR determines absolute telomere length in kb units from single cells. Accuracy and precision o... more ΩqPCR determines absolute telomere length in kb units from single cells. Accuracy and precision of ΩqPCR were assessed using 800 bp and 1600 bp synthetic telomeres inserted into plasmids, which were measured to be 819 ± 19.6 and 1590 ± 42.3 bp, respectively. This is the first telomere length measuring method verified in this way. The approach uses Ω-probes, a DNA strand containing sequence information that enables: (i) hybridization with the telomere via the 3′ and 5′ ends that become opposed; (ii) ligation of the hybridized probes to circularize the Ω-probes and (iii) circularized-dependent qPCR due to sequence information for a forward primer, and for a reverse primer binding site, and qPCR hydrolysis probe binding. Read through of the polymerase during qPCR occurs only in circularized Ω-probes, which quantifies their number that is directly proportional to telomere length. When used in concert with information about the cell cycle stage from a single-copy gene, and ploidy, the MT...
An asymmetric, fully-connected 8-city traveling salesman problem (TSP) was solved by DNA computin... more An asymmetric, fully-connected 8-city traveling salesman problem (TSP) was solved by DNA computing using the ordered node pair abundance (ONPA) approach through the use of pair ligation probe quantitative real time polymerase chain reaction (PLP-qPCR). The validity of using ONPA to derive the optimal answer was confirmed by in silico computing using a reverse-engineering method to reconstruct the complete tours in the feasible answer set from the measured ONPA. The high specificity of the sequence-tagged hybridization, and ligation that results from the use of PLPs significantly increased the accuracy of answer determination in DNA computing. When combined with the high throughput efficiency of qPCR, the time required to identify the optimal answer to the TSP was reduced from days to 25 min.
F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Esch... more F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αβ)3δab2. Subunits c10 or (αβ)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a ...
The two opposed rotary molecular motors of the F 0 F 1-ATP synthase work together to provide the ... more The two opposed rotary molecular motors of the F 0 F 1-ATP synthase work together to provide the majority of ATP in biological organisms. Rotation occurs in 120°power strokes separated by dwells when F 1 synthesizes or hydrolyzes ATP. F 0 and F 1 complexes connect via a central rotor stalk and a peripheral stator stalk. A major unresolved question is the mechanism in which the interaction between subunit-a and rotating subunitc-ring in the F 0 motor uses the flux of H ؉ across the membrane to induce clockwise rotation against the force of counterclockwise rotation driven by the F 1-ATPase. In single-molecule measurements of F 0 F 1 embedded in lipid bilayer nanodiscs, we observed that the ability of the F 0 motor to form transient dwells increases with decreasing pH. Transient dwells can halt counterclockwise rotation powered by the F 1-ATPase in steps equivalent to the rotation of single c-subunits in the c-ring of F 0 , and can push the common axle shared by the two motors clockwise by as much as one c-subunit. Because the F 0 proton half-channels that access the periplasm and the cytoplasm are exposed to the same pH, these data are consistent with the conclusion that the periplasmic half-channel is more easily protonated in a manner that halts ATPase-driven rotation by blocking ATPase-dependent proton pumping. The fit of transient dwell occurrence to the sum of three Gaussian curves suggests that the asymmetry of the three ATPase-dependent 120°power strokes imposed by the relative positions of the central and peripheral stalks affects c-subunit stepping efficiency. The F-ATP synthases, located in eubacteria, mitochondria, and chloroplasts, are members of a family of rotary molecular motors that also include archaeal A-ATP synthases, bacterial A/V-like ATP synthases, and eukaryotic vacuolar V-ATPases (1). The F-ATP synthases are composed of two opposed rotary motors known as the membrane-embedded F 0 , and the extrinsic membrane protein complex F 1 (Fig. 1). These motors are connected by a central stalk that serves as the rotor and a peripheral stalk that is a component of the stator (2). In Escherichia coli, as in most organisms, the largest single source of cellular ATP is that made by the F 0 F 1-ATP synthase.
The F1FO ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causativ... more The F1FO ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). In addition to their synthase function most F-ATP synthases possess an ATP-hydrolase activity, which is coupled to proton-pumping activity. However, the mycobacterial enzyme lacks this reverse activity, but the reason for this deficiency is unclear. Here, we report that a Mycobacterium-specific, 36 amino acid long C-terminal domain in the nucleotide-binding subunit α (Mtα) of F-ATP synthase suppresses its ATPase activity and determined the mechanism of suppression. First, we employed vesicles to show that in intact membrane-embedded mycobacterial F-ATP synthases deletion of the C-terminal domain enabled ATPase and proton-pumping activity. We then generated a heterologous F-ATP synthase model system, which demonstrated that transfer of the mycobacterial C-terminal domain to a standard F-ATP synthase α subunit suppresses ATPase activity. Single-molecule rotati...
The angular velocities of ATPase-dependent power strokes as a function of the rotational position... more The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF from the Methanosarcina mazei Goe1 A-ATP synthase, and the thermophilic motor α3β3γ from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase are resolved at 5 μsec resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and differ only in the magnitude of their velocities. M. mazei A3B3DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, since ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP-binding to A3B3DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwel...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014
Nano/micron-sized reaction chamber arrays (femtoliter-chamber arrays) enable highly sensitive and... more Nano/micron-sized reaction chamber arrays (femtoliter-chamber arrays) enable highly sensitive and quantitative biological assays, such as single-molecule enzymatic assays [1, 2], digital PCR [3, 4], and digital ELISA [5]. However, the versatility of femtoliter-chamber arrays has been limited to reactions in aqueous solutions. In this presentation, I will introduce an arrayed lipid bilayer chamber system (ALBiC) that displays a sub-million femtoliter chambers, each sealed with a stable 4-μm diameter lipid bilayer membrane with extremely high efficiency (yield:~99%). When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or FoF1-ATP synthase, the chambers of the ALBiC exhibited stochastic and quantized transporting activities, demonstrating that the single molecule analysis of passive and active membrane transports is achievable with the ALBiC system. Thus, this new platform has vastly extended the versatility of femtoliter chamber arrays and could contribute to the understanding of the working mechanism of membrane proteins as well as to further analytical and pharmacological applications. If time allows, I would like to talk about new versions of the ALBiC that we recently developed.
Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient ... more Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the Fo motor uses a transmembrane electrochemical ion gradient to create clockwise (CW) torque that overcomes F1ATPase-driven counterclockwise (CCW) torque at high ATP is a major unresolved question. Using single FoF1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of Fo-dependent rotation of the c10-ring in the ATP synthase (CW) direction against the CCW force of ATPase-driven rotation that occurs upon formation of a leash with Fo-stator subunit-a. Mutational studies indicate that the leash is important for ATP synthase activity, and support a mechanism in which residues aE196 and cR50 participate in the cytoplasmic proton half-channel to promote leash formation.
Although single-molecule experiments have provided mechanistic insight for several molecular moto... more Although single-molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F o F 1-ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F o has been particularly hampered by signal-to-noise and time resolution. Here, we show the presence of a transient dwell between F o subunits a and c by improving the time resolution to 10 ls at unprecedented S/N, and by using Escherichia coli F o F 1 embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 ls to form and 175 ls to dissociate, is independent of proton transport residues aR210 and cD61, and behaves as a leash that allows rotary motion of the c-ring to a limit of B361 while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the F o motor where c-ring rotational diffusion is limited to 361.
Proceedings of the National Academy of Sciences, 2014
Significance We present a description of the angular velocity of the power stroke as a function o... more Significance We present a description of the angular velocity of the power stroke as a function of rotational position for the F 1 -ATPase molecular motor. The angular velocity of this motor, which rotates in 120° power strokes separated by catalytic dwells, is part of the F o F 1 ATP synthase in oxidative phosphorylation, and has been thought not to vary. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur, providing direct evidence that angular velocity depends on substrate binding affinity. The data support a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F 1 structures 2HLD1 and 1H8E (Protein Data Bank).
Brief saturating light fashes were used to probe the mechanism of inactivation of 02 evolution by... more Brief saturating light fashes were used to probe the mechanism of inactivation of 02 evolution by Tris in chloroplasts. Maximum inactiation with a single flash and an oscillation with period of four on subsequent flashes was observed. Analyses of the oscillations suggested that only the charge-collecting 02-evolving catalyst of photosystem H (S2-state) was a target of inactivation by Tris. This conclusion was supported by the following observations: (a) hydroxylamine preequWlibration caused a threeflash delay in the inactivation pattern; (b) the lifetimes of the Tris-inactivable and S2-states were similar, and (c) reagents acceleratin S2 deactivation decreased the lifetime of the inactivable state. Inactivation proved to be moderated by F, the precursor of Signal II., as shown by a one flash delay with chloroplasts having high abundance of F. Evidence was obtained for cooperativity effects in inactivation and NH3 was shown to be a competitive inhibitor of the Tris-induced inactivation. Sdependent inactivation was inhibited by glutaraldehyde fixation of chkoroplasts, possibly suggesting that inactivation proceeds via confornational changes of the S2-state. A previous report (11) on the mechanism of Tris inactivation of ' This work was supported in part by Grant PCM78-07694 from the National Science Foundation to G. M. C. This paper (79-3-153) is published with the approval of the Director of the Kentucky Agricultural Experiment Station. 2 In memory of Bessel, who didn't suffer fools gladly and never compromised his high standards in science.
Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abund... more Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg-20 ng linear detection range between thermal cycle threshold (C t value) and target content. The C t values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology.
Journal of Radioanalytical and Nuclear Chemistry Articles, 1987
A method for the quantitative determination of small amounts of protein samples was developed emp... more A method for the quantitative determination of small amounts of protein samples was developed employing neutron activation analysis. Current methods of protein concentration determination are severely limited as a result of differences in the specific :characteristics'. of each protein. Silver binding.has been used as a sensitive colorimetric method to indicate the presence of protein. However, silver-protein complexes can have a variety of absorbance spectra unique to each protein, which complicate the analysis. Various amounts of specific proteins were equilibrated in an excess of silver nitrate prior to the reduction of the silver by the addition of NaBI-h, HCHO, and NaOH. The protein-silver complex was rapidly separated from the unbound silver by centrifugation chromatography and the amount of bound silver was determined by INAA. The amount of silver was proportional to the amount of protein present in each sample. When the silver was not reduced prior to removal of the unbound silver by chromatography, only negl!gible amounts of silver remained bound to the protein. The stoiehiometry of bound silver to protein on a molar basis showed relatively small differences for the proteins that were examined. This ratio was found to depend on the conditions of the binding and reduction of the silver. The results suggest that *Ae binding of silver is not specific t~ any charged or polar groups on these proteins and may, fl~e~'efore, provide a means of determination of the concentration of protein that has general a?plication for all proteins.
Site-directed mutations were made to the phosphatebinding loop threonine in the -subunit of the ... more Site-directed mutations were made to the phosphatebinding loop threonine in the -subunit of the chloroplast F 1-ATPase in Chlamydomonas (T168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from T168D, T168C, and T168L mutants had <10% of the wild type rates, as did rates of Mg 2؉-ATPase activity of purified chloroplast F 1-ATPase (CF 1). The EPR spectra of VO 2؉-ATP bound to Site 3 of CF 1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF 1 from each mutant in both signal intensity and in 51 V hyperfine parameters that depend on the equatorial VO 2؉ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphatebinding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg 2؉-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF 1 with entrapped Mg 2؉-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.
Site-directed mutations D262C, D262H, D262N, and D262T were made to the  subunit Walker Homology... more Site-directed mutations D262C, D262H, D262N, and D262T were made to the  subunit Walker Homology B aspartate of chloroplast F 1-ATPase in Chlamydomonas. Photoautotrophic growth and photophosphorylation rates were 3-14% of wild type as were ATPase activities of purified chloroplast F 1 indicating that D262 is an essential residue for catalysis. The EPR spectrum of vanadyl bound to Site 3 of chloroplast F 1 as VO 2؉-ATP gave rise to two EPR species designated B and C in wild type and mutants. 51 V-hyperfine parameters of species C, present exclusively in the activated enzyme state, did not change significantly by the mutations examined indicating that it is not an equatorial ligand to VO 2؉ , nor is it hydrogen-bonded to a coordinated water at an equatorial position. Every mutation changed the ratio of EPR species C/B and/or the 51 V-hyperfine parameters of species B, the predominant conformation of VO 2؉-nucleotide bound to Site 3 in the latent (down-regulated) state. The results indicate that the Walker Homology B aspartate coordinates the metal of the predominant metal-nucleotide conformation at Site 3 in the latent state but not in the conformation present exclusively upon activation and elucidates one of the specific changes in metal ligation involved with activation.
Removal of the ability to form a salt bridge or hydrogen bonds between the  subunit catch loop (... more Removal of the ability to form a salt bridge or hydrogen bonds between the  subunit catch loop (Y297-D305) and the ␥ subunit of Escherichia coli F 1 F o-ATP synthase significantly altered the ability of the enzyme to hydrolyze ATP and the bacteria to grow via oxidative phosphorylation. Residues T304, D305, D302, ␥Q269, and ␥R268 were found to be very important for ATP hydrolysis catalyzed by soluble F 1-ATPase, and the latter four residues were also very important for oxidative phosphorylation. The greatest effects on catalytic activity were observed by the substitution of side chains that contribute to the shortest and/or multiple H-bonds as well as the salt bridge. Residue D305 would not tolerate substitution with Val or Ser and had extremely low activity as D305E, suggesting that this residue is particularly important for synthesis and hydrolysis activity. These results provide evidence that tight winding of the ␥ subunit coiled-coil is important to the rate-limiting step in ATP hydrolysis and are consistent with an escapement mechanism for ATP synthesis in which ␣␥ intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven ␥ subunit rotation.
Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and AT... more Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and ATP synthases. The V 1 complex consumes ATP to drive rotation of a central rotor that pumps protons across membranes via the V o complex. Eukaryotic V-ATPases are regulated by reversible disassembly of subunit C, V 1 without C, and V O. ATP hydrolysis is thought to generate an unknown rotary state that initiates regulated disassembly. Dissociated V 1 is inhibited by subunit H that traps it in a specific rotational position. Here, we report the first single-molecule studies with high resolution of time and rotational position of Saccharomyces cerevisiae V 1-ATPase lacking subunits H and C (V 1 ΔHC), which resolves previously elusive dwells and angular velocity changes. Rotation occurred in 120°power strokes separated by dwells comparable to catalytic dwells observed in other rotary ATPases. However, unique V 1 ΔHC rotational features included: 1) faltering power stroke rotation during the first 60°; 2) a dwell often occurring~45°after the catalytic dwell, which did not increase in duration at limiting MgATP; 3) a second dwell,~2-fold longer occurring 112°that increased in duration and occurrence at limiting MgATP; 4) limiting MgATP-dependent decreases in power stroke angular velocity where dwells were not observed. The results presented here are consistent with MgATP binding to the empty catalytic site at 112°and MgADP released at~45°, and provide important new insight concerning the molecular basis for the differences in rotary positions of substrate binding and product release between V-type and F-type ATPases.
A novel method for detecting F 1-ATPase rotation in a manner sufficiently sensitive to achieve ac... more A novel method for detecting F 1-ATPase rotation in a manner sufficiently sensitive to achieve acquisition rates with a time resolution of 2.5 μs (equivalent to 400 000 fps) is reported. This is sufficient for resolving the rate at which the γ-subunit travels from one dwell state to another (transition time). Rotation is detected via a gold nanorod attached to the rotating γ-subunit of an immobilized F 1-ATPase. Variations in scattered light intensity allow precise measurement of changes in the angular position of the rod below the diffraction limit of light. Using this approach, the transition time of Escherichia coli F 1-ATPase γ-subunit rotation was determined to be 7.62 ± 0.15 (standard deviation) rad/ms. The average rate-limiting dwell time between rotation events observed at the saturating substrate concentration was 8.03 ms, comparable to the observed Mg 2+-ATPase k cat of 130 s −1 (7.7 ms). Histograms of scattered light intensity from ATP-dependent nanorod rotation as a function of polarization angle allowed the determination of the nanorod orientation with respect to the axis of rotation and plane of polarization. This information allowed the drag coefficient to be determined, which implied that the instantaneous torque generated by F 1 was 63.3 ± 2.9 pN nm. The high temporal resolution of rotation allowed the measurement of the instantaneous torque of F 1 , resulting in direct implications for its rotational mechanism. The F 1 F o-ATP synthase couples the energy provided by a transmembrane proton gradient to the production of ATP from ADP and phosphate. The intrinsic membrane complex of ab 2 c 10-14 subunits known as F o 1 functions as a proton channel, and the F 1 peripheral membrane complex of α 3 β 3 γδϵ subunits contains one site for ATP synthesis and/or hydrolysis per αβ heterodimer. When F 1 is purified from F o and the membrane, it retains the ability to hydrolyze ATP (1-3). There are three catalytic sites which are coupled such that the binding of the substrate to one site induces conformational changes that result in product
The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, ... more The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F1 (αβ)3 ring stator contains three catalytic sites. Single-molecule F1 rotation studies revealed that ATP hydrolysis at each catalytic site (0°) precedes a power-stroke that rotates subunit-γ 120° with angular velocities that vary with rotational position. Catalytic site conformations vary relative to subunit-γ position (βE, empty; βD, ADP bound; βT, ATP-bound). During a power stroke, βE binds ATP (0°–60°) and βD releases ADP (60°–120°). Årrhenius analysis of the power stroke revealed that elastic energy powers rotation via unwinding the γ-subunit coiled-coil. Energy from ATP binding at 34° closes βE upon subunit-γ to drive rotation to 120° and forcing the subunit-γ to exchange its tether from βE to βD, which changes catalytic site conformations. In F1FO, the membrane-bound FO complex contains a ring of...
Most cellular ATP is made by rotary F 1 F O ATP synthases using proton translocationgenerated clo... more Most cellular ATP is made by rotary F 1 F O ATP synthases using proton translocationgenerated clockwise torque on the F O c-ring rotor, while F 1-ATP hydrolysis can force counterclockwise rotation and proton pumping. The F O torque-generating mechanism remains elusive even though the F O interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F 1 F O structures. Here, single-molecule F 1 F O rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of F O to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through F O operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the Escherichia coli c 10-ring of F 1 F O against the torque of F 1-ATPase-dependent rotation that result from H + transfer events from F O subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c 10-ring occur to sustain F 1 F O ATP synthesis. Editor's evaluation This manuscript describes single molecule spectroscopic work to probe the mechanisms by which protonation/deprotonation steps produce torque between the a-subunit and the C10 ring, which is subsequently conveyed to F1 to couple to ATP synthesis. This is an important bioenergetics question and the approach yields some tantalizing clues as to which protonation steps are involved. In principle, this knowledge could lead to direct experimental or computational tests to resolve the overall mechanism.
ΩqPCR determines absolute telomere length in kb units from single cells. Accuracy and precision o... more ΩqPCR determines absolute telomere length in kb units from single cells. Accuracy and precision of ΩqPCR were assessed using 800 bp and 1600 bp synthetic telomeres inserted into plasmids, which were measured to be 819 ± 19.6 and 1590 ± 42.3 bp, respectively. This is the first telomere length measuring method verified in this way. The approach uses Ω-probes, a DNA strand containing sequence information that enables: (i) hybridization with the telomere via the 3′ and 5′ ends that become opposed; (ii) ligation of the hybridized probes to circularize the Ω-probes and (iii) circularized-dependent qPCR due to sequence information for a forward primer, and for a reverse primer binding site, and qPCR hydrolysis probe binding. Read through of the polymerase during qPCR occurs only in circularized Ω-probes, which quantifies their number that is directly proportional to telomere length. When used in concert with information about the cell cycle stage from a single-copy gene, and ploidy, the MT...
An asymmetric, fully-connected 8-city traveling salesman problem (TSP) was solved by DNA computin... more An asymmetric, fully-connected 8-city traveling salesman problem (TSP) was solved by DNA computing using the ordered node pair abundance (ONPA) approach through the use of pair ligation probe quantitative real time polymerase chain reaction (PLP-qPCR). The validity of using ONPA to derive the optimal answer was confirmed by in silico computing using a reverse-engineering method to reconstruct the complete tours in the feasible answer set from the measured ONPA. The high specificity of the sequence-tagged hybridization, and ligation that results from the use of PLPs significantly increased the accuracy of answer determination in DNA computing. When combined with the high throughput efficiency of qPCR, the time required to identify the optimal answer to the TSP was reduced from days to 25 min.
F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Esch... more F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αβ)3δab2. Subunits c10 or (αβ)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a ...
The two opposed rotary molecular motors of the F 0 F 1-ATP synthase work together to provide the ... more The two opposed rotary molecular motors of the F 0 F 1-ATP synthase work together to provide the majority of ATP in biological organisms. Rotation occurs in 120°power strokes separated by dwells when F 1 synthesizes or hydrolyzes ATP. F 0 and F 1 complexes connect via a central rotor stalk and a peripheral stator stalk. A major unresolved question is the mechanism in which the interaction between subunit-a and rotating subunitc-ring in the F 0 motor uses the flux of H ؉ across the membrane to induce clockwise rotation against the force of counterclockwise rotation driven by the F 1-ATPase. In single-molecule measurements of F 0 F 1 embedded in lipid bilayer nanodiscs, we observed that the ability of the F 0 motor to form transient dwells increases with decreasing pH. Transient dwells can halt counterclockwise rotation powered by the F 1-ATPase in steps equivalent to the rotation of single c-subunits in the c-ring of F 0 , and can push the common axle shared by the two motors clockwise by as much as one c-subunit. Because the F 0 proton half-channels that access the periplasm and the cytoplasm are exposed to the same pH, these data are consistent with the conclusion that the periplasmic half-channel is more easily protonated in a manner that halts ATPase-driven rotation by blocking ATPase-dependent proton pumping. The fit of transient dwell occurrence to the sum of three Gaussian curves suggests that the asymmetry of the three ATPase-dependent 120°power strokes imposed by the relative positions of the central and peripheral stalks affects c-subunit stepping efficiency. The F-ATP synthases, located in eubacteria, mitochondria, and chloroplasts, are members of a family of rotary molecular motors that also include archaeal A-ATP synthases, bacterial A/V-like ATP synthases, and eukaryotic vacuolar V-ATPases (1). The F-ATP synthases are composed of two opposed rotary motors known as the membrane-embedded F 0 , and the extrinsic membrane protein complex F 1 (Fig. 1). These motors are connected by a central stalk that serves as the rotor and a peripheral stalk that is a component of the stator (2). In Escherichia coli, as in most organisms, the largest single source of cellular ATP is that made by the F 0 F 1-ATP synthase.
The F1FO ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causativ... more The F1FO ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). In addition to their synthase function most F-ATP synthases possess an ATP-hydrolase activity, which is coupled to proton-pumping activity. However, the mycobacterial enzyme lacks this reverse activity, but the reason for this deficiency is unclear. Here, we report that a Mycobacterium-specific, 36 amino acid long C-terminal domain in the nucleotide-binding subunit α (Mtα) of F-ATP synthase suppresses its ATPase activity and determined the mechanism of suppression. First, we employed vesicles to show that in intact membrane-embedded mycobacterial F-ATP synthases deletion of the C-terminal domain enabled ATPase and proton-pumping activity. We then generated a heterologous F-ATP synthase model system, which demonstrated that transfer of the mycobacterial C-terminal domain to a standard F-ATP synthase α subunit suppresses ATPase activity. Single-molecule rotati...
The angular velocities of ATPase-dependent power strokes as a function of the rotational position... more The angular velocities of ATPase-dependent power strokes as a function of the rotational position for the A-type molecular motor A3B3DF from the Methanosarcina mazei Goe1 A-ATP synthase, and the thermophilic motor α3β3γ from Geobacillus stearothermophilus (formerly known as Bacillus PS3) F-ATP synthase are resolved at 5 μsec resolution for the first time. Unexpectedly, the angular velocity profile of the A-type was closely similar in the angular positions of accelerations and decelerations to the profiles of the evolutionarily distant F-type motors of thermophilic and mesophilic origins, and differ only in the magnitude of their velocities. M. mazei A3B3DF power strokes occurred in 120° steps at saturating ATP concentrations like the F-type motors. However, since ATP-binding dwells did not interrupt the 120° steps at limiting ATP, ATP-binding to A3B3DF must occur during the catalytic dwell. Elevated concentrations of ADP did not increase dwells occurring 40° after the catalytic dwel...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014
Nano/micron-sized reaction chamber arrays (femtoliter-chamber arrays) enable highly sensitive and... more Nano/micron-sized reaction chamber arrays (femtoliter-chamber arrays) enable highly sensitive and quantitative biological assays, such as single-molecule enzymatic assays [1, 2], digital PCR [3, 4], and digital ELISA [5]. However, the versatility of femtoliter-chamber arrays has been limited to reactions in aqueous solutions. In this presentation, I will introduce an arrayed lipid bilayer chamber system (ALBiC) that displays a sub-million femtoliter chambers, each sealed with a stable 4-μm diameter lipid bilayer membrane with extremely high efficiency (yield:~99%). When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or FoF1-ATP synthase, the chambers of the ALBiC exhibited stochastic and quantized transporting activities, demonstrating that the single molecule analysis of passive and active membrane transports is achievable with the ALBiC system. Thus, this new platform has vastly extended the versatility of femtoliter chamber arrays and could contribute to the understanding of the working mechanism of membrane proteins as well as to further analytical and pharmacological applications. If time allows, I would like to talk about new versions of the ALBiC that we recently developed.
Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient ... more Living organisms rely on the FoF1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the Fo motor uses a transmembrane electrochemical ion gradient to create clockwise (CW) torque that overcomes F1ATPase-driven counterclockwise (CCW) torque at high ATP is a major unresolved question. Using single FoF1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of Fo-dependent rotation of the c10-ring in the ATP synthase (CW) direction against the CCW force of ATPase-driven rotation that occurs upon formation of a leash with Fo-stator subunit-a. Mutational studies indicate that the leash is important for ATP synthase activity, and support a mechanism in which residues aE196 and cR50 participate in the cytoplasmic proton half-channel to promote leash formation.
Although single-molecule experiments have provided mechanistic insight for several molecular moto... more Although single-molecule experiments have provided mechanistic insight for several molecular motors, these approaches have proved difficult for membrane bound molecular motors like the F o F 1-ATP synthase, in which proton transport across a membrane is used to synthesize ATP. Resolution of smaller steps in F o has been particularly hampered by signal-to-noise and time resolution. Here, we show the presence of a transient dwell between F o subunits a and c by improving the time resolution to 10 ls at unprecedented S/N, and by using Escherichia coli F o F 1 embedded in lipid bilayer nanodiscs. The transient dwell interaction requires 163 ls to form and 175 ls to dissociate, is independent of proton transport residues aR210 and cD61, and behaves as a leash that allows rotary motion of the c-ring to a limit of B361 while engaged. This leash behaviour satisfies a requirement of a Brownian ratchet mechanism for the F o motor where c-ring rotational diffusion is limited to 361.
Proceedings of the National Academy of Sciences, 2014
Significance We present a description of the angular velocity of the power stroke as a function o... more Significance We present a description of the angular velocity of the power stroke as a function of rotational position for the F 1 -ATPase molecular motor. The angular velocity of this motor, which rotates in 120° power strokes separated by catalytic dwells, is part of the F o F 1 ATP synthase in oxidative phosphorylation, and has been thought not to vary. This paper reports the unexpected discovery that a series of angular accelerations and decelerations occur, providing direct evidence that angular velocity depends on substrate binding affinity. The data support a model in which rotation is powered by Van der Waals repulsive forces during the final 85° of rotation, consistent with a transition from F 1 structures 2HLD1 and 1H8E (Protein Data Bank).
Brief saturating light fashes were used to probe the mechanism of inactivation of 02 evolution by... more Brief saturating light fashes were used to probe the mechanism of inactivation of 02 evolution by Tris in chloroplasts. Maximum inactiation with a single flash and an oscillation with period of four on subsequent flashes was observed. Analyses of the oscillations suggested that only the charge-collecting 02-evolving catalyst of photosystem H (S2-state) was a target of inactivation by Tris. This conclusion was supported by the following observations: (a) hydroxylamine preequWlibration caused a threeflash delay in the inactivation pattern; (b) the lifetimes of the Tris-inactivable and S2-states were similar, and (c) reagents acceleratin S2 deactivation decreased the lifetime of the inactivable state. Inactivation proved to be moderated by F, the precursor of Signal II., as shown by a one flash delay with chloroplasts having high abundance of F. Evidence was obtained for cooperativity effects in inactivation and NH3 was shown to be a competitive inhibitor of the Tris-induced inactivation. Sdependent inactivation was inhibited by glutaraldehyde fixation of chkoroplasts, possibly suggesting that inactivation proceeds via confornational changes of the S2-state. A previous report (11) on the mechanism of Tris inactivation of ' This work was supported in part by Grant PCM78-07694 from the National Science Foundation to G. M. C. This paper (79-3-153) is published with the approval of the Director of the Kentucky Agricultural Experiment Station. 2 In memory of Bessel, who didn't suffer fools gladly and never compromised his high standards in science.
Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abund... more Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg-20 ng linear detection range between thermal cycle threshold (C t value) and target content. The C t values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology.
Journal of Radioanalytical and Nuclear Chemistry Articles, 1987
A method for the quantitative determination of small amounts of protein samples was developed emp... more A method for the quantitative determination of small amounts of protein samples was developed employing neutron activation analysis. Current methods of protein concentration determination are severely limited as a result of differences in the specific :characteristics'. of each protein. Silver binding.has been used as a sensitive colorimetric method to indicate the presence of protein. However, silver-protein complexes can have a variety of absorbance spectra unique to each protein, which complicate the analysis. Various amounts of specific proteins were equilibrated in an excess of silver nitrate prior to the reduction of the silver by the addition of NaBI-h, HCHO, and NaOH. The protein-silver complex was rapidly separated from the unbound silver by centrifugation chromatography and the amount of bound silver was determined by INAA. The amount of silver was proportional to the amount of protein present in each sample. When the silver was not reduced prior to removal of the unbound silver by chromatography, only negl!gible amounts of silver remained bound to the protein. The stoiehiometry of bound silver to protein on a molar basis showed relatively small differences for the proteins that were examined. This ratio was found to depend on the conditions of the binding and reduction of the silver. The results suggest that *Ae binding of silver is not specific t~ any charged or polar groups on these proteins and may, fl~e~'efore, provide a means of determination of the concentration of protein that has general a?plication for all proteins.
Site-directed mutations were made to the phosphatebinding loop threonine in the -subunit of the ... more Site-directed mutations were made to the phosphatebinding loop threonine in the -subunit of the chloroplast F 1-ATPase in Chlamydomonas (T168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from T168D, T168C, and T168L mutants had <10% of the wild type rates, as did rates of Mg 2؉-ATPase activity of purified chloroplast F 1-ATPase (CF 1). The EPR spectra of VO 2؉-ATP bound to Site 3 of CF 1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF 1 from each mutant in both signal intensity and in 51 V hyperfine parameters that depend on the equatorial VO 2؉ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphatebinding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg 2؉-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF 1 with entrapped Mg 2؉-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.
Site-directed mutations D262C, D262H, D262N, and D262T were made to the  subunit Walker Homology... more Site-directed mutations D262C, D262H, D262N, and D262T were made to the  subunit Walker Homology B aspartate of chloroplast F 1-ATPase in Chlamydomonas. Photoautotrophic growth and photophosphorylation rates were 3-14% of wild type as were ATPase activities of purified chloroplast F 1 indicating that D262 is an essential residue for catalysis. The EPR spectrum of vanadyl bound to Site 3 of chloroplast F 1 as VO 2؉-ATP gave rise to two EPR species designated B and C in wild type and mutants. 51 V-hyperfine parameters of species C, present exclusively in the activated enzyme state, did not change significantly by the mutations examined indicating that it is not an equatorial ligand to VO 2؉ , nor is it hydrogen-bonded to a coordinated water at an equatorial position. Every mutation changed the ratio of EPR species C/B and/or the 51 V-hyperfine parameters of species B, the predominant conformation of VO 2؉-nucleotide bound to Site 3 in the latent (down-regulated) state. The results indicate that the Walker Homology B aspartate coordinates the metal of the predominant metal-nucleotide conformation at Site 3 in the latent state but not in the conformation present exclusively upon activation and elucidates one of the specific changes in metal ligation involved with activation.
Removal of the ability to form a salt bridge or hydrogen bonds between the  subunit catch loop (... more Removal of the ability to form a salt bridge or hydrogen bonds between the  subunit catch loop (Y297-D305) and the ␥ subunit of Escherichia coli F 1 F o-ATP synthase significantly altered the ability of the enzyme to hydrolyze ATP and the bacteria to grow via oxidative phosphorylation. Residues T304, D305, D302, ␥Q269, and ␥R268 were found to be very important for ATP hydrolysis catalyzed by soluble F 1-ATPase, and the latter four residues were also very important for oxidative phosphorylation. The greatest effects on catalytic activity were observed by the substitution of side chains that contribute to the shortest and/or multiple H-bonds as well as the salt bridge. Residue D305 would not tolerate substitution with Val or Ser and had extremely low activity as D305E, suggesting that this residue is particularly important for synthesis and hydrolysis activity. These results provide evidence that tight winding of the ␥ subunit coiled-coil is important to the rate-limiting step in ATP hydrolysis and are consistent with an escapement mechanism for ATP synthesis in which ␣␥ intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven ␥ subunit rotation.
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Papers by Wayne Frasch