Research & Publications
Forces hold everything together and determine the structures and dynamics of macromolecules. We have broad interests and fine skills in measuring the intra- and inter-molecular forces and the forces generated by molecular machines as a crucial step to understand their biological functions. Our primary tool is combined optical trapping and single-molecule fluorescence spectroscopy, which allows us to simultaneously manipulate and visualize single molecules in real time. As a result, dynamic structures of proteins inaccessible by other experimental methods are obtained. Our primary interest is folding dynamics of soluble and membrane proteins involved in fundamental biological processes and human diseases. In particular, we focus on SNARE proteins and their regulators essential for intracellular vesicular fusion and regulated exocytosis associated with release of neurotransmitters and insulin.
We have developed a unique single-molecule manipulation approach to characterize the folding intermediates, energy and kinetics of various SNARE complexes. We have helped to establish that SNARE proteins are truly molecular engines for membrane fusion and gained important insights into their regulatory mechanisms.
Specialized Terms: Single-molecule biophysics and biochemistry; Optical tweezers; SNAREs; SNARE assembly; Munc18-1; Sec1/Munc18 (SM) proteins; Munc13-1; Synaptotagmins; Extended synaptotagmins; Complexin; Membrane fusion; Neurotransmitter release; Lipid exchange; Protein folding
Extensive Research Description
We are focused on understanding the molecular mechanisms that underlie three important biological processes:
1. Regulated SNARE folding and assembly.
The membrane fusion machinery contains SNARE proteins, Sec1/Munc18 (SM) proteins, synaptotagmin, complexin, NSF, SNAP, and Munc13. Among these proteins, SNAREs are key players. They couple their dynamical assembly and disassembly to membrane fusion in a precisely controlled manner. Specifically, SNARE assembly generates force to draw two membranes into proximity and use their folding energy to lower the energy barrier of membrane fusion. SM proteins, synaptotagmin, and complexin regulate SNARE assembly and enable membrane fusion to occur at right time and location. After membrane fusion, NSF and SNAP disassemble the fully assembled SNARE complexes in an ATP-dependent manner, recycling SNAREs for next round of fusion. We plan to understand how the above mentioned proteins work together to control exocytosis and how malfunctions of the fusion machinery cause diseases.
Sudhof, T.C., and Rothman, J.E. (2009). Membrane fusion: Grappling with SNARE and SM proteins. Science 323, 474-477.
Y. Gao, S. Zorman, G. Gundersen, Z. Q. Xi, G. Sirinakis, J. E. Rothman*, Y. L. Zhang*, Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science 337: 1340-1343 (2012).
L. Ma, A. A. Rebane, G. Yang, Z. Xi, Y. Kang, Y. Gao, Y. L. Zhang, Munc18-1-regulated stage-wise SNARE assembly underlying synaptic exocytosis. eLIFE 4, e09580 (2015).
S. Zorman, A. A. Rebane, L. Ma, G. Yang, M. A. Molski, J. Coleman, F. Pincet, J. E. Rothman*, Y. L. Zhang*, Common intermediates and kinetics, but different energetics, in the assembly of SNARE proteins. eLIFE 3, e03348 (2014).
Y. L. Zhang, Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers, Protein Sci. 26, 1252-1265 (2017).
A. A. Rebane, B. Wang, L. Ma, H. Qu, J. Coleman, S. S. Krishnakumar, J. E. Rothman*, Y. L. Zhang*, Two disease-causing SNAP-25B mutations selectively impair SNARE C-terminal assembly. J. Mol. Biol. 430, 479 (2018).
J. Jiao, M. He, S. A. Port, R. W. Baker, Y. Xu, H. Qu, Y. Xiong, Y. Wang, H. Jin, T. J. Eisemann, F. M. Hughson*, Y. L. Zhang*, Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association. Elife 7, e41771 (2018).
* Co-corresponding authors.
2. Membrane protein folding, stability, and protein-membrane interactions.
Optical tweezers have been widely applied to study folding dynamics of soluble proteins, but not membrane proteins so far. We have been developing novel approaches to measure the folding energy and kinetics of membrane proteins using high-resolution optical tweezers. We are also interested in proteins that help membrane proteins get in and out membranes.
L. Ma, Y. Cai, Y. Li, J. Jiao, Z. Wu, B. O'Shaughnessy, P. De Camilli*, E. Karatekin*, Y. L. Zhang*, Single-molecule force spectroscopy of protein-membrane interactions. Elife 6, e30493 (2017)
3. Development of new single-molecule methods.
We have been developing new instruments or upgrading our machines by combining high-resolution optical tweezers, single-molecule fluorescence detection, and microfluidics to better study single proteins or protein complexes. We have been also developing new methods or algorithms to analyze data from single molecule experiments.
G. Sirinakis, Y. X. Ren, Y. Gao, Z. Q. Xi, Y. L. Zhang, Combined and versatile high-resolution optical tweezers and single-molecule fluorescence microscopy. Rev Sci Instrum. 83: 093708-(1-9) (2012).
Y. L. Zhang, J. Jiao, A. A. Rebane, Hidden Markov modeling with detailed balance and its application to single protein folding Biophys J 111, 2110 (2016).
A. A. Rebane, L. Ma, Y. L. Zhang, Structure-based derivation of protein folding intermediates and energies from optical tweezers. Biophys J 110, 441 (2016).
4. Molecular mechanism of the mechanosensitive ion channel NOMPC.
NOMPC is involved in mechanosensation of touch and hearing in flies. Unlike many mechanosensitive ion channels that sense membrane tension or force in the membrane, NOMPC has been proposed to sense force out of the membrane through a gating spring. In collaboration with groups of Yifan Cheng and Yuh-Nung Jan in UCSF, we have been investigating whether and how force modulates the ion conductance of NOMPC, using optical tweezers and fluorescence imaging.
- P. Jin, D. Bulkley, Y. M. Guo, W. Zhang, Z. H. Guo, W. Huynh, S. P. Wu, S. Meltzer, T. Cheng, L. Y. Jan, Y. N. Jan, Y. F. Cheng, Electron cryo-microscopy structure of the mechanotransduction channel NOMPC. Nature 547, 118 (2017).
- W. Zhang, L. E. Cheng, M. Kittelmann, J. F. Li, M. Petkovic, T. Cheng, P. Jin, Z. H. Guo, M. C. Gopfert, L. Y. Jan, Y. N. Jan, Ankyrin repeats convey force to gate the NOMPC mechanotransduction channel. Cell 162, 1391 (2015).
Exocytosis; Membrane Fusion; Synaptic Transmission; Protein Folding; Chromatin Assembly and Disassembly; SNARE Proteins; Synaptotagmins; Munc18 Proteins; Optical Tweezers
Public Health Interests
- Alpha-SNAP enhances SNARE zippering by stabilizing the SNARE four-helix bundleL. Ma, Y. Kang, J. Y. Jiao, A. A. Rebane, H. K. Cha, Z. Xi, H. Qu, Y. L. Zhang, Alpha-SNAP enhances SNARE zippering by stabilizing the SNARE four-helix bundle. Cell Reports 15: 531-539 (2016).