Tweezing out the SNARE Complex

Using new technology dubbed “optical tweezers,” Yale researchers, led by Professors of Cell Biology Yongli Zhang and Jim Rothman, have discovered intricate details about the workings of a protein complex that is the engine of membrane fusion in mammals and yeast.

SNARE proteins, or Soluble NSF Attachment Protein Receptors, fuse membranes through a process known to scientists as the “zippering model.” A SNARE complex consists of two sets of proteins, T-SNARE and V-SNARE, which are located on two membranes and join to “zipper” the membranes together. SNARE protein complexes are involved in all intercellular trafficking processes and are also key players in many diseases, such as those in which pathogens take advantage of the SNARE mechanism to infect cells.

The SNARE complex before completing membrane fusion. Courtesy of Nature.

In the late 1990s, the crystal structure of this important protein complex was solved, largely due to the contributions of A.T. Brunger and colleagues at the Yale Department of Molecular Biophysics and Biochemistry. At that time, Zhang was a graduate student in Brunger’s lab. Years later, after completing his postdoctoral work and starting his own lab, Zhang realized that the SNARE complex could be a prime target for optical tweezer technology.

“An optical tweezer basically extends our hand such that we can grab a polystyrene bead attached to a single molecule and move the bead,” said Zhang. “I decided that the SNARE protein would be the perfect subject to study with optical tweezers because in this protein, mechanical force is very significant. It takes a lot of force to draw two membranes together for fusion, and using optical tweezers, we can directly measure this force.”

Using optical tweezers, Zhang and his team pull on a single SNARE complex to measure the force it takes to unzip the complex and the extension change to study how it re-zippers. The force is related to the strength of the complex and the folding energy of the protein, and the extension is related to its structure. From these data, the researchers can deduce the amount of energy produced by the SNARE zippering process and the process’s intermediate states.

Their single-molecule experiments led Zhang and his team to confirm that the SNARE engine is indeed a powerful molecular motor that is well-suited for membrane fusion. They found that a single SNARE complex can generate up to 65 kBT of free energy, which is likely the most energy generated by the folding of a single protein complex.

Zhang and Rothman also identified several intermediate states of the SNARE zippering process and published their results in the September issue of Science. The half-zippered state they identified has a particularly significant biological role because the SNARE complex is only partially zippered at first in a primed state. Only once a membrane fusion-triggering action potential arrives can the half-zippered state continue to fold rapidly and finish fusing the membranes.

An example of a cellular process involving SNARE membrane fusion. Courtesy of Nature Reviews: Molecular Cell Biology.

“The fully-folded SNARE complex has a very beautiful four-helix structure,” said Zhang. “What was missing was how it folds, how it assembles, and using optical tweezers, we investigated how a single SNARE complex assembles and folds in real time.”

Applying optical tweezer technology to the SNARE complex did not come without its challenges, however. At first, the researchers had trouble forming the complex and attaching it to the polystyrene beads. “There were a lot of challenges in the molecular biology of forming these kinds of linkages before we got to the optical tweezers,” said Zhang. “After we found a way to attach a single SNARE complex between the two beads, the rest was quite straightforward.”

Now that details of the energetics and kinetics of the SNARE zippering process have been elucidated, a large focus of SNARE research is to understand how SNARE zippering is regulated. Synaptical membrane fusion, an important role of SNARE zippering, occurs only after an action potential arrives — thus, there are many proteins regulating calcium signals sent to the SNARE zipper.

Although throughout the course of his research, Zhang used optical tweezers primarily on the SNARE protein complex, he stresses what he believes is the wide applicability of this technology. “Many people don’t know about optical tweezers or consider them to be a very specialized tool,” said Zhang. “However, I think one can find a wide application for them in biology and especially in molecular biology. At Yale I have been trying to let more and more people know about optical tweezers –— they are one of the few tools that allow us to catch a single biomolecule and play with it.”