A novel strategy developed at Rice University allows scientists to zoom in on tiny segments of proteins inside living cells, revealing localized environmental changes that could indicate the earliest stages of diseases such as Alzheimer's, Parkinson's and cancer. The study, published in Nature Chemical Biology on Sept. 10, also shows promise for drug screening that targets protein aggregation diseases.
The research team engineered a fluorescent probe, known as AnapTh, into precise subdomains of proteins, creating a tool that monitors microenvironmental shifts in real time. Unlike conventional techniques that provide only broad signals, this approach reveals how distinct regions of the same protein behave differently during the aggregation process. The discovery led by Han Xiao , professor of chemistry and director of Rice's SynthX Center , enhances the basic understanding of disease mechanisms and lays the groundwork for identifying drug targets and screening potential therapeutics at an earlier stage.
"We essentially built a molecular magnifying glass," Xiao said. "This allows us to visualize subtle environmental changes that previously went unnoticed, and those early changes often hold the key to understanding protein-related diseases."
Precision lighting of protein subdomains
To explore how individual protein segments behave during aggregation, the researchers hypothesized that local environmental changes precede visible clumping. Based on that hypothesis, they designed AnapTh, a small fluorescent amino acid whose emission spectrum shifts based on its microenvironment. Using genetic code expansion, the team inserted this probe at specific sites without altering protein folding or function.
By tracking changes in fluorescence within living cells, the research team monitored the real-time responses of targeted subdomains. This technique provided a level of spatial resolution and temporal monitoring unmatched by existing tools.
"We wanted a method to light up just one spot in a protein and watch what happens around it in live cells," said Mengxi Zhang , a graduate student and co-first author of the study. "When aggregation starts, some parts become denser and more hydrophobic, while others remain unchanged. Our tool detects those distinctions nearly instantly."
Uneven aggregation revealed in living cells
When applying their technique to disease-related proteins, the researchers discovered that aggregation is far from uniform. Specific subdomains exhibited increased fluorescence intensity and spectral shifts, indicating heightened crowding and altered chemical surroundings, while other regions remained stable. These findings suggest that aggregation initiates at discrete "hot spots" before spreading.
This uneven process challenges traditional models that view protein aggregation as a homogeneous phenomenon. Instead, it highlights a more nuanced progression, where early localized misfolding events could serve as biomarkers or therapeutic entry points — results that provide a new perspective for studying protein aggregation at the molecular level.
Real-time monitoring of drug efficacy in protein aggregation diseases
The ability to detect early subdomain-specific changes opens opportunities to monitor neurodegenerative and protein misfolding disease progression more sensitively and to identify small molecules that intervene before aggregation spreads. The implications span from molecular biology to pharmaceutical innovation.
"This platform gives us a jump start," said Shudan Yang , a graduate student and co-first author of the study. "Now we can test potential inhibitors and see at the very first sign of trouble whether they prevent local misfolding. That kind of precision screening is what drug discovery needs."
Co-authors of the study include Rice scientists Shikai Jin, Yuda Chen, Yiming Guo, Yu Hu and Peter Wolynes .
This study was supported by the Robert A. Welch Foundation, Rice's SynthX Seed Award, the Cancer Prevention Research Institute of Texas, the National Institutes of Health, the U.S. Department of Defense, the John S. Dunn Foundation Collaborative Research Award, the Hamill Innovation Award, the National Science Foundation and the D.R. Bullard Welch Chair at Rice.
