Session L10: Focus Session: Single Molecule Studies of Enzymes

Identifying Single Molecule Dynamics in Real Time via Time-Resolved Coherent Anti-Stokes Raman Scattering

By enhancing the local response of a single molecule with a dipolar nano-antenna, the vibrational dynamics at the single molecule limit can be measured in real time, by Time-Resolved Coherent Anti-Stokes Raman Scattering (TR-CARS). Such measurements involve the preparation and subsequent probing of vibrational wavepackets on the ground electronic state. In contrast to ensemble measurements, the vibrational coherence of a single molecule is not subject to dephasing; it exhibits dynamic phase and amplitude noise due to the~collapse of the wavepacket upon measurement. Continuous measurements of the amplitude noise distribution as a function of phase delay, allows the complete reconstruction of the state of the system. Under ambient conditions, repeated measurements of a single molecule coherence reduces to a statistical state of a system coupled to the thermal bath. The signature of the statistical state of a single molecule is characteristic, distinct from that of two, or three, or many; and this can be directly demonstrated through measurements.   

Impact of the Diffusion of Microtubule-Associated Protein EB1 on Kinesin Translocation \textit{in Vitro}

Using the slowly hydrolyzable GTP analog GMPCPP, we polymerize microtubules that recapitulate the end binding behavior of EB1 along their entire length, and investigate the impact of EB1 on kinesin translocation. Through direct observation of single molecules of EB1 fused to GFP, we find that EB1 diffuses along the microtubule lattice, and that the presence of taxol affects the rate of diffusion. To test whether EB1 presence and diffusion has an effect on kinesin-driven cargo transport, we observe quantum dot labeled kinesins walking on microtubules assembled with GMPCPP and taxol and coated with EB1. We find that the addition of EB1 significantly reduces kinesin speed compared to the no EB1 condition, but when microtubules stabilized by both taxol and GMPCPP are used, the speed reduction is nearly abolished. Our data suggest a new possible mechanism for the regulation of kinesin function by EB1 in which kinesin speed is directly modulated through the interference of EB1 diffusion. Our results also raise important questions about the effects of taxol on microtubule-MAP interactions.   

Acceleration of molecular complex dissociation by occlusion of rapid rebinding

Molecular complexes in biology are held together by non-covalent interactions. Explicit consideration of molecular diffusion suggests that the dissociation kinetics of these systems are not adequately explained by simple distinctions between ``bound'' and ``free'' states of its molecular components. We formulated physical models that describe the effect of competitor molecules on the dissociation of a complex. The models show that competitor acceleration of complex dissociation by occluding rapid re-association is a natural feature of a molecular competition that could play a significant role in a wide variety of biological regulatory processes. We use single-molecule fluorescence colocalization experiments on a model complex to test this prediction and show that the effect is observed in biologically relevant ranges of competitor concentration. The results also demonstrate that single-molecule colocalization experiments can accurately measure dissociation rates despite their limited spatiotemporal resolution.   

Single molecule analysis of cytoplasmic dynein motility

Cytoplasmic dynein is a homodimeric AAA$+$ motor that transports a multitude of cargos towards the microtubule (MT) minus end. The mechanism of dynein motility remains unclear, due to its large size (2.6 MDa) and the complexity of its structure. By tracking the stepping motion of both heads at nanometer resolution, we observed that dynein heads move independently along the MT, in contrast to hand over hand movement of kinesins and myosin. Stepping behavior of the heads varies as a function of interhead separation and establishing the basis of high variability in dynein step size. By engineering the mechanical and catalytic properties of the dynein motor domain, we show that a rigid linkage between monomers and dimerization between N-terminal tail domains are not essential for processive movement. Instead, dynein processivity minimally requires the linker domain of one active monomer to be attached to an inert MT tether retaining only the MT-binding domain. The release of a dynein monomer from the MT can be mediated either by nucleotide binding or external load. Nucleotide dependent release is inhibited by the tension on the linker domain at high interhead separations. Tension dependent release is highly asymmetric, with faster release towards the minus-end. Reversing the asymmetry of the MT binding interface results in plus end directed motility, even though the force was generated by the dynein motor activity. On the basis of these measurements, we propose a model that describes the basis of dynein processivity, directionality and force generation.   

Single-molecule comparison of DNA Pol I activity with native and analog nucleotides

DNA polymerases are critical enzymes for DNA replication, and because of their complex catalytic cycle they are excellent targets for investigation by single-molecule experimental techniques. Recently, we studied the Klenow fragment (KF) of DNA polymerase I using a label-free, electronic technique involving single KF molecules attached to carbon nanotube transistors [1]. The electronic technique allowed long-duration monitoring of a single KF molecule while processing thousands of template strands. Processivity of up to 42 nucleotide bases was directly observed, and statistical analysis of the recordings determined key kinetic parameters for the enzyme's open and closed conformations. Subsequently, we have used the same technique to compare the incorporation of canonical nucleotides like dATP to analogs like 1-thio-2'-dATP. The analog had almost no affect on duration of the closed conformation, during which the nucleotide is incorporated. On the other hand, the analog increased the rate-limiting duration of the open conformation by almost 40{\%}. We propose that the thiolated analog interferes with KF's recognition and binding, two key steps that determine its ensemble turnover rate. [1] T. J. Olsen, et. al., JACS 135, 7855 (2013).   

Harmonic force spectroscopy reveals a force-velocity curve from a single human beta cardiac myosin motor

A muscle contracts rapidly under low load, but slowly under high load. Its molecular mechanisms remain to be elucidated, however. During contraction, myosins in thick filaments interact with actin in thin filaments in the sarcomere, cycling between a strongly bound (force producing) state and a weakly bound (relaxed) state. Huxley et al. have previously proposed that the transition from the strong to the weak interaction can be modulated by a load. We use a new method we call ``harmonic force spectroscopy'' to extract a load-velocity curve from a single human beta cardiac myosin II motor. With a dual-beam optical trap, we hold an actin dumbbell over a myosin molecule anchored to the microscope stage that oscillates sinusoidally. Upon binding, the motor experiences an oscillatory load with a mean that is directed forward or backward, depending on binding location We find that the bound time at saturating [ATP] is exponentially correlated with the mean load, which is explained by Arrhenius transition theory. With a stroke size measurement, we obtained a load-velocity curve from a single myosin. We compare the curves for wild-type motors with mutants that cause hypertrophic cardiomyopathies, to understand the effects on the contractile cycle   

Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.   

Molecular motors are stymied by microtubule lattice defects

The microtubule surface provides the tracks that molecular motors use to transport cargo throughout the cell. Much like any surface lattice, the microtubule surface may have surface defects such as dislocations or step edges caused by missing tubulin dimers or shifts in the number of protofilaments, respectively. It is an open question as to how microtubule lattice defects affect molecular motors walking along microtubule surfaces. We used the kinesin-1 motor that walks along a single protofilament and has a short step size of only 8 nm to test how lattice defects affect transport. We created microtubule lattice defects by end-to-end annealing microtubules with different protofilament numbers and differential fluorescence labeling, creating a transition in microtubule radius at the annealed site that is directly visualizable. Surprisingly, we observed that kinesin-1 motors are significantly inhibited by protofilament shift defects. GFP-tagged kinesin-1 motors detach at the defect site during at least 70{\%} of encounters with the defect. We find end-to-end annealed microtubules without the additional change in protofilament number at the defect site inhibit at least 50{\%} of kinesin-1 motors at the defect, suggesting that the process of end-to-end annealing creates defects within the lattice. Our results imply that defects within the microtubule lattice can inhibit motility, and must be corrected. Our work sheds light on the biological importance of removing and correcting lattice defects, an activity known to occur by multiple methods in cells.   

Interplay between Velocity and Travel Distance of Kinesin-based Transport in the Presence of Tau

Although the disease-relevant microtubule-associated protein tau is known to severely inhibit kinesin-based transport in vitro, potential mechanisms for reversing this detrimental effect to maintain healthy transport in cells remain unknown. Here we report the unambiguous up-regulation of multiple-kinesin travel distance despite the presence of tau, via decreased single-kinesin velocity. Intriguingly, the presence of tau also modestly reduced velocity in multiple-kinesin transport. Our stochastic simulations indicate that the tau-mediated reduction in single-kinesin travel is sufficient for the observed reduction in multiple-kinesin velocity. Taken together, our observations suggest that single-kinesin velocity is a promising experimental handle for tuning the effect of tau on multiple-kinesin travel distance, and uncover a previously unexplored role of tau for inhibiting multiple-kinesin velocity via reducing single-kinesin travel distance.   

Molecular motors and the 2$^{\mathrm{nd}}$ law of thermodynamics

Molecular motors from biology and nanotechnology often operate on chemical energy of fuel molecules in an isothermal environment, unlike macroscopic heat engines that draw energy from a heat flow between two temperatures. Nevertheless, isothermal molecular motors are still subject to the 2$^{\mathrm{nd}}$ law of thermodynamics in a fundamental way: their directional motion must cost a finite amount of energy other than the environmental heat even though no work is done; otherwise the 2$^{\mathrm{nd}}$ law would be violated. Hence the 2$^{\mathrm{nd}}$ law requires a finite energy price for pure direction of molecular motors. But what is the lowest price of direction allowed by the 2$^{\mathrm{nd}}$ law? And how does the 2$^{\mathrm{nd}}$ law-decreed price of direction limit performance of molecular motors? In the talk, I shall present our theoretical study of the 2$^{\mathrm{nd}}$ law-molecular motor link on basis of the accumulated biomotor phenomenology, and also introduce our experimental effort to develop biomimetic DNA bipedal nanomotors following the mechanistic guidelines out of the theoretical study. [Main contents of this talk are from references: J. Chem. Phys. 139, 035105 (2013); Phys. Rev. E 88, 022703 (2013); Phys. Rev. Lett. 109, 238104 (2012)]   

A propagating ATPase gradient drives transport of surface-confined cellular cargo

The process of DNA segregation is of central importance for all organisms. Although eukaryotic mitosis is relatively well established, the most common mechanism employed for bacterial DNA segregation has been unclear. ParA ATPases form dynamic patterns on the bacterial nucleoid, to spatially organize plasmids, chromosomes and other large cellular cargo, but the force generating mechanism has been a source of controversy and debate. A dominant view proposes that ParA-mediated transport and cargo positioning occurs via a filament-based mechanism that resembles eukaryotic mitosis. Here we present direct evidence against such models. Our cell-free reconstitution supports a non-filament-based mode of transport that may be as widely found in nature as actin filaments and microtubules.