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

Probing osmolyte participation in the unfolding transition state of a protein

1. Lorna Dougan, 
2. Georgi Z. Genchev, 
3. Hui Lu, and 
4. Julio M. Fernandez
Understanding the molecular mechanisms of osmolyte protection in protein stability has proved to be challenging. In particular, little is known about the role of osmolytes in the structure of the unfolding transition state of a protein, the main determinant of its dynamics. We have developed an experimental protocol to directly probe the transition state of a protein in a range of osmolyte environments. We use an atomic force microscope in force-clamp mode to apply mechanical forces to the protein I27 and obtain force-dependent rate constants of protein unfolding. We measure the distance to the unfolding transition state, Δxu, along a 1D reaction coordinate imposed by mechanical force. We find that for the small osmolytes, ethylene glycol, propylene glycol, and glycerol, Δxuscales with the size of the molecule, whereas for larger osmolytes, sorbitol and sucrose, Δxu remains the same as that measured in water. These results are in agreement with steered molecular dynamics simulations that show that small osmolytes act as solvent bridges in the unfolding transition state structure, whereas only water molecules act as solvent bridges in large osmolyte environments. These results demonstrate that novel force protocols combined with solvent substitution can directly probe angstrom changes in unfolding transition state structure. This approach creates new opportunities to gain molecular level understanding of the action of osmolytes in biomolecular processes.
1. DOI
1. Journal: 
Proceedings of the National Academy of Sciences

Latent TGF-b structure and activation

Minlong Shi, Jianghai Zhu, Rui Wang, Xing Chen, Lizhi Mi, Thomas Walz, and Timothy A. Springer


Transforming growth factor (TGF)-β is stored in the extracellular matrix as a latent complex with its prodomain. Activation of TGF-β1 requires the binding of αv integrin to an RGD sequence in the prodomain and exertion of force on this domain, which is held in the extracellular matrix by latent TGF-β binding proteins. Crystals of dimeric porcine proTGF-β1 reveal a ring-shaped complex, a novel fold for the prodomain, and show how the prodomain shields the growth factor from recognition by receptors and alters its conformation. Complex formation between αvβ6 integrin and the prodomain is insufficient for TGF-β1 release. Force-dependent activation requires unfastening of a ‘straitjacket’ that encircles each growth-factor monomer at a position that can be locked by a disulphide bond. Sequences of all 33 TGF-β family members indicate a similar prodomain fold. The structure provides insights into the regulation of a family of growth and differentiation factors of fundamental importance in morphogenesis and homeostasis.


DOI


Journal: Nature

The RSC chromatin remodelling ATPase translocates DNA with high force and small step size

George Sirinakis, Cedric R Clapier, Ying Gao, Ramya Viswanathan, Bradley R Cairns, and Yongli Zhang


ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to reposition and reconfigure nucleosomes. Despite their diverse functions, all remodellers share highly conserved ATPase domains, many shown to translocate DNA. Understanding remodelling requires biophysical knowledge of the DNA translocation process: how the ATPase moves DNA and generates force, and how translocation and force generation are coupled on nucleosomes. Here, we characterize the real-time activity of a minimal RSC translocase ‘motor’ on bare DNA, using high-resolution optical tweezers and a ‘tethered’ translocase system. We observe on dsDNA a processivity of ~35 bp, a speed of ~25 bp/s, and a step size of 2.0 (±0.4, s.e.m.) bp. Surprisingly, the motor is capable of moving against high force, up to 30 pN, making it one of the most force-resistant motors known. We also provide evidence for DNA ‘buckling’ at initiation. These observations reveal the ATPase as a powerful DNA translocating motor capable of disrupting DNA–histone interactions by mechanical force.


DOI


Journal: The EMBO Journal

Ultrahigh-resolution optical trap with single-fluorophore sensitivity

Matthew J Comstock, Taekjip Ha, and Yann R Chemla


We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore–labeled DNA oligonucleotides to bind and unbind complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (for example, single-base-pair stepping of DNA translocases) along with the detection of properties of fluorescently labeled protein (for example, internal configuration).


DOI


Journal: Nature Methods

Single Molecule Force Spectroscopy Reveals that Electrostatic Interactions Affect the Mechanical Stability of Proteins

Peng Zheng, Yi Cao, Tianjia Bu, Suzana K. Straus, and Hongbin Li


It is well known that electrostatic interactions play important roles in determining the thermodynamic stability of proteins. However, the investigation into the role of electrostatic interactions in mechanical unfolding of proteins has just begun. Here we used single molecule atomic force microscopy techniques to directly evaluate the effect of electrostatic interactions on the mechanical stability of a small protein GB1. We engineered a bi-histidine motif into the force-bearing region of GB1. By varying the pH, histidine residues can switch between protonated and deprotonated states, leading to the change of the electrostatic interactions between the two histidine residues. We found that the mechanical unfolding force of the engineered protein decreased by ∼34% (from 115 pN to 76 pN) on changing the pH from 8.5 to 3, due to the increased electrostatic repulsion between the two positively charged histidines at acidic pH. Our results demonstrated that electrostatic interactions can significantly affect the mechanical stability of elastomeric proteins, and modulating the electrostatic interactions of key charged residues can become a promising method for regulating the mechanical stability of elastomeric proteins.


DOI


Journal: Biophysical Journal

Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit

Ned Van Eps, Anita M. Preininger, Nathan Alexander, Ali I. Kaya, Scott Meier, Jens Meiler, Heidi E. Hammb, and Wayne L. Hubbell


In G-protein signaling, an activated receptor catalyzes GDP/GTP exchange on theGα subunit of a heterotrimeric G protein. In an initial step, receptor interaction with Gα acts to allosterically trigger GDP release from a binding site located between the nucleotide binding domain and a helical domain, but the molecular mechanism is unknown. In this study, site-directed spin labeling and double electron–electron resonance spectroscopy are employed to reveal a large-scale separation of the domains that provides a direct pathway for nucleotide escape. Cross-linking studies show that the domain separation is required for receptor enhancement of nucleotide exchange rates. The interdomain opening is coupled to receptor binding via the C-terminal helix of Gα, the extension of which is a high-affinity receptor binding element.


DOI


Journal: Proceedings of the National Academy of Sciences

Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

Derrick W. Meinhold and Peter E. Wrigh

1. Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use ¹⁵N,
2. ¹H
^N, and ¹³CO NMR R₂ relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R₂ dispersion for residues contacted by the F-helix region in the native (N) structure reveal a transient state formed by local unfolding of helix F and undocking from the protein core. A similar state was detected at pH 4.75–4.95 and determined to be an on-pathway intermediate (I1) in a linear three-state unfolding scheme (N⇆I1⇆MG) leading to a transiently populated molten globule (MG) state. The slowest steps in unfolding and refolding are N → I1 (36 s^-1) and MG → I1 (26 s^-1), respectively. Differences in chemical shift between N and I1 are very small, except in regions adjacent to helix F, showing that their core structures are similar. Chemical shift changes between the N and MG states, obtained from R₂dispersion, reveal that the transient MG state is structurally similar to the equilibrium MG observed previously at high temperature and low pH. Analysis of MG state chemical shifts shows the location of residual helical structure in the transient intermediate and identifies regions that unfold or rearrange into nonnative structure during the N → MG transition. The experiments also identify regions of energetic frustration that “crack” during unfolding and impede the refolding process.

DOI

Journal: Proceedings of the National Academy of Sciences

Probing cellular protein complexes using single-molecule pull-down

Ankur Jain, Ruijie Liu, Biswarathan Ramani, Edwin Arauz, Yuji Ishitsuka, Kaushik Ragunathan, Jeehae Park, Jie Chen, Yang K. Xiang, and Taekjip H


Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.


DOI


Journal: Nature

Collective cell guidance by cooperative intercellular forces

Dhananjay T. Tambe, C. Corey Hardin, Thomas E. Angelini, Kavitha Rajendran, Chan Young Park, Xavier Serra-Picamal, Enhua H. Zhou, Muhammad H. Zaman, James P. Butler, David A. Weitz, Jeffrey J. Fredberg, and Xavier Trepat


Cells comprising a tissue migrate as part of a collective. How collective processes are coordinated over large multi-cellular assemblies has remained unclear, however, because mechanical stresses exerted at cell–cell junctions have not been accessible experimentally. We report here maps of these stresses within and between cells comprising a monolayer. Within the cell sheet there arise unanticipated fluctuations of mechanical stress that are severe, emerge spontaneously, and ripple across the monolayer. Within that stress landscape, local cellular migrations follow local orientations of maximal principal stress. Migrations of both endothelial and epithelial monolayers conform to this behaviour, as do breast cancer cell lines before but not after the epithelial–mesenchymal transition. Collective migration in these diverse systems is seen to be governed by a simple but unifying physiological principle: neighbouring cells join forces to transmit appreciable normal stress across the cell–cell junction, but migrate along orientations of minimal intercellular shear stress.


DOI


Journal: Nature Materials

Conformational capture of the SAM-II riboswitch

Andrea Haller, Ulrike Rieder, Michaela Aigner, Scott C Blanchard, and Ronald Micura


Riboswitches are gene regulation elements in mRNA that function by specifically responding to metabolites. Although the metabolite-bound states of riboswitches have proven amenable to structure determination efforts, knowledge of the structural features of riboswitches in their ligand-free forms and their ligand-response mechanisms giving rise to regulatory control is lacking. Here we explore the ligand-induced folding process of the S-adenosylmethionine type II (SAM-II) riboswitch using chemical and biophysical methods, including NMR and fluorescence spectroscopy, and single-molecule fluorescence imaging. The data reveal that the unliganded SAM-II riboswitch is dynamic in nature, in that its stem-loop element becomes engaged in a pseudoknot fold through base-pairing with nucleosides in the 3′ overhang containing the Shine-Dalgarno sequence. Although the pseudoknot structure is highly transient in the absence of its ligand, S-adenosylmethionine (SAM), it becomes conformationally restrained upon ligand recognition, through a conformational capture mechanism. These insights provide a molecular understanding of riboswitch dynamics that shed new light on the mechanism of riboswitch-mediated translational regulation.


DOI


Journal: Nature Chemical Biology

Single-molecule paleoenzymology probes the chemistry of resurrected enzymes

Raul Perez-Jimenez, Alvaro Inglés-Prieto, Zi-Ming Zhao, Inmaculada Sanchez-Romero, Jorge Alegre-Cebollada, Pallav Kosuri, Sergi Garcia-Manyes, T Joseph Kappock, Masaru Tanokura, Arne Holmgren, Jose M Sanchez-Ruiz, Eric A Gaucher, and Julio M Fernandez


It is possible to travel back in time at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx) dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32 °C more stable than modern enzymes, and the oldest show markedly higher activity than extant ones at pH 5. We probed the mechanisms of reduction of these enzymes using single-molecule force spectroscopy. From the force dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs use chemical mechanisms of reduction similar to those of modern enzymes. Although Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over 4 Gyr to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth.


DOI


Journal: Nature Structural and Molecular Biology

Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins

Madeleine B. Borgia, Alessandro Borgia,  Robert B. Best, Annette Steward, Daniel Nettels, Bengt Wunderlich, Benjamin Schuler, and Jane Clarke


A large range of debilitating medical conditions is linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains. Seventy-five per cent of the eukaryotic proteome consists of multidomain proteins, yet it is not understood how interdomain misfolding is avoided. It has been proposed that maintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding. Here we use single-molecule Förster resonance energy transfer to detect and quantify rare misfolding events in tandem immunoglobulin domains from the I band of titin under native conditions. About 5.5 per cent of molecules with identical domains misfold during refolding in vitro and form an unexpectedly stable state with an unfolding half-time of several days. Tandem arrays of immunoglobulin-like domains in humans show significantly lower sequence identity between neighbouring domains than between non-adjacent domains. In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40 per cent. We observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24 per cent), whereas misfolding occurs between domains that are 42 per cent identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures that are in excellent agreement with the observed transfer efficiency of the misfolded species. We infer that the interactions underlying misfolding are very specific and result in a sequence-specific domain-swapping mechanism. Diversifying the sequence between neighbouring domains seems to be a successful evolutionary strategy to avoid misfolding in multidomain proteins.


DOI


Journal: Nature