The small protein called transthyretin can lead to significant health issues when it misfolds after being released from cells. In its healthy form, transthyretin is responsible for transporting hormones through the bloodstream and spinal fluid. However, when it misfolds, it creates harmful aggregates that can accumulate in the heart and nerves, resulting in a serious and progressive illness known as transthyretin amyloidosis (ATTR). Research indicates that nearly 25% of men over 80 years old may be affected by some level of ATTR, which can manifest as symptoms like shortness of breath, dizziness, and tingling or numbness in the limbs.
The small protein called transthyretin can lead to significant health issues when it misfolds after being released from cells. In its healthy form, transthyretin is responsible for transporting hormones through the bloodstream and spinal fluid. However, when it misfolds, it creates harmful aggregates that can accumulate in the heart and nerves, resulting in a serious and progressive illness known as transthyretin amyloidosis (ATTR). Research indicates that nearly 25% of men over 80 years old may be affected by some level of ATTR, which can manifest as symptoms like shortness of breath, dizziness, and tingling or numbness in the limbs.
Recently, researchers from Scripps Research have discovered new forms of transthyretin. Their study, published in Nature Structural & Molecular Biology on January 22, 2025, reveals how the uneven three-dimensional shape of the protein may affect its stability. This breakthrough could help in creating new treatments for ATTR.
“We’ve uncovered a level of molecular intricacy that researchers have been unaware of for years, which allows us to design better medications aimed at stabilizing transthyretin,” explained co-senior author Gabriel Lander, PhD, a professor at Scripps Research.
“The new findings highlight differences between two thyroid hormone binding sites that were previously thought to be identical, and clarify why a drug that binds to one site can alter the binding ability at the other site,” added Jeffery Kelly, PhD, the Lita Annenberg Hazen Professor of Chemistry at Scripps Research and a co-senior author of the research.
To visualize the 3D structure of small proteins like transthyretin, scientists often utilize crystallography, a technique where proteins are organized into large continuous crystals before imaging. However, the arrangement of a crystallized protein may not accurately reflect that of freely moving proteins in the body.
Another technique, called cryo-electron microscopy (cryo-EM), quickly freezes proteins to capture them in their more natural state. Unfortunately, during this process, smaller proteins like transthyretin tend to adhere to the air-liquid interface instead of remaining completely submerged, which can compromise both their structural integrity and the clarity of the obtained images.
To address this issue, Lander’s team devised a thin grid coated with graphene, allowing transthyretin molecules to attach themselves naturally. They then rapidly plunged this grid into liquid ethane to freeze the sample, effectively securing the transthyretin molecules on the graphene surface and preserving their natural shapes, simulating how they would appear in the bloodstream or other bodily fluids.
“We built upon the 2019 research done by the Yan lab at Princeton while creating our grids. It’s critical to get the surface chemistry right for studies like this. For small proteins such as transthyretin, making a quality sample is just the start; analyzing the results also poses a significant challenge,” stated Benjamin Basanta, PhD, a former research associate in Lander’s lab and the lead author of the new paper.
Upon applying this approach to study transthyretin, the team found that the protein exhibits asymmetric structures with two differently shaped binding pockets. Their earlier research, which had identified over 200 crystallized structures, had assumed these sites were the same. They demonstrated that this difference arises because the transthyretin complex continuously oscillates between two states, akin to a molecular form of “breathing,” according to Lander. This asymmetry in transthyretin’s native structure presents a hypothesis for how dissociation and misfolding may lead to protein clumping and subsequently cause disease.
By attaching tafamidis—a drug developed by the Kelly lab—to one or both of the transthyretin binding sites, they were able to stabilize the molecule and reduce this movement.
Moving forward, Lander and his team intend to investigate how this structure and its stabilization relate to ATTR, as well as how drugs interacting with transthyretin could aid in treating the disorder. They also believe that their graphene grid technique could be applicable to identify the structures of other small and unstable proteins, including the amyloid-beta peptide that accumulates in the brain of Alzheimer’s patients.
“The methods we’ve created have opened new pathways for potential treatments that could one day shield patients from not only TTR amyloidosis but other related amyloid diseases as well,” Lander concluded.
This research was funded by the National Institutes of Health (GM142196, AG067594, DK046335) and a Postdoctoral Fellowship from the George E. Hewitt Foundation for Medical Research.
