Unfolding and melting of DNA (RNA) hairpins: the concept of structure-specific 2D dynamic landscapes

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In many physical and biological systems the transition from an amorphous to ordered native structure involves complex energy landscapes, and understanding such transformations requires not only their thermodynamics but also the structural dynamics during the process. Here, we extend our 4D visualization method with electron imaging to include the study of irreversible processes with a single pulse in the same ultrafast electron microscope (UEM) as used before in the single-electron mode for the study of reversible processes. With this augmentation, we report on the transformation of amorphous to crystalline structure with silicon as an example. A single heating pulse was used to initiate crystallization from the amorphous phase while a single packet of electrons imaged selectively in space the transformation as the structure continuously changes with time. From the evolution of crystallinity in real time and the changes in morphology, for nanosecond and femtosecond pulse heating, we describe two types of processes, one that occurs at early time and involves a nondiffusive motion and another that takes place on a longer time scale. Similar mechanisms of two distinct time scales may perhaps be important in biomolecular folding.diffraction ͉ imaging ͉ structural dynamics ͉ ultrafast electron microscopy T he transformation of amorphous structures, such as liquids or random-coiled proteins, into ordered structures involves complex dynamical processes that ultimately lead to the final native state. The mechanism is determined by the scales of time, length, and energy as they define the nature of the elementary steps involved. For example, an amorphous bulk liquid crystallizes depending on the degree of initial (nanoscale) nucleation, the time scale of heat diffusion, and the latent energy acquired. Similarly, for a protein, the funneling toward the native structure requires the balance of the entropic and enthalpic free energy contributions, as well as the ''diffusion'' through many energy barriers, possibly with nucleation on the path to the final state.To observe such processes on the time-length scale of the phenomena, our method of choice has been 4D space-time visualization (ref. 1 and references therein, and ref. 2) developed in ultrafast electron microscopy (UEM) and diffraction for imaging with a wide scope (3) of applications (1-10). For microscopy, the concept of femtosecond single (or few) electron packets was introduced to allow for the study of structural dynamics in the temporal, space-charge-free regime and to obtain the atomic-scale spatial resolution. In this mode of UEM operation, a train of electron packets coherently ''builds up'' and coheres into the final image. However, for nonequilibrium irreversible processes, such as crystallization, the transformation must be visualized through single-pulse imaging.In this contribution, we augment the UEM apparatus to include this single-pulse capability, thus covering domains of femtoseconds (fs) to seconds. Using this mode of imaging, here reported is the ...

Section: Resultsmentioning

confidence: 99%

4D visualization of embryonic, structural crystallization by single-pulse microscopy

Kwon

Barwick

Park

et al. 2008

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“…1). Being an important postulate of the equilibrium and "kinetic zipper" models (5,18,19) most commonly used to describe macromolecular transformations, the SSA is also an approximation used in our KIS model of helix-coil transitions in biopolymers such as DNA (20)(21)(22) and α-helical polypeptides. Significantly, this makes all of the relevant (partially folded) states of the macromolecule describable by just two variables (i and j; see below).…”

Section: Significancementioning

confidence: 99%

Dominance of misfolded intermediates in the dynamics of α-helix folding

Lin

Shorokhov

Zewail

2014

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Helices are the "hydrogen atoms" of biomolecular complexity; the DNA/RNA double hairpin and protein α-helix ubiquitously form the building blocks of life's constituents at the nanometer scale. Nevertheless, the formation processes of these structures, especially the dynamical pathways and rates, remain challenging to predict and control. Here, we present a general analytical method for constructing dynamical free-energy landscapes of helices. Such landscapes contain information about the thermodynamic stabilities of the possible macromolecular conformations, as well as about the dynamic connectivity, thus enabling the visualization and computation of folding pathways and timescales. We elucidate the methodology using the folding of polyalanine, and demonstrate that its α-helix folding kinetics is dominated by misfolded intermediates. At the physiological temperature of T = 298 K and midfolding time t = 250 ns, the fraction of structures in the native-state (α-helical) basin equals 22%, which is in good agreement with time-resolved experiments and massively distributed, ensemble-convergent molecular-dynamics simulations. We discuss the prominent role of β-strandlike intermediates in flight toward the native fold, and in relation to the primary conformational change precipitating aggregation in some neurodegenerative diseases.protein folding | misfolding intermediates I f there is one signature characteristic of biological macromolecules, it is the presence of helices. The double helix is the dominant feature of DNA and RNA, and, in proteins, the α-helix is the most ubiquitous structural motif. The formation and decay of α-helices play a pivotal role in protein folding (and misfolding). Indeed, even for proteins containing little native helical structure, helix formation, concomitant with hydrophobic collapse, seems to be a universal precursor of the folding process (1), much as the nonnative conversion of helical (or disordered) to β-strand-rich structures is the precursor to protein aggregation diseases such as Alzheimer's (2). Although the thermodynamics of such transformations was well understood beginning with the 1950s, the dynamical pathways and rates involved have remained an area of active research. Thus, relatively little was known about the associated kinetics until the end of the 1990s (3). With the advent of temperature-jump (T-jump) experimental techniques, the rates of the helix−coil transitions became readily measurable (4-6), whereas the progress in temporal resolution spanning both the nanosecond (7) and picosecond (8) regimes elucidated the early steps of folding/unfolding. Importantly, studies of the elementary steps accompanying such processes can now uncover the nature of kinetic-intermediate states, as well as their characteristic lifetimes (8, 9).Here, we introduce a statistical mechanical method of constructing dynamical free-energy landscapes of helices. The dynamical landscape allows folding pathways and rates to be visualized and computed in terms of elementary structural steps. For a r...

“…By contrast with proteins, relatively few studies have probed the energy landscapes of nucleic acids, particularly via calorimetric techniques (27)(28)(29)(30)(31)(32)(33)(34)(35). As a result, the potential energetic/ biological role(s), if any, of kinetically stable (metastable) microstates that make up the time-averaged, native state ensembles of macroscopic nucleic acid states remains to be determined.…”

mentioning

confidence: 99%

DNA energy landscapes via calorimetric detection of microstate ensembles of metastable macrostates and triplet repeat diseases

Völker

Klump

Breslauer

2008

Biopolymers exhibit rough energy landscapes, thereby allowing biological processes to access a broad range of kinetic and thermodynamic states. In contrast to proteins, the energy landscapes of nucleic acids have been the subject of relatively few experimental investigations. In this study, we use calorimetric and spectroscopic observables to detect, resolve, and selectively enrich energetically discrete ensembles of microstates within metastable DNA structures. Our results are consistent with metastable, ''native'' DNA states being composed of an ensemble of discrete and kinetically stable microstates of differential stabilities, rather than exclusively being a single, discrete thermodynamic species. This conceptual construct is important for understanding the linkage between biopolymer conformational/configurational space and biological function, such as in protein folding, allosteric control of enzyme activity, RNA and DNA folding and function, DNA structure and biological regulation, etc. For the specific DNA sequences and structures studied here, the demonstration of discrete, kinetically stable microstates potentially has biological consequences for understanding the development and onset of DNA expansion and triplet repeat diseases.calorimetry ͉ nucleic acid conformations T he concept of rough biopolymer energy landscapes is important for understanding biopolymer properties and function. Complex landscapes allow partial population of discrete, kinetically stable (metastable) species (1-12). These substates, in turn, may serve as biologically significant ligands, beyond the traditional, thermodynamically most stable, time-averaged, ''native'' macrostrate. Although it is well recognized that macroscopic states are composed of microscopic substates, it has been customary for experimentalists to think of a biologically relevant native state as a singular macrostate with a unique, well defined structure characterized by a homogeneous/smooth energy profile. Many biophysical measurements probe properties that are averaged over the ensemble of microstates that collectively correspond to a macroscopic native state. However, experimental evidence for the presence of, and dynamic interchange between, multiple states that constitute a global macrostate is provided by direct time-resolved measurements, and by structural dispersions in crystallographic and NMR studies (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Such results underscore the potential importance as ligands or receptors in biological mechanisms of kinetically stable, trapped microstates embedded within the rough energy landscapes of purportedly singular macrostates. For example, the detection of transiently populated structural states in the native unbound protein ensemble that resemble those of the proteins in their bound state provides evidence for the so-called ''conformational selection model'' of protein binding, thereby suggesting significant biological functions for microstates in proteins (23)(24)(25)(26).By contrast with proteins, relatively few stu...