New findings on protein folding in bacteriorhodopsin

When it comes to drug development, membrane proteins play a crucial role, with approximately 50% of drugs targeting these molecules. Understanding the function of these membrane proteins, which connect to cell membranes, is important for designing the next line of powerful drugs. To do this, scientists study model proteins, such as bacteriorhodopsin (bR), which, when triggered by light, pump protons across the cell membrane.

While bR has been studied for half a century, physicists have recently developed techniques to observe its folding mechanisms and energy in the native environment of the cell’s lipid bilayer membrane.

In a new study published by Proceedings of the National Academy of Sciences (PNAS), JILA member Thomas Perkins and his team advanced these methods by combining atomic force microscopy (AFM), a conventional nanoscience measurement tool, with precisely timed light triggers to study function functionality. proteins in real time.

“The energy of membrane proteins is difficult to study and therefore not well understood,” Perkins explained. “Using AFM and other methods, we can create ways to explore this question further.” Armed with a better understanding of the energy of these proteins, chemists can design more powerful drugs to treat specific symptoms and diseases caused by protein dysfunction.

Measuring protein dynamics in milliseconds

Although bR is a microscopic protein, it can be seen with the naked eye, and even in satellite images, when archaeal microorganisms bloom, they leave large amounts as residue in saltwater ponds . “The ponds are filling up with something called Halobacterium salinarum, the parent organism of bacteriorhodopsin,” Perkins said. “These ponds are used to harvest salt, and because they are warm and salty, bacteria loves to grow there.”

At the microscopic level, bR works with other membrane proteins to produce energy for the cell by creating a proton gradient on one side of the cell membrane, which passes the proton to the other side of the membrane. To do this, bR bends and unfolds its helices into specific shapes to control the number of protons that pass through the membrane. During this process, migrating protons produce chemical energy in the form of adenosine tri-phosphate (ATP).

For Perkins and his co-author David Jacobson (former postdoctoral researcher at JILA and now assistant professor at Clemson University), bR presented an opportunity to design a new experimental method to study functional energetics in real time.

To study proteins like bR, Jacobson and Perkins use AFM, which acts like a little finger to gently pull on the protein, which helps AFM feel the protein’s surface, map its structure, and better understand how the protein folds.

Since bR folding processes are triggered by light, Perkins and Jacobson added an illumination element to the AFM procedure. “We had this clever idea of ​​sticking ultra-thin green LEDs, which trigger bacteriorhodopsin, onto a metal washer that we can attach to the AFM,” Perkins said. “These green LEDs are also inexpensive, between $1.00 each or $1.50 each. Compared to our AFM cantilever, which costs around $80 each, throwing away a $1.50 LED is not hardly a concern that concerns us.”

With this inexpensive add-on to their AFM, Perkins and Jacobson could trick the bR into folding and unfolding with millisecond precision. After collecting their data, the researchers found that the protein folded correctly 60% of the time, allowing protons to pass through the membrane.

To check the real-time energy and function of protein folding, the scientists mutated the bR protein so that it always remained in the “open” or unfolded state. Thanks to their new experimental setup, they were able to reproduce results similar to those previously observed in the “open” phase of the bR photocycle.

“In biology, you can see something, but you have to ask yourself: Am I seeing what I think I am seeing?” » said Perkins. “So by performing a mutation and seeing the effect we expected, we have increased confidence that we are actually studying the process we think we are studying.”

The mystery of the misfolded protein

While Perkins and Jacobson observed correct folding in 60% of cases, the remaining 40% surprised them, because the protein misfolded but could still pump a proton across the membrane. “The bad folding is stabilizing,” Perkins added. “And it was really surprising.” In many cases, protein misfolding does not result in stabilization.

Due to energy stabilization, Perkins and Jacobson hypothesized that the structural helices of the bR were not separating properly to provide a completely open tunnel for the proton, even though it was still wriggling, a process difficult to detect with the AFM imaging.

Trying to better understand the mechanisms underlying misfolding, Perkins and Jacobson lowered the force on the AFM tensile test to zero to see if that would allow the protein to fold correctly. However, the results remained the same: 40% of cases resulted in misfolding.

These results, with the same number of folding errors, intrigued the researchers. Although Perkins and Jacobson were unable to identify the cause of these misfolded cases, they hope to investigate further. They now want to see what the rest of the biophysics community thinks of these results.

“There could be more subtle effects, or perhaps new scientific data,” Perkins added. “There might be a path that people might not have been able to see before.”