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By Paul Muhlrad

Barely six months into his new job, Joel Kralj is already making his mark. Kralj, an assistant professor in CU-Boulder’s Department of Molecular, Cellular and Developmental Biology and member of the BioFrontiers Institute, is one of 15 newly named Searle Scholars, selected among nominees from 153 of the top research institutions in the U.S. The prestigious designation, given to “exceptional young faculty in the biomedical sciences and chemistry“ comes with a 3-year $300,000 research grant. Kralj will use his grant to investigate the new field of biology that he discovered: bacterial electrophysiology.

Kralj arrived in Boulder last September from Harvard University, where, as a postdoctoral fellow, he engineered proteins that can serve as cellular voltage meters by making living cells fluoresce when they undergo a change in voltage. He dubbed the high-tech voltage sensors VIPs, for Voltage Indicator Proteins. Before Kralj invented VIPs, scientists had to poke tiny glass electrodes into cells to record their electrical activities. That technique, called patch clamp, earned a Nobel Prize for the two scientists who developed it. While powerful, patch clamp is a technically challenging and invasive procedure, and can only be performed on large cells, one cell at a time. Kralj, who completed his undergraduate and master’s degrees in engineering physics, developed optics, engineered proteins, and image processing technologies so that he can not only measure the voltage changes in thousands of cells at a time without the size constraints imposed by patch clamp.

The Benefits of Beer

The idea for voltage-sensing proteins came at a happy hour when Kralj was a graduate student at Boston University. He had been collaborating with a student in the lab of Harvard professor Adam Cohen on a project involving a class of colored proteins called rhodopsins. In animals, rhodopsin is the main light-sensing pigment in the retina. Certain photosynthetic bacteria and algae use different forms of the protein to capture sunlight and convert it into chemical energy. The rhodopsin protein is embedded in the membrane surrounding the cell. When light shines on the cell, the protein changes shape and pumps positively charged hydrogen ions, or protons, through the plasma membrane into the cell, creating a charge gradient, or voltage, between the inside and outside of the cell. Over happy hour beers, Cohen posed a question: “Do you think rhodopsins could be used to sense voltage instead of creating voltage?“

Scientists had previously tried to adapt other proteins as fluorescent voltage indicators, with only marginal success. Their sensor proteins weren’t sensitive enough to detect small voltage changes, or they didn’t work in neurons, the cells where people were most eager to see electrical impulses. One journal review article called the problem “the Holy Grail of neuronal circuit imaging.“ Kralj was intrigued by the challenge. “I thought about it for a little while and I came up with a scheme that made sense,“ he recalls. The first trick would be to make a mutation to jam the proton-pumping part of the protein so that it couldn’t generate voltage in the cell. The second task would be to tweak another part of the protein so that small changes in voltage would trigger the conformational change, leading to a color shift, which would be visible under the microscope. Soon after their conversation, Kralj graduated and joined Cohen’s lab as a post-doc to put his ideas to the test.

Kralj and Cohen started their experiments with a protein called proteorhodopsin because it had the many desirable optical properties to form the backbone of a voltage sensor. “We knew that it would change colors in response to pH at a physiological pH. That was the most important thing in terms of what we thought would work as a voltage-sensing scheme.“ Proteorhodopsin was also easy to express in E. coli bacteria, which multiply rapidly. “We could turn around experiments very fast. We could make mutations, and then see how they looked very very quickly,“ Kralj explained.

Blinking Bacteria

Almost immediately, Kralj and Cohen hit pay dirt. Microbiologists had known for years that bacteria maintain a constant negative voltage across their cell membranes. It’s easy to neutralize, or depolarize, this voltage by exposing bacteria to a chemical called sodium azide. When Kralj added sodium azide to a population of E. coli containing his custom-tailored proteorhodopsin, he saw the optical change that he expected. “So I had strong evidence that I was seeing a change in voltage.“

But then, Kralj explains, something unexpected happened. “I had been running all of my experiments in PBS (phosphate-buffered saline-a dilute salt solution), and Adam just suggested adding glucose to see what happens. So, that way the bacteria had an energy source. And as soon as I did that, they all started blinking!“

While it was well known that bacteria maintain a constant voltage, nobody had ever observed them changing their voltage. Kralj and Cohen were astounded, and at first didn’t believe their own eyes. “We thought we were seeing some sort of artifact.“ They repeated the experiment many times, and every time, the bacteria started blinking. “We talked to a lot of E. coli experts at Harvard and asked if they’d seen anything like this. They said that this was completely unknown. After we talked to them, we had this hunch that we might have actually done it. We might have actually found a voltage sensor and discovered an entirely new phenomenon. So, for the next six months we just did experiments to confirm that we actually had both of these things in our hands.“

The team next tried to express the proteorhodopsin voltage sensor (which Kralj called PROPS, for “PRoteorhodopsin Optical Proton Sensor“) into mammalian cells. The cells made the protein, but instead of getting inserted into the plasma membrane, where it needed to be, all of the protein ended up in another part of the cell-the endoplasmic reticulum. All kinds of tricks to coax the protein to go to the plasma membrane failed. Then, another lab published a paper on a closely related protein, called archaerhodopsin, which localized to the plasma membrane in mammalian cells. So Kralj engineered into archaerhodopsin all the mutations that had successfully converted proteorhodopsin into a voltage sensor. He expressed the new protein in mammalian cells, and it worked perfectly. The mammalian sensor also gave Kralj and Cohen definitive proof that their engineered proteins were really doing what they thought they were doing. “It was very easy to tell if we had a voltage sensor because we could just patch clamp onto cells and change the voltage,“ Kralj explains. It was like switching a light bulb on and off.

Kralj and his colleagues have now developed VIPs from four different rhodopsin varieties, with hundreds of different mutant variations, each having slightly different optical and biochemical properties. They’ve sent their VIPs to hundreds of other labs around the world, which Kralj finds gratifying. “People have used these to look at voltages in single synapses, and they also did it inside a live fly brain. And so, people are actually using these to learn about biology, which is really exciting for us.“

Bacterial Electrophysiology

Getting the Searle Scholars award, Kralj says, means he’ll have the flexibility-in this era of tight research funding and risk-averse funding agencies-to really explore “bacterial electrophysiology,“ a term that never existed before he invented VIPs . “I want to ask how and why bacteria are modulating their voltage. This involves looking for the proteins and small molecules that are involved. How do these voltage transients arise? What are the actual components that cause them?“ That’s the how. The why is a deeper set of questions, and Kralj has some interesting ideas. Might bacteria use voltage to defend against antibiotics, or to communicate with each other, or to conduct warfare against other microbial species?

To figure out the parts list-all the proteins E. coli uses to change their voltage-Kralj and his students will build a microscope that can image many samples of bacteria under many different conditions. “The first thing that we want to do is look at genome-wide knockouts. As we knock out every single protein in the E. coli genome, how does that affect electrophysiology?“ The experiment will require around 4000 different E. coli strains, each one containing the VIP but missing one of its own genes. Kralj then plans to repeat the experiment, but instead of imaging different gene knockout strains, he’ll add different chemicals to test their effect on bacterial voltage.

Kralj is the seventh Searle Scholar at CU-Boulder. Other former Searle Scholars at CU-Boulder are Natalie Ahn (Chemistry & Biochemistry and BioFrontiers); Arthur Pardi, Roy Parker (Chemistry & Biochemistry); Min Han, Gia Voeltz and Ding Xue (Molecular, Cellular and Developmental Biology).

Photo provided by Patrick Campbell, University of Colorado