Single-molecule imaging reveals the translocation and DNA looping dynamics of hepatitis C virus NS3 helicase

Chang-Ting Lin, Felix Tritschler, Kyung Suk Lee, Meigang Gu, Charles M. Rice, Taekjip Ha

Non-structural protein 3 (NS3) is an essential enzyme and a therapeutic target of hepatitis C virus (HCV). Compared to NS3-catalyzed nucleic acids unwinding, its translation on single stranded nucleic acids have received relatively little attention. To investigate the NS3h translocation with single-stranded nucleic acids substrates directly, we have applied a hybrid platform of single-molecule fluorescence detection combined with optical trapping. With the aid of mechanical manipulation and fluorescence localization, we probed the translocase activity of NS3h on laterally stretched, kilobase-size single-stranded DNA and RNA. We observed that the translocation rate of NS3h on ssDNA at a rate of 24.4 nucleotides per second, and NS3h translocates about three time faster on ssRNA, 74 nucleotides per second. The translocation speed was minimally affected by the applied force. A subpopulation of NS3h underwent a novel translocation mode on ssDNA where the stretched DNA shortened gradually and then recovers its original length abruptly before repeating the cycle repetitively. The speed of this mode of translocation was reduced with increasing force. With corroborating data from single-molecule fluorescence resonance energy transfer (smFRET) experiments, we proposed that NS3h can cause repetitive looping of DNA. The smFRET dwell time analysis showed similar translocation time between sole translocation mode versus repetitive looping mode, suggesting that the motor domain exhibits indistinguishable enzymatic activities between the two translocation modes. We propose a potential secondary nucleic acids binding site at NS3h which might function as an anchor point for translocation-coupled looping.

DOI

The ribosome destabilizes native and non-native structures in a nascent multidomain protein

Kaixian Liu, Joseph E. Rehfus, Elliot Mattson, Christian M. Kaiser

Correct folding is a prerequisite for the biological activity of most proteins. Folding has largely been studied using in vitro refolding assays with isolated small, robustly folding proteins. A substantial fraction of all cellular proteomes is composed of multidomain proteins that are often not amenable to this approach, and their folding remains poorly understood. These large proteins likely begin to fold during their synthesis by the ribosome, a large molecular machine that translates the genetic code. The ribosome affects how folding proceeds, but the underlying mechanisms remain largely obscure. We have utilized optical tweezers to study the folding of elongation factor G, a multidomain protein composed of five domains. We find that interactions among unfolded domains interfere with productive folding in the full-length protein. The N-terminal G-domain constitutes an independently folding unit that, upon in vitro refolding, adopts two similar states that correspond to the natively folded and a non-native, possibly misfolded structure. The ribosome destabilizes both of these states, suggesting a mechanism by which terminal misfolding into highly stable, non-native structures is avoided. The ribosome may thus directly contribute to efficient folding by modulating the folding of nascent multidomain proteins.

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The chaperone toolbox at the single-molecule level: From clamping to confining

Mario J. Avellaneda, Eline J. Koers, Mohsin M. Naqvi, Sander J. Tans

Protein folding is well known to be supervised by a dedicated class of proteins called chaperones. However, the core mode of action of these molecular machines has remained elusive due to several reasons including the promiscuous nature of the interactions between chaperones and their many clients, as well as the dynamics and heterogeneity of chaperone conformations and the folding process itself. While troublesome for traditional bulk techniques, these properties make an excellent case for the use of single-molecule approaches. In this review, we will discuss how force spectroscopy, fluorescence microscopy, FCS, and FRET methods are starting to zoom in on this intriguing and diverse molecular toolbox that is of direct importance for protein quality control in cells, as well as numerous degenerative conditions that depend on it.

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Nucleosome mobility and the regulation of gene expression: Insights from single-molecule studies

Sergei Rudnizky, Omri Malik, Adaiah Bavly, Lilach Pnueli, Philippa Melamed, Ariel Kaplan

Nucleosomes at the promoters of genes regulate the accessibility of the transcription machinery to DNA, and function as a basic layer in the complex regulation of gene expression. Our understanding of the role of the nucleosome's spontaneous, thermally driven position changes in modulating expression is lacking. This is the result of the paucity of experimental data on these dynamics, at high-resolution, and for DNA sequences that belong to real, transcribed genes. We have developed an assay that uses partial, reversible unzipping of nucleosomes with optical tweezers to repeatedly probe a nucleosome's position over time. Using the nucleosomes at the promoters of two model genes, Cga and Lhb, we show that the mobility of nucleosomes is modulated by the sequence of DNA and by the use of alternative histone variants, and describe how the mobility can affect transcription, at the initiation and elongation phases.

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Single-molecule mechanochemical characterization of E. coli pol III core catalytic activity

M. Nabuan Naufer, David A. Murison, Ioulia Rouzina, Penny J. Beuning, Mark C. Williams

Pol III core is the three-subunit subassembly of the E. coli replicative DNA polymerase III holoenzyme. It contains the catalytic polymerase subunit α, the 3′ → 5′ proofreading exonuclease ε, and a subunit of unknown function, θ. We employ optical tweezers to characterize pol III core activity on a single DNA substrate. We observe polymerization at applied template forces F < 25 pN and exonucleolysis at F > 30 pN. Both polymerization and exonucleolysis occur as a series of short bursts separated by pauses. For polymerization, the initiation rate after pausing is independent of force. In contrast, the exonucleolysis initiation rate depends strongly on force. The measured force and concentration dependence of exonucleolysis initiation fits well to a two-step reaction scheme in which pol III core binds bimolecularly to the primer-template junction, then converts at rate k2 into an exo-competent conformation. Fits to the force dependence of kinit show that exo initiation requires fluctuational opening of two base pairs, in agreement with temperature- and mismatch-dependent bulk biochemical assays. Taken together, our results support a model in which the pol and exo activities of pol III core are effectively independent, and in which recognition of the 3′ end of the primer by either α or ε is governed by the primer stability. Thus, binding to an unstable primer is the primary mechanism for mismatch recognition during proofreading, rather than an alternative model of duplex defect recognition.

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Single molecule force spectroscopy reveals the effect of BiP chaperone on protein folding

María Paz Ramírez, Maira Rivera, Diego Quiroga-Roger, Andrés Bustamante, Marcela Vega, Mauricio Baez, Elias M. Puchner, Christian A.M. Wilson

BiP (Immunoglobulin Binding Protein) is a member of the Hsp70 chaperones that participates in protein folding in the endoplasmic reticulum. The function of BiP relies on cycles of ATP hydrolysis driving the binding and release of its substrate proteins. It still remains unknown how BiP affects the protein folding pathway and there has been no direct demonstration showing which folding state of the substrate protein is bound by BiP, as previous work has used only peptides. Here, we employ optical tweezers for single molecule force spectroscopy experiments to investigate how BiP affects the folding mechanism of a complete protein and how this effect depends on nucleotides. Using the protein MJ0366 as the substrate for BiP, we performed pulling and relaxing cycles at constant velocity to unfold and refold the substrate. In the absence of BiP, MJ0366 unfolded and refolded in every cycle. However, when BiP was added, the frequency of folding events of MJ0366 significantly decreased, and the loss of folding always occurred after a successful unfolding event. This process was dependent on ATP and ADP, since when either ATP was decreased or ADP was added, the duration of periods without folding events increased. Our results show that the affinity of BiP for the substrate protein increased in these conditions, which correlates with previous studies in bulk. Therefore, we conclude that BiP binds to the unfolded state of MJ0366 and prevents its refolding, and that this effect is dependent on both the type and concentration of nucleotides.

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Force generation by titin folding

Zsolt Mártonfalvi, Pasquale Bianco, Katalin Naftz, György G. Ferenczy, Miklós Kellermayer

Titin is a giant protein that provides elasticity to muscle. As the sarcomere is stretched, titin extends hierarchically according to the mechanics of its segments. Whether titin's globular domains unfold during this process and how such unfolded domains might contribute to muscle contractility are strongly debated. To explore the force-dependent folding mechanisms, here we manipulated skeletal-muscle titin molecules with high-resolution optical tweezers. In force-clamp mode, after quenching the force (<10 pN), extension fluctuated without resolvable discrete events. In position-clamp experiments the time-dependent force trace contained rapid fluctuations and a gradual increase of average force, indicating that titin can develop force via dynamic transitions between its structural states en route to the native conformation. In 4 M urea, which destabilizes H-bonds hence the consolidated native domain structure, the net force increase disappeared but the fluctuations persisted. Thus, whereas net force generation is caused by the ensemble folding of the elastically-coupled domains, force fluctuations arise due to a dynamic equilibrium between unfolded and molten-globule states. Monte-Carlo simulations incorporating a compact molten-globule intermediate in the folding landscape recovered all features of our nanomechanics results. The ensemble molten-globule dynamics delivers significant added contractility that may assist sarcomere mechanics, and it may reduce the dissipative energy loss associated with titin unfolding/refolding during muscle contraction/relaxation cycles.

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Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers

Yongli Zhang

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers (OTs). We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain (NTD) association initiates SNARE assembly; a middle domain (MD) suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain (CTD) and the linker domain (LD) directly drive membrane fusion. In addition, the kinetics and pathway of the stage-wise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins.

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