A ribosome’s work: synthesizing and destabilizing one protein strand at a time

Despite over 50 years of research, how proteins fold continues to perplex researchers. Now, new tools show that the ribosome not only synthesizes proteins, it also helps them fold.

The protein energy landscape represents the energy levels of a protein as it explores different conformations while folding. Adapted from (7).

Pulse proteolysis: Protein samples are chemically denatured after which a protease degrades the unfolded portion of the protein before analyzed on a gel.

Left to Right: Avi Samelson, Susan Marqusee, and Madeleine Jensen found that the ribosome destabilizes the protein strand during translation.

Madeleine Jensen found that the ribosome destabilizes the protein strand during translation.

 

Claire Wineland, an 18-year old living with the life-threatening genetic disease cystic fibrosis, lists the perks of her frequent hospital stays, including room service, laundry service, and nurses who listen to the latest boy drama, in her series of YouTube videos for the Clairity Project, an organization aimed to increase awareness of cystic fibrosis.

Genetic diseases such as cystic fibrosis result from mutations that alter protein folding, causing protein malfunction. According to Patricia Clark from the University of Notre Dame, “It’s becoming increasingly clear that many of what are considered protein-folding diseases are diseases not necessarily of the final folded structure of the protein or mutations that destabilize the folded structure of the protein. Many of these mutations seem to alter the process of folding.”

In 1972, Christian Anfinsen won The Nobel Prize in Chemistry for proposing that a protein’s amino acid sequence predicts its structure (1). However, even Anfinsen acknowledged that there is more to protein folding that just the sequence. Now, researchers including Susan Marqusee of the University of California at Berkeley are building tools to look beyond how the amino acid sequence encodes the protein’s structure by examining the whole energy landscape of the protein-folding process.

A Simple Tool for a Complex Problem

A protein’s energy landscape describes the conformational energetics and dynamics of the whole molecule. As it folds, the protein samples different conformations along the way before finally reaching an energetically favorable state. Genetic variation and environmental changes can alter this landscape. Typically, measuring a protein’s energy landscape requires large amounts of pure protein and specialized spectroscopic assays, making study of the folding process difficult.

In 2005, Chiwook Park, then a post-doctoral researcher in Marqusee’s group, developed an easily accessible quantitative method to measure protein stability (2). “It actually came about in a sort of an odd way,” said Marqusee.

At the time, Park pondered about an unusual characteristic of the protein alpha lytic protease (3): it doesn’t get proteolyzed or degraded because, unlike most other proteins, it never undergoes natural conformational fluctuations. While thinking about this phenomenon, Park realized that since proteins are only cleaved when fluctuating away from the folded state, monitoring chemical denaturation by proteolysis would allow him to measure protein stability.

Based on that idea, Park created an assay he called “pulse proteolysis,” where he gradually increased the concentration of chemical denaturant and measured the undenatured portion of proteins on a gel to quantify their stability. Pulse proteolysis is a simple assay that provides quantitative information about a protein’s stability and energy landscape using tools available in any biology lab. “It could’ve been done 25 years earlier. It didn’t require anything that wasn’t around. You really just needed to think of it,” said Marqusee.

Marqusee initially envisioned using pulse proteolysis for drug discovery by screening protein stability changes upon binding to potentially therapeutic ligands. “Most assays for drugs require knowledge of the function, so you do some sort of functional assay. This assay requires no knowledge of the active site of the protein. You could throw in a bag of inhibitors and see which ones shifted the stability,” said Marqusee.

When Park tested the assay, however, he serendipitously used a protein prep discarded as too impure for other experiments. Park didn’t mind the impure protein because pulse proteolysis only required following a band on a gel. When showing Marqusee the results, both Park and Marqusee realized that this property makes pulse proteolysis a viable tool for studying protein folding in biologically complex systems, which are normally too messy for most protein-folding experiments.

Over the years, the protein-folding community has adapted pulse proteolysis to study protein folding in complex systems such as membrane proteins (4) and knotted proteins (5).

A Return to Simple Tools

With pulse proteolysis in hand, potential new applications for the assay constantly buzzed in the back of Marqusee’s mind. Meanwhile, her team moved towards measuring protein energetics in complex cellular-like environments. “It would be naïve to think that the cellular environment didn’t also change the properties of the protein,” said Marqusee.

However, according to Clark, who is not associated with the Marqusee group, studying protein folding in a complicated intracellular environment rather than isolated in a test tube is challenging. “The vast majority of techniques that we would normally use to study protein folding don’t work,” she said.

Then in 2012, Avi Samelson, a graduate student in Marqusee’s group, began using complicated single-molecule fluorescence tools to study co-translational folding—protein folding as the ribosome nascent chain (RNC) emerges from the ribosome. Others also started studying co-translational folding using NMR, but this approach does not provide information concerning the energy landscape.

In the midst of these challenges, Marqusee came across an article published by researchers at the University of Cambridge who uniquely combined pulse proteolysis with in vitro translation and fluorescent labeling of the protein (5). This prompted Samelson and other members of the team to develop a modified version of the assay by combining pulse proteolysis with a commercially made in vitro transcription/translation system and fluorescent labeling to measure the stability of RNCs emerging from the ribosome (6).

Using pulse proteolysis, the team verified that protein stability didn’t change with fluorescent labeling. They also tested the sensitivity of the ribosome and the attached RNC to denaturant using sucrose gradient ultracentrifugation and fluorescence correlation spectroscopy to establish the maximum concentration of denaturant suitable for the assay. Finally, the tool was ready to measure how the ribosome changes the stability of the RNC during translation.

Destabilize to Stabilize

Marqusee and her team probed the stability of the RNC by s¬talling translation and performing pulse proteolysis. Using three well-characterized proteins to ensure that any observations weren’t unique to a single protein, they found that the ribosome destabilized the nascent chain compared to proteins folded after release from the ribosome.

Furthermore, by performing the same experiment on RNCs farther away from the ribosome by attaching linkers of increasing length to the RNC, the team found that RNC stability increased with increasing distance from the ribosome. Together, these results indicate that the ribosome destabilizes and prevents protein folding of the nascent chain.

These results initially surprised Samelson since previous co-translational studies reported increased protein-folding efficiency in the presence of the ribosome. However, according to Samelson, because protein folding can occur on a much faster timescale than translation, a partially translated protein could potentially become trapped in an unproductive conformation that inhibits formation of the final folded structure. Ribosome destabilization potentially serves as a method to prevent any premature structures from forming, thus enabling efficient protein folding.

Although verifying their assay was technically challenging, the use of commercially-made components combined with an already simple assay makes this version of pulse proteolysis easily accessible for researchers who want to study protein folding in complex environments.

“This is a significant step forward,” said Clark, “It’s going to help me appreciate how the process of folding, not necessarily the product of the final of folding, but the process of folding may be different in the cell,” she said.

Although Anfinsen may not have realized the far-reaching influence of protein-folding when he won the Nobel Prize, patients such as Wineland who suffer from cystic fibrosis or other protein-folding diseases can benefit from the new tools as discoveries about how proteins fold offer hope for future therapies.