Zhiqun Xi

Single Reconstituted Neuronal SNARE Complexes Zipper in Three Distinct Stages

Dissecting SNARE Zippering The SNARE complex is critical for vesicle fusion, notably during release of neurotransmitters at synapses. Understanding the biophysics of SNARE assembly has been the object of several structural studies, and yet much remains to be understood about the mechanisms. Now, Gao et al. (p. 1340, published online 16 August; see the Perspective by Rizo) describe the results of cell-free experiments using optical tweezers to elucidate assembly and disassembly of the SNARE complex. Direct observations of SNARE intermediates revealed multiple steps of the assembly process, along with the associated energetics and kinetics. Applying forces similar to those occurring during fusion, an intermediate was stabilized, and the derived mechanism indicates how neurotransmitter release may be regulated. Zippering of a single SNARE complex generates high force and energy that can potentially drive synaptic membrane fusion. Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins drive membrane fusion by assembling into a four-helix bundle in a zippering process. Here, we used optical tweezers to observe in a cell-free reconstitution experiment in real time a long-sought SNARE assembly intermediate in which only the membrane-distal amino-terminal half of the bundle is assembled. Our findings support the zippering hypothesis, but suggest that zippering proceeds through three sequential binary switches, not continuously, in the amino- and carboxyl-terminal halves of the bundle and the linker domain. The half-zippered intermediate was stabilized by externally applied force that mimicked the repulsion between apposed membranes being forced to fuse. This intermediate then rapidly and forcefully zippered, delivering free energy of 36 kBT (where kB is Boltzmann’s constant and T is temperature) to mediate fusion.

Single-molecule observation of helix staggering, sliding, and coiled coil misfolding

The biological functions of coiled coils generally depend on efficient folding and perfect pairing of their α-helices. Dynamic changes in the helical registry that lead to staggered helices have only been proposed for a few special systems and not found in generic coiled coils. Here, we report our observations of multiple staggered helical structures of two canonical coiled coils. The partially folded structures are formed predominantly by coiled coil misfolding and occasionally by helix sliding. Using high-resolution optical tweezers, we characterized their energies and transition kinetics at a single-molecule level. The staggered states occur less than 2% of the time and about 0.1% of the time at zero force. We conclude that dynamic changes in helical registry may be a general property of coiled coils. Our findings should have broad and unique implications in functions and dysfunctions of proteins containing coiled coils.

Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy

Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level.

Munc18-1-regulated stage-wise SNARE assembly underlying synaptic exocytosis

Synaptic-soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins couple their stage-wise folding/assembly to rapid exocytosis of neurotransmitters in a Munc18-1-dependent manner. The functions of the different assembly stages in exocytosis and the role of Munc18-1 in SNARE assembly are not well understood. Using optical tweezers, we observed four distinct stages of assembly in SNARE N-terminal, middle, C-terminal, and linker domains (or NTD, MD, CTD, and LD, respectively). We found that SNARE layer mutations differentially affect SNARE assembly. Comparison of their effects on SNARE assembly and on exocytosis reveals that NTD and CTD are responsible for vesicle docking and fusion, respectively, whereas MD regulates SNARE assembly and fusion. Munc18-1 initiates SNARE assembly and structures t-SNARE C-terminus independent of syntaxin N-terminal regulatory domain (NRD) and stabilizes the half-zippered SNARE complex dependent upon the NRD. Our observations demonstrate distinct functions of SNARE domains whose assembly is intimately chaperoned by Munc18-1. DOI: http://dx.doi.org/10.7554/eLife.09580.001

α-SNAP Enhances SNARE Zippering by Stabilizing the SNARE Four-Helix Bundle

Intracellular membrane fusion is mediated by dynamic assembly and disassembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs). α-SNAP guides NSF to disassemble SNARE complexes after membrane fusion. Recent experiments showed that α-SNAP also dramatically enhances SNARE assembly and membrane fusion. How α-SNAP is involved in these opposing activities is not known. Here, we examine the effect of α-SNAP on the stepwise assembly of the synaptic SNARE complex using optical tweezers. We found that α-SNAP destabilized the linker domain (LD) of the SNARE complex but stabilized its C-terminal domain (CTD) through a conformational selection mechanism. In contrast, α-SNAP minimally affected assembly of the SNARE N-terminal domain (NTD), indicating that α-SNAP barely bound the partially assembled trans-SNARE complex. Thus, α-SNAP recognizes the folded CTD for SNARE disassembly with NSF and subtly modulates membrane fusion by altering the stabilities of the SNARE CTD and LD.

DNA Translocation of ATP-Dependent Chromatin Remodeling Factors Revealed by High-Resolution Optical Tweezers

ATP-dependent chromatin remodeling complexes (remodelers) use the energy of ATP hydrolysis to regulate chromatin structures by repositioning and reconfiguring nucleosomes. Ensemble experiments have suggested that remodeler ATPases are DNA translocases, molecular motors capable of processively moving along DNA. This concept of DNA translocation has become a foundation for understanding the molecular mechanisms of ATP-dependent chromatin remodeling and its biological functions. However, quantitative characterizations of DNA translocation by representative remodelers are rare. Furthermore, it is unclear how a unified theory of chromatin remodeling is built upon this foundation. To address these problems, high-resolution optical tweezers have been applied to investigate remodeler translocation on bare DNA and nucleosomal DNA substrates at a single-molecule level. Our strategy is to hold two ends of a single DNA molecule and measure remodeler translocation by detecting the end-to-end extension and tension changes of the DNA molecule in response to chromatin remodeling. These single-molecule assays can reveal detailed kinetics of remodeler translocation, including velocity, processivity, stall force, pauses, direction changes, and even step size. Here we describe instruments, reagents, sample preparations, and detailed protocols for the single-molecule experiments. We show that optical tweezer force microscopy is a powerful and friendly tool for studies of chromatin structures and remodeling.

Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA

We have investigated the anomalously weak binding of human papillomavirus (HPV) regulatory protein E2 to a DNA target containing the spacer sequence TATA. Experiments in magnesium (Mg2+) and calcium (Ca2+) ion buffers revealed a marked reduction in cutting by DNase I at the CpG sequence in the protein-binding site 3′ to the TATA spacer sequence, Studies of the cation dependence of DNA-E2 affinities showed that upon E2 binding the TATA sequence releases approximately twice as many Mg2+ ions as the average of the other spacer sequences. Binding experiments for TATA spacer relative to ATAT showed that in potassium ion (K+) the E2 affinity of the two sequences is nearly equal, but the relative dissociation constant (Kd) for TATA increases in the order K+ < Na+ < Ca2+ < Mg2+. Except for Mg2+, Kd for TATA relative to ATAT is independent of ion concentration, whereas for Mg2+ the affinity for TATA drops sharply as ion concentration increases. Thus, ions of increasing positive charge density increasingly distort the E2 binding site, weakening the affinity for protein. In the case of Mg2+, additional ions are bound to TATA that require displacement for protein binding. We suggest that the TATA sequence may bias the DNA structure towards a conformation that binds the protein relatively weakly.