Discovery shows that even neutral molecules take sides when it comes to biochemistry

A new study led by a pair of researchers at the University of Massachusetts Amherst turns long-held conventional wisdom about a certain type of polymer on its head, greatly expanding understanding of how some of biochemistry’s fundamental forces work. The study, released recently in Nature Communications, opens the door for new biomedical research running the gamut from analyzing and identifying proteins and carbohydrates to drug delivery.

The work involves a kind of polymer made up of neutral polyzwitterions. Because they have a neutral electrical charge, polyzwitterions are not expected to respond to an electric field. However, the team found not only that certain neutral polyzwitterions behave as if they were charged, but also that the electric field surrounding polyzwitterions, once thought to be uniform, varies in strength.

“My interest is in the proteins and amino acids, which are the building blocks for protein, inside our body’s cells,” says Yeseul Lee, lead author and graduate student in polymer science and engineering at UMass Amherst.

“Proteins are biopolymers and a biopolymer is a series of charged and neutral elements. Biopolymers are crowded throughout the cellular environment. It is of great significance to understand how these molecules move from one location to another and communicate in such crowded environments, because we cannot live without their movement and communication.”

“We know a good deal about the movement of the charged parts of biopolymers,” says Murugappan Muthukumar, the Wilmer D. Barrett Distinguished Professor in Polymer Science and Engineering at UMass Amherst and the study’s senior author.

“But until now, there hasn’t been much interest in the neutral parts. The assumption was that they were essentially silent, that they didn’t play much of a role in the way that proteins transport themselves under electrical stimuli.”

All those biopolymers jumbled together in our cells generate local electric fields, which means that protein molecules containing neutrally charged parts are floating around in an electrical field inside our bodies.

But how do these biopolymers get where they need to go? Are the neutral units merely burdensome for their movement? And is that electrical field inside our cells uniform?

To answer these questions, Lee and Muthukumar chose polyzwitterions as a model polymer, because polyzwitterions consist of only neutral units. Polyzwitterions are built of zwitterions, single molecules in which one part has a positive charge and another a negative. The two charges cancel each other out, leaving the zwitterion with a net neutral charge.

In addition, the team turned to a tool called single-molecule electrophoresis, which is a method of identifying and sequencing macromolecules based on their charge distribution. One way to understand how single-molecule electrophoresis works is to imagine a desktop-sized swimming pool, only instead of water, it’s filled with an electrolyte solution—potassium chloride.

And instead of a line of buoys separating the shallow and deep ends, imagine a wall with a hole in it. A teeny hole. In this case, a hole 3.5 nanometers in diameter, small enough that only one polymer strand can move through at a time, giving the researchers a chance to identify it.

When an electric field is applied across the “swimming pool,” all the negatively charged parts, including the polymers, flow through the hole to the deep end, and all the positively charged parts flock to the shallow end, while the researchers watch, measure and learn.

For their experiment, Lee and Muthukumar separately dumped two different kinds of polyzwitterions into the swimming pool—PSBMA and PMPC. What they expected to see was that, once the electric current was switched on, the neutral polyzwitterions stayed put.

But that’s not what happened.

Instead, PSBMA acted as if it had a net negative charge and migrated to the deep end, while PMPC acted as if it had a net positive charge and moved to the shallow end, a phenomenon that has never before been observed.

What explains this movement is that some polyzwitterions, which look something like a rib, carry one charge at one extremity—at the tip of the rib—while the other, opposite charge is somewhere closer to the middle of the rib, near where it connects to the biopolymer backbone. PSBMA carries a negative charge at its tip, and PMPC a positive one.

And this is where Lee and Muthukumar made their second discovery.

“It has long been thought that the cellular electrolyte solution surrounding the polyzwitterions and biopolymers has a uniform dielectric constant,” says Lee.

In such solutions, the electrolyte ions reduce the polymer unit’s charges depending on the local dielectric constant. The lower the local dielectric constant, the lower the polymer unit’s charge. If, as had been previously thought, the local dielectric constant was uniform, then each polymer unit’s electric charge should decrease equally.

Yet, Lee and Muthukumar observed the polyzwitterions moving in only one direction during their electrophoresis experiment, which means that the charge reduction in the positively and negatively charged units of the polyzwitterion must be unequal. In other words, and contrary to the belief in a uniform dielectric constant, the local dielectric constant is different around the positively charged and negatively charged units.

In reality, the dielectric constant is far weaker the closer one gets to the biopolymer backbone. Neutrally charged polyzwitterions act like charged particles because the dielectric constant is much higher at the tip of the zwitterionic rib than where it connects to the backbone. In effect, only the charge at the tip “matters”—the other charge, closer to the backbone, winds up “hidden,” or shielded.

“This is a new contribution to our understanding of fundamental forces in biochemistry,” says Muthukumar. “No one knew that the dielectric constant varied as one moves away from the polymer backbone. Here we could also quantify its consequences.”

Knowing more about how human proteins assemble and move around opens up all sorts of avenues for disease detection and drug delivery.