Ultrafast structural fluctuations and rearrangements of water's hydrogen bonded network

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2007."December 2006." Vita.Includes bibliographical references.Aqueous chemistry is strongly influenced by water's ability to form an extended network of hydrogen bonds. It is the fluctuations and rearrangements of this network that stabilize reaction products and drive the transport of excess protons through solution. Experimental observations of the dynamics of the hydrogen bonded network are difficult because (1) the timescales are exceedingly fast with relevant fluctuations occurring on a tens of femtosecond period and (2) the experimental probe must be sensitive to the local hydrogen bonded structure. In this thesis I address these experimental challenges through the development of ultrafast nonlinear infrared spectroscopy of the OH stretch of HOD in D20. The frequency of the OH stretch, OH, is sensitive to the configuration of the hydrogen bonded pair. Therefore, time-dependent changes in OoH can be correlated with changes in the hydrogen bonded geometry. I describe how broadband homodyne echo and polarization-dependent pump-probe experiments can be utilized to separate the contributions of spectral diffusion, vibrational relaxation and molecular reorientation. These experiments observe the underdamped motion of the hydrogen bonded pair and the librational motion of the OH dipole on the 180 and 50 fs timescales, respectively.(cont.) These dynamics occur on a relatively local (i.e. molecular) length scale. At times greater than ~300 fs the experiments observe signatures of a kinetic regime. No longer can the spectral relaxation be ascribed to a clear molecular motion. Instead, the decay originates from the collective reorganization of many molecules. Two dimensional infrared spectroscopy (2D IR) is applied to further investigate the mechanism of hydrogen bond rearrangement. 2D IR is an optical analogue of multidimensional NMR. As a correlation spectroscopy, time dependent changes in 2D IR line shapes track how vibrational oscillators relax from one frequency to another. I describe two methods of acquiring high fidelity 2D line shapes at wavelengths of 3 gtm. Both methods utilize a HeNe laser as a frequency standard and balanced detection of the signal field. Spectral diffusion is found to dominate the evolution of the 2D line shapes of the OH stretch up to the vibrational lifetime of 700 fs. At times beyond this point the line shapes change substantially, indicating population relaxation out of the v= 1 state and the formation of a spectroscopically distinct vibrationally excited ground state. Frequency dependent relaxation of the 2D IR line shapes reveals that molecules in hydrogen bonded and non-bonded configurations experience qualitatively different fluctuations.(cont.) Non-bonded configurations are found to return to band center on -100 fs timescale indicating that these configurations are inherently unstable. Hydrogen bonded oscillators undergo underdamped oscillations at the hydrogen bond stretching frequency before subsequent barrier crossing. Hydrogen bonding not only affects COOH. The transition dipole, gi, is modulated by the hydrogen bonding interaction, resulting in higher oscillator strength for strong hydrogen bonds. I describe how modeling the temperature dependent behavior of IR and Raman line shapes in combination with nonlinear IR spectroscopies can extract the frequency dependent magnitude of gi. The variation in the transition dipole with frequency is found to be roughly linear on resonance but is found to be strongly nonlinear for weak hydrogen bonds on the high frequency side of the OH line shape.by Joseph J. Loparo.Ph.D

Exchange between Escherichia coli polymerases II and III on a processivity clamp

Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, β. Single-molecule experiments reveal that the interactions of Pol II and Pol III with β allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a β-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

Disaccharide topology induces slow down in local water dynamics

Molecular level insight into water structure and structural dynamics near proteins, lipids and nucleic acids is critical to the quantitative understanding of many biophysical processes. Un- fortunately, understanding hydration and hydration dynamics around such large molecules is challenging because of the necessity of deconvoluting the effects of topography and chemical heterogeneity. Here we study, via classical all atom simulation, water structure and structural dynamics around two biologically relevant solutes large enough to have significant chemical and topological heterogeneity but small enough to be computationally tractable: the disaccharides Kojibiose and Trehalose. We find both molecules to be strongly amphiphilic (as quantified from normalized local density fluctuations) and to induce nonuniform local slowdown in water translational and rotational motion. Detailed analysis of the rotational slowdown shows that while the rotational mechanism is similar to that previously identified in other aqueous systems by Laage, Hynes and coworkers, two novel characteristics are observed: broadening of the transition state during hydrogen bond exchange (water rotation) and a subpopulation of water for which rotation is slowed because of hindered access of the new accepting water molecule to the transition state. Both of these characteristics are expected to be generic features of water rotation around larger biomolecules and, taken together, emphasize the difficulty in transferring insight into water rotation around small molecules to much larger amphiphilic solutes.This work is part of the research program of the “Stichting voor Fundamenteel Onderzoek der Materie (FOM)” which is financially supported by the “Nederlandse organisatie voor Wetenschap- pelijk Onderzoek (NWO)”. Further financial support was provided by a Marie Curie Incoming International Fellowship (RKC). We gratefully acknowledge SARA, the Dutch center for high- performance computing, for computational time and Huib Bakker and Daan Frenkel for useful critical reviews on an earlier version of this work. We thank two anonymous reviewers for their excellent work, especially for bringing to our attention calculations done on the transition state geometry of dimers and the overstructuring of the O-O radial distribution function of SPC/E water

Real-time single-molecule observation of rolling-circle DNA replication

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors

Average flow constraints and stabilizability in uncertain production-distribution systems

We consider a multi-inventory system with controlled flows and uncertain demands (disturbances) bounded within assigned compact sets. The system is modelled as a first-order one integrating the discrepancy between controlled flows and demands at different sites/nodes. Thus, the buffer levels at the nodes represent the system state. Given a long-term average demand, we are interested in a control strategy that satisfies just one of two requirements: (i) meeting any possible demand at each time (worst case stability) or (ii) achieving a predefined flow in the average (average flow constraints). Necessary and sufficient conditions for the achievement of both goals have been proposed by the authors. In this paper, we face the case in which these conditions are not satisfied. We show that, if we ignore the requirement on worst case stability, we can find a control strategy driving the expected value of the state to zero. On the contrary, if we ignore the average flow constraints, we can find a control strategy that satisfies worst case stability while optimizing any linear cost on the average control. In the latter case, we provide a tight bound for the cost

Guidelines for DNA recombination and repair studies: Mechanistic assays of DNA repair processes

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies