Hidden Markov Modeling with Detailed Balance and Its Application to Single Protein Folding

Zhang, Yongli; Jiao, Junyi; Rebane, Aleksander A.

doi:10.1016/j.bpj.2016.09.045

Cited by 38 publications

(49 citation statements)

References 45 publications

(109 reference statements)

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“…We found that the t-SNARE complex folded and unfolded among states 2, 3, and 4 with distinct average extensions. We analyzed the extension trajectories using three-state hidden-Markov modeling (HMM) (8,26,27), which revealed idealized extension transitions ( Fig. 2A) and best-fit model parameters, including state probabilities and transition rates (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Our methods are described in detail elsewhere (9,26,27). Briefly, the extension trajectories were analyzed by two-or three-state HMM, which yielded the probability, extension, force, lifetime, and transition rates for each state (27). To relate the experimental measurements to the conformations and energy (or the energy landscape) of the t-SNARE complex at zero force, we constructed a structural model for t-SNARE folding (26).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

Zhang

Rebane

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 k B T, where k B is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission.t-SNARE complex | SNARE four-helix bundle | SNARE assembly | membrane fusion | optical tweezers S ynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate fast and calcium-triggered fusion of synaptic vesicles to presynaptic plasma membranes required for neurotransmission (1). They consist of VAMP2 (vesicle associated membrane protein 2, also called synaptobrevin 2) anchored on vesicles (v-SNARE) and syntaxin and SNAP-25 (synaptosome associated protein 25) located on target plasma membranes (t-SNAREs) (2). These SNAREs contain characteristic SNARE motifs of ∼60 amino acids (3) (Fig. 1A). Syntaxin and SNAP-25 can form a 1:1 t-SNARE complex (4-6). During membrane fusion, the t-and v-SNAREs join to form an extraordinarily stable four-helix bundle (3, 7-10). In the core of the bundle are 15 layers of hydrophobic amino acids and a central ionic layer containing three glutamines and one arginine. Whereas the zippering energy and kinetics between t-and v-SNAREs have recently been measured (8, 9), the structure, stability, and dynamics of the t-SNARE complex have not been well understood.The structure and dynamics of the t-SNARE complex are crucial for SNARE assembly and membrane fusion. Formation of the t-SNARE complex is likely an obligate intermediate before SNARE zippering (6,(11)(12)(13)(14). A preformed t-SNARE complex docks the vesicles to plasma membranes (15) and boosts the speed, strength, and accuracy of SNARE zippering (5, 9, 16). Furthermore, the t-SNARE complex is an important target for proteins that regulate SNAR...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

Zhang

Rebane

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Hidden-Markov modeling (HMM) and derivations of the energy and kinetics at zero force Methods and algorithms of HMM and energy and structural modeling are detailed elsewhere (Zhang et al, 2016b;Jiao et al, 2017;Rebane et al, 2016). The MATLAB codes used for these calculations can be found in Ref.…”

Section: Membrane Coating On Silica Beadsmentioning

confidence: 99%

“…To quantify the kinetics of C2 domain-membrane binding, we fit the extension trajectories using two-state hidden-Markov modeling (Zhang et al, 2016b) ( these observations are characteristic of two-state transitions (Bustamante et al, 2004;Rebane et al, 2016). We simultaneously fit unbinding probabilities, transition rates, and extension changes using a nonlinear model similar to the force-dependent protein folding transitions (Rebane et al, 2016), which included effects of the polypeptide linker and the DNA handles on the observed binding and unbinding transitions (see Materials and methods).…”

Section: Energetics and Kinetics Of C2 Domain-membrane Bindingmentioning

confidence: 99%

Single-molecule force spectroscopy of protein-membrane interactions

Cai

et al. 2017

Preprint

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Many biological processes rely on protein-membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach, single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2-7 pN and had binding energies of 4-14 k B T per C2 domain. Regulation by bilayer composition or Ca 2+ recapitulated known properties of both proteins. The method can be widely applied to study protein-membrane interactions.

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

Zhang

2017

Protein Science

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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. 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 association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise 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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Hidden Markov Modeling with Detailed Balance and Its Application to Single Protein Folding

Cited by 38 publications

References 45 publications

Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

Single-molecule force spectroscopy of protein-membrane interactions

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

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