Molecular Origins of DNA Flexibility: Sequence Effects on Conformational and Mechanical Properties

Vanessa Ortiz and Juan J. de Pablo


A central question in biophysics is whether DNA sequence affects its mechanical properties, which are thought to influence nucleosome positioning and gene expression. Previous attempts to answer this question have been hindered by an inability to resolve DNA structure and dynamics at the base-pair level. Here we use a model to measure the effects of sequence on the stability of DNA under bending. Sequence is shown to influence DNA’s flexibility and its ability to form kinks, which arise when certain motifs slide past others to form non-native contacts. A mechanism for nucleosome positioning is proposed in which sequence influences DNA-histone binding by altering the local base-pair-level structure when subject to the curvature necessary for binding.


DOI


Journal: Physical Review Letters

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins

1. Fabien Pinaud and 
2. Maxime Dahan

1. Single-molecule (SM) microscopy allows outstanding insight into biomolecular mechanisms in cells. However, selective detection of single biomolecules in their native environment remains particularly challenging. Here, we introduce an easy methodology that combines specific targeting and nanometer accuracy imaging of individual biomolecules in living cells. In this method, named complementation-activated light microscopy (CALM), proteins are fused to dark split-fluorescent proteins (split-FPs), which are activated into bright FPs by complementation with synthetic peptides. Using CALM, the diffusion dynamics of a controlled subset of extracellular and intracellular proteins are imaged with nanometer precision, and SM tracking can additionally be performed with fluorophores and quantum dots. In cells, site-specific labeling of these probes is verified by coincidence SM detection with the complemented split-FP fusion proteins or intramolecular single-pair Förster resonance energy transfer. CALM is simple and combines advantages from genetically encoded and synthetic fluorescent probes to allow high-accuracy imaging of single biomolecules in living cells, independently of their expression level and at very high probe concentrations.

1. DOI

1. Journal: Proceedings of the National Academy of Sciences

Minimum energy compact structures in force-quench polyubiquitin folding are domain swapped

Fei Xia, D. Thirumalai, and Frauke Gräter

Single molecule experiments that initiate folding using mechanical force are uniquely suited to reveal the nature of populated states in the folding process. Using a strategy proposed on theoretical grounds, which calls for repeated cycling of force from high to low values using force pulses, it was demonstrated in atomic force spectroscopy (AFM) experiments that an ensemble of minimum energy compact structures (MECS) are sampled during the folding of polyubiquitin. The structures in the ensemble are mechanically resistant to a lesser extent than the native state. Remarkably, forced unfolding of the populated intermediates reveals a broad distribution of extensions including steps up to 30 nm and beyond. We show using molecular simulations that favorable interdomain interactions leading to domain swapping between adjacent ubiquitin modules results in the formation of the ensemble of MECS, whose unfolding leads to an unusually broad distribution of steps. We obtained the domain-swapped structures using coarse-grained ubiquitin dimer models by exchanging native interactions between two monomeric ubiquitin molecules. Brownian dynamics force unfolding of the proposed domain-swapped structures, with mechanical stability that is approximately 100-fold lower than the native state, gives rise to a distribution of extensions from 2 to 30 nm. Our results, which are in quantitative agreement with AFM experiments, suggest that domain swapping may be a general mechanism in the assembly of multi-sub-unit proteins.

DOI

Journal: Proceedings of the National Academy of Sciences

Supramolecular fishing for plasma membrane proteins using an ultrastable synthetic host–guest binding pair

Don-Wook Lee, Kyeng Min Park, Mainak Banerjee, Sang Hoon Ha, Taehoon Lee, Kyungwon Suh, Somak Paul, Hyuntae Jung, Jaeyoon Kim, Narayanan Selvapalam, Sung Ho Ryu and Kimoon Kim


Membrane proteomics, the large-scale global analysis of membrane proteins, is often constrained by the efficiency of separating and extracting membrane proteins. Recent approaches involve conjugating membrane proteins with the small molecule biotin and using the receptor streptavidin to extract the labelled proteins. Despite the many advantages of this method, several shortcomings remain, including potential contamination by endogenously biotinylated molecules and interference by streptavidin during analytical stages. Here, we report a supramolecular fishing method for membrane proteins using the synthetic receptor–ligand pair cucurbit[7]uril–1-trimethylammoniomethylferrocene (CB[7]–AFc). CB[7]-conjugated beads selectively capture AFc-labelled proteins from heterogeneous protein mixtures, and AFc-labelling of cells results in the efficient capture of membrane proteins by these beads. The captured proteins can be recovered easily at room temperature by treatment with a strong competitor such as 1,1′-bis(trimethylammoniomethyl)ferrocene. This synthetic but biocompatible host–guest system may be a useful alternative to streptavidin–biotin for membrane proteomics as well as other biological and biotechnological applications.


DOI


Journal: Nature Chemistry

Human cryptochrome exhibits light-dependent magnetosensitivity

Lauren E. Foley, Robert J. Gegear and Steven M. Reppert




Humans are not believed to have a magnetic sense, even though many animals use the Earth's magnetic field for orientation and navigation. One model of magnetosensing in animals proposes that geomagnetic fields are perceived by light-sensitive chemical reactions involving the flavoprotein cryptochrome (CRY). Here we show using a transgenic approach that human CRY2, which is heavily expressed in the retina, can function as a magnetosensor in the magnetoreception system of Drosophila and that it does so in a light-dependent manner. The results show that human CRY2 has the molecular capability to function as a light-sensitive magnetosensor and reopen an area of sensory biology that is ready for further exploration in humans.

DOI

Journal: Nature Communications

Collapse of DNA in AC Electric Fields

Chunda Zhou, Walter W. Reisner, Rory J. Staunton, Amir Ashan, Robert H. Austin, and Robert Riehn


We report that double-stranded DNA collapses in the presence of ac electric fields at frequencies of a few hundred Hertz, and does not stretch as commonly assumed. In particular, we show that confinement-stretched DNA can collapse to about one quarter of its equilibrium length. We propose that this effect is based on finite relaxation times of the counterion cloud, and the subsequent partitioning of the molecule into mutually attractive units. We discuss alternative models of those attractive units.


DOI


Journal: Physical Review Letters

Protein unfolding accounts for the unusual mechanical behavior of fibrin networks

Prashant K. Purohit, Rustem I. Litvinov, Andre E.X. Brown, Dennis E. Discher,and John W. Weisel


We describe the mechanical behavior of isotropic fibrin networks at the macroscopic scale in terms of the nanoscale force response of fibrin molecules that are its basic building blocks. We show that the remarkable extensibility and compressibility of fibrin networks have their origins in the unfolding of fibrin molecules. The force–stretch behavior of a single fibrin fiber is described using a two-state model in which the fiber has a linear force–stretch relation in the folded phase and behaves like a worm-like-chain in the unfolded phase. The nanoscale force–stretch response is connected to the macro-scale stress–stretch response by means of the eight-chain model. This model is able to capture the macroscopic response of a fibrin network in uniaxial tension and appears remarkably simple given the molecular complexity. We use the eight-chain model to explain why fibrin networks have negative compressibility and Poisson’s ratio greater than 1 due to unfolding of fibrin molecules.

DOI

Journal: Acta Biomaterialia