Free energy landscape and characteristic forces for the initiation of DNA unzipping

DNA unzipping, the separation of its double helix into single strands, is crucial in modulating a host of genetic processes. Although the large-scale separation of double-stranded DNA has been studied with a variety of theoretical and experimental techniques, the minute details of the very first steps of unzipping are still unclear. Here, we use atomistic molecular dynamics (MD) simulations, coarse-grained simulations and a statistical-mechanical model to study the initiation of DNA unzipping by an external force. The calculation of the potential of mean force profiles for the initial separation of the first few terminal base pairs in a DNA oligomer reveal that forces ranging between 130 and 230 pN are needed to disrupt the first base pair, values of an order of magnitude larger than those needed to disrupt base pairs in partially unzipped DNA. The force peak has an "echo," of approximately 50 pN, at the distance that unzips the second base pair. We show that the high peak needed to initiate unzipping derives from a free energy basin that is distinct from the basins of subsequent base pairs because of entropic contributions and we highlight the microscopic origin of the peak. Our results suggest a new window of exploration for single molecule experiments.Comment: 25 pages, 6 figures , Accepted for publication in Biophysical Journa

Consequences of local inter-strand dehybridization for large-amplitude bending fluctuations of double-stranded DNA

The wormlike chain (WLC) model of DNA bending accurately reproduces single-molecule force-extension profiles of long (kilobase) chains. These bending statistics over large scales do not, however, establish a unique microscopic model for elasticity at the 1-10 bp scale, which holds particular interest in biological contexts. Here we examine a class of microscopic models which allow for disruption of base pairing (i.e., a `melt' or `kink', generically an `excitation') and consequently enhanced local flexibility. We first analyze the effect on the excitation free energy of integrating out the spatial degrees of freedom in a wormlike chain. Based on this analysis, we present a formulation of these models that ensures consistency with the well-established thermodynamics of melting in long chains. Using a new method to calculate cyclization statistics of short chains from enhanced-sampling Monte Carlo simulations, we compute J-factors of a meltable wormlike chain (MWLC) over a broad range of chain lengths, including very short molecules (30 bp) that have not yet been explored experimentally. For chains longer than about 120 bp, including most molecules studied to date in the laboratory, we find that melting excitations have little impact on cyclization kinetics. Strong signatures of melting, which might be resolved within typical experimental scatter, emerge only for shorter chains.Comment: 13 pages, 5 figure

Biomolecular simulations: From dynamics and mechanisms to computational assays of biological activity

Biomolecular simulation is increasingly central to understanding and designing biological molecules and their interactions. Detailed, physics‐based simulation methods are demonstrating rapidly growing impact in areas as diverse as biocatalysis, drug delivery, biomaterials, biotechnology, and drug design. Simulations offer the potential of uniquely detailed, atomic‐level insight into mechanisms, dynamics, and processes, as well as increasingly accurate predictions of molecular properties. Simulations can now be used as computational assays of biological activity, for example, in predictions of drug resistance. Methodological and algorithmic developments, combined with advances in computational hardware, are transforming the scope and range of calculations. Different types of methods are required for different types of problem. Accurate methods and extensive simulations promise quantitative comparison with experiments across biochemistry. Atomistic simulations can now access experimentally relevant timescales for large systems, leading to a fertile interplay of experiment and theory and offering unprecedented opportunities for validating and developing models. Coarse‐grained methods allow studies on larger length‐ and timescales, and theoretical developments are bringing electronic structure calculations into new regimes. Multiscale methods are another key focus for development, combining different levels of theory to increase accuracy, aiming to connect chemical and molecular changes to macroscopic observables. In this review, we outline biomolecular simulation methods and highlight examples of its application to investigate questions in biology. This article is categorized under: Molecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo Methods Structure and Mechanism > Computational Biochemistry and Biophysics Molecular and Statistical Mechanics > Free Energy Method

Structure energy relationship of biological halogen bonds

2012 Summer.Includes bibliographical references.The primary goal of the studies in this thesis is to derive a set of mathematical models to describe the anisotropic atomic nature of covalent bound halogens and by extension their molecular interactions. We use a DNA Holliday junctions as a experimental model system to assay the structure energy relationship of halogen bonds (X-bonds) in a complex biological environment. The first chapter of this dissertation is reserved for a review on DNA structure and the Holliday Junction in context of other DNA conformations. The conformational isomerization of engineered Holliday junctions will be established as a means to assay the energies of bromine X-bonds both in crystal and in solution. The experimental data are then used in the development of anisotropic force fields for use in the mathematical modeling of bromine halogen bonds, serving as a foundation to model all biological halogen interactions. The DNA Holliday junction experimental system is expanded to compare and contrast halogens from fluorine to iodine. This comprehensive study is used to determine the effects of polarization on the structure-energy relationship of biological X-bonds in solid state and solution phase. The culmination of the work in this thesis, in addition to previously published studies, provides a growing set of principles to guide knowledge-based application of halogens in drug design. These principles are applied to the selection of X-bond acceptors in a protein binding pocket, optimal placement of the halogen on the lead compound, and which halogen is best suited for a particular interaction

TOWARDS ELUCIDATION OF THE MECHANISM OF BIOLOGICAL NANOMOTORS

Biological functions such as cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging, and cell entry all involve biomotor-driven DNA translocation. In the past, the ubiquitous biological nanomotors were classified into two categories: linear and rotation motors. In 2013, we discovered a third type of biomotor, revolving motor without rotation. The revolving motion is further found to be widespread among many biological systems. In addition, the detailed sequential action mechanism of the ATPase ring in the phi29 dsDNA packaging motor has been elucidated: ATP binding induces a conformational entropy alternation of ATPase to a high affinity toward dsDNA; ATP hydrolysis triggers another conformational entropy change in ATPase to a low DNA affinity, by which the dsDNA substrate is pushed toward an adjacent ATPase subunit. The subunit communication is regulated by an arginine finger that extends from one ATPase subunit to the adjacent unit, resulting in an asymmetrical hexameric organization. Continuation of this process promotes the movement and revolving of the dsDNA within the hexameric ATPase ring. Coordination of all the motor components facilitate the motion direction control of the viral DNA packaging motors, and make it unusually powerful and effective

Re-evaluation of analytical chemistry techniques in studying DNA structures

This work describes the use of analytical chemistry techniques to examine the structural changes that DNA adopts when subjected to a number of external/internal factors. A self-complementary sequence, d(CG)9, and a non-self-complementary sequence (mixed sequence) were used to study the conformational effects displayed by each type of oligonucleotide sequence. The structural changes adopted by DNA was examined using a variety of analytical techniques, such as: nuclear magnetic resonance imaging (NMR), differential scanning calorimetry (DSC), ultra violet visible (UV-Vis) spectroscopy, circular dichroism (CD) spectroscopy, and high-performance liquid chromatography (HPLC). 1) d(CG)9 and a mixed sequence in the B- and Z-DNA conformation was examined by CD and UV-Vis at a concentration of 1mM using a home-made cuvette called a Flexicell with a minimum pathlength of 0.129± 0.015 mm. The CD and UV-Vis spectra’s produced were found to be reliable when compared to commercial cuvettes with a pathlength of 1 cm and sample concentration of 10 µM. 2) d(CG)9 was lyophilized and reconstituted using either water or buffer to determine if d(CG)9 adopts a different structure when reconstituted using different conditions. It was determined that lyophilized d(CG)9 adopts a hairpin conformation when reconstituted with water, and a B-DNA duplex when reconstituted with a buffer containing NaCl. 3) d(CG)9 was thermally denatured using DSC to determine if DSC can be a viable method to study oligonucleotides. It was determined that d(CG)9 undergoes a two-state unfolding pathway. 4) Nuclear Overhauser Effect spectroscopy (NOESY) and correlation spectroscopy (COSY) were used to examine the conformational differences of 2’-deoxyadenosine when incubated in water. From the distance and torsion angle constraints obtained from NOESY and COSY respectively, and from existing crystal structures, it was found the structures that were determined by NMR spectroscopy were misleading because of spectral artifacts. 5) A mixed sequence was treated with organic modifying agents to determine the minimal condition required for DNA denaturation when different modifiers were used. It was determined that urea at a concentration of 8 M and at a pH of 12.5 is sufficient to denature the mixed sequence duplex