Acceleration of DNA Replication of Klenow Fragment by Small Resisting Force

DNA polymerases are an essential class of enzymes or molecular motors that catalyze processive DNA syntheses during DNA replications. A critical issue for DNA polymerases is their molecular mechanism of processive DNA replication. We have proposed a model for chemomechanical coupling of DNA polymerases before, based on which the predicted results have been provided about the dependence of DNA replication velocity upon the external force on Klenow fragment of DNA polymerase I. Here, we performed single molecule measurements of the replication velocity of Klenow fragment under the external force by using magnetic tweezers. The single molecule data verified quantitatively the previous theoretical predictions, which is critical to the chemomechanical coupling mechanism of DNA polymerases. A prominent characteristic for the Klenow fragment is that the replication velocity is independent of the assisting force whereas the velocity increases largely with the increase of the resisting force, attains the maximum velocity at about 3.8 pN and then decreases with the further increase of the resisting force.

Modeling Lithium Transport and Electrodeposition in Ionic-Liquid Based Electrolytes

Purely ionic electrolytes—wherein ionic liquids replace neutral solvents—have been proposed to improve lithium-ion-battery performance, on the basis that the unique microscopic characteristics of polarized ionic-liquid/electrode interfaces may improve the selectivity and kinetics of interfacial lithium-exchange reactions. Here we model a “three-ion” ionic-liquid electrolyte, composed of a traditional ionic liquid and a lithium salt with a common anion. Newman's concentrated-solution theory is extended to account for space charging and chemomechanical coupling. We simulate electrolytes in equilibrium and under steady currents. We find that the local conductivity and lithium transference number in the diffuse double layers near interfaces differ considerably from their bulk values. The mechanical coupling causes ion size to play a crucial role in the interface's electrical response. Interfacial kinetics and surface charge on the electrodes both affect the apparent transport properties of purely ionic electrolytes near interfaces. Larger ionic-liquid cations and anions may facilitate interfacial lithium-exchange kinetics.

A model of processive walking and slipping of kinesin-8 molecular motors

Kinesin-8 molecular motor can move with superprocessivity on microtubules towards the plus end by hydrolyzing ATP molecules, depolymerizing microtubules. The available single molecule data for yeast kinesin-8 (Kip3) motor showed that its superprocessive movement is frequently interrupted by brief stick–slip motion. Here, a model is presented for the chemomechanical coupling of the kinesin-8 motor. On the basis of the model, the dynamics of Kip3 motor is studied analytically. The analytical results reproduce quantitatively the available single molecule data on velocity without including the slip and that with including the slip versus external load at saturating ATP as well as slipping velocity versus external load at saturating ADP and no ATP. Predicted results on load dependence of stepping ratio at saturating ATP and load dependence of velocity at non-saturating ATP are provided. Similarities and differences between dynamics of kinesin-8 and that of kinesin-1 are discussed.

Clutch mechanism of chemomechanical coupling in a DNA resecting motor nuclease

Mycobacterial AdnAB is a heterodimeric helicase–nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3′ to 5′ translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet–like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB–DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch.

Mechanochemical properties of human myosin 1C are modulated by isoform-specific differences in the N-terminal extension

Myosin-1C is a single-headed, short-tailed member of the myosin class I subfamily that supports a variety of actin-based functions in the cytosol and nucleus. In vertebrates, alternative splicing of the MYO1C gene leads to the production of three isoforms, myosin-1C0, myosin-1C16 and myosin-1C35, that carry N-terminal extensions of different length. However, it is not clear how these extensions affect the chemomechanical coupling of human myosin-1C isoforms. Here, we report on the motor activity of the different myosin-1C isoforms measuring the unloaded velocities of constructs lacking the C-terminal lipid binding domain on nitrocellulose-coated glass surfaces and full-length constructs on reconstituted, supported lipid bilayers. The higher yields of purified protein obtained with constructs lacking the lipid binding domain allowed a detailed characterization of the individual kinetic steps of human myosin-1C isoforms in their productive interaction with nucleotides and filamentous actin. Isoform-specific differences include 18-fold changes in the maximum power output per myosin-1C motor and 4-fold changes in the velocity and the resistive force at which maximum power output occurs. Our results support a model in which the isoform-specific N-terminal extensions affect chemomechanical coupling by combined steric and allosteric effects, thereby reducing both the length of the working stroke and the rate of ADP release in the absence of external loads by a factor of two for myosin-1C35. As the large change in maximum power output shows, the functional differences between the isoforms are further amplified by the presence of external loads.

Bridging biochemical activities with conformational dynamics observed in atomic force microscopy

This dissertation was written with the intention to provide future investigators with general information sufficient to start their own investigations in biological atomic force microscopy. It is noted that background information from both physics and biology has been included for overall clarity. A main emphasis of my thesis work was on modifying traditional assays to measure biochemical activities of membrane proteins adsorbed on surfaces prepared in an identical manner for atomic force microscopy (AFM) measurements. Additional projects included probing conformational dynamics of enzymes and utilizing atomic force spectroscopy to probe peptide-lipid interactions at enhanced temporal resolution via focused ion beam (FIB) modified AFM cantilevers. The experimental procedures in the appendix were purposefully written in a step by step format, with detailed notes of important or tricky aspects and precautions. Thus, these sections could serve as practical templates to construct future protocols and experiments. A chapter on future directions serves as suggestions of possible avenues of research. AFM measurements can shed light on membrane protein conformational dynamics and folding at a single molecule level. However, the unavoidable close proximity of the supporting surface to AFM specimens raises questions about the viability and preservation of biochemical activities. We quantified activities of the translocase from the general secretory (Sec) system of Escherichia coli, (E. coli), via two biochemical assays in surface supported bilayers: ATP hydrolysis and translocation. The ATP hydrolysis assays revealed distinct levels of activation ranging from low (basal), to medium (translocase-activated), to high (translocation-associated) corresponding to binding partners of SecA, the ATPase enzyme that hydrolyzes ATP. The measured on surface ATP hydrolysis activity levels were similar to traditional solution experiments. Furthermore, the surface activity assays uncovered characteristics of conformational hysteresis of SecA. Translocation assays displayed turn over numbers that were comparable to solution but with a reduction in the apparent rate constant. Despite a 10-fold difference in kinetics, the chemomechanical coupling (ATP hydrolyzed per residue translocated) only varied twofold on glass compared to solution. The activity changed with the topography of the supporting surface underneath the lipid bilayer. Glass cover slips have higher surface roughness than that of mica; this roughness can provide extra submembrane space. In turn, this extra space could lower the frictional coupling between the translocating polypeptide and the supporting surface. For these reasons, glass surfaces were favored over mica. Neutron reflectometry corroborated the results and provided characterization of the integral and peripheral components, as well as the submembrane space between the surface and the lower bilayer leaflet. Overall, surface activity assays had sufficient sensitivity to distinguish different levels of ATP hydrolysis and translocation activities of surface adsorbed systems, albeit with a slower rate-limiting step than observed in solution assays. Equipped with biochemical activity information for the surface-adsorbed proteins, we could then more strongly correlate conformational dynamics of the proteins observed in AFM measurements to their biochemical activities. We conducted AFM investigations on conformational dynamics of SecA on mica surfaces yielding fruitful information to specify the domain responsible for conformational dynamics during the ATP hydrolysis cycle. We also investigated the dynamics of translocase complexes engaging in translocation of precursor proteins across the membrane surface. These experiments brought to light previously underappreciated precursor species dependent conformational dynamics of the translocase.