New small-molecule probes enable measurement of folded versus misfolded proteins in cells
Researchers at The Scripps Research Institute (TSRI) have developed small-molecule “folding probes” that can selectively label and quantify the fraction of a target protein that is correctly folded and functional versus the misfolded, disease-associated conformations inside cells. This innovation provides a practical way to monitor the folding status of specific proteins in cellular samples and under differing biological conditions.
Accurate tools for measuring protein folding in cells have been in high demand because protein misfolding underlies many human disorders. Excessive misfolding and aggregation damage tissues and contribute to a range of conditions including Alzheimer’s disease, Parkinson’s disease, systemic amyloidoses and prion-related infections, as well as certain inherited enzyme deficiencies. The new probes are designed to help researchers better understand these processes and to accelerate discovery of therapies that restore or preserve normal protein folding.
“This probe technology should enable more precise measurement of how proteins fold in cells,” said Jeffery W. Kelly, chair of TSRI’s Department of Molecular and Experimental Medicine and Lita Annenberg Hazen Professor of Chemistry. “A simple fluorescence-based readout of the functional, folded fraction will speed efforts to develop drugs that reduce misfolding.”
The study, led by Kelly and his laboratory, appears in the online Early Edition of the Proceedings of the National Academy of Sciences.
Why distinguishing folded from misfolded proteins matters
Proteins that share the same amino acid sequence can adopt very different three-dimensional shapes. A correctly folded protein performs its biological function, while a misfolded protein often loses activity and can expose hydrophobic surfaces that promote aggregation. Aggregates can disrupt cellular processes and damage tissues that regenerate poorly, producing severe clinical consequences.
Diseases caused by loss of normal protein function or by toxic gain-of-function from aggregates can shorten healthy lifespan. Cellular systems that assist folding and maintain protein homeostasis are therefore major determinants of whether a protein stays functional or becomes harmful. The new folding probes allow researchers to quantify how interventions that bolster the cell’s protein quality-control machinery change the proportion of correctly folded protein.
Design and validation of folding probes
Kelly’s team set out to tag only the folded, active conformations of specific proteins. For proof-of-principle they used a designed enzyme called retroaldolase, produced by collaborator David Baker’s laboratory, and transthyretin (TTR), a clinically important protein whose misfolding and aggregation cause cardiomyopathies and polyneuropathies. (Kelly’s lab contributed to the development of targeted therapies for TTR-related polyneuropathy.)
Graduate students Yu Liu and Yun Lei Tan, together with Research Associate Xin Zhang, led the experimental work to synthesize folding probes that covalently label the properly folded, functional states of target proteins but not their misfolded forms. When probe solutions were added to lysates from cells expressing the proteins of interest, the probes reacted selectively with the folded fraction and produced a fluorescent signal that could be quantified to determine the concentration of functional protein present at the time of lysis.
Strategies to avoid bias in measurements
Previous covalent probes that react with folded proteins raised concerns because the act of labeling can stabilize the folded state and inflate the apparent folded fraction. To minimize this bias, the researchers combined probe labeling with protocols that prevent further folding after cell lysis. Specifically, by depleting cellular ATP they caused chaperones to hold onto unfolded proteins, effectively freezing the folding equilibrium and giving a snapshot of the folded, functional population while limiting probe-induced overrepresentation.
Additionally, the team designed probes with “turn-on” fluorescent beacons that remain dark until covalently attached to the folded target. This eliminates the need for time-consuming removal of unreacted probe or separation of probe–protein conjugates, enabling a rapid readout suitable for high-throughput formats.
Applications: drug discovery and disease research
One of the most promising uses of these folding probes is in high-throughput screening of large compound libraries to identify molecules that increase the amount of properly folded, functional protein by enhancing cellular folding capacity or altering protein homeostasis. By quantifying the functional fraction directly, screens can select compounds that genuinely improve protein quality rather than merely binding or stabilizing in vitro.
If translated into drug discovery pipelines, this approach could accelerate development of therapies that reduce protein misfolding and aggregation, potentially preventing or slowing age-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease and treating inherited enzyme-deficiency disorders. The research team notes that anti-misfolding strategies may have broad therapeutic potential given how common misfolded proteins can be in cells and the many ways misfolding may cause harm.
Study contributors and support
The paper, “Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts,” lists authors Yu Liu, Yun Lei Tan, Xin Zhang, Gira Bhabha, Damian C. Ekiert, Joseph C. Genereux, Younhee Cho, Yakov Kipnis, Sinisa Bjelic, David Baker and Jeffery W. Kelly. Additional contributors include scientists from the University of California, San Francisco, and the Baker Laboratory at the University of Washington, Seattle.
The work was supported by the National Institutes of Health, the Skaggs Institute for Chemical Biology and the Lita Annenberg Hazen Foundation.
Contact: Mika Ono – Scripps Research Institute
Source: Scripps Research Institute press release
Original Research: Abstract for “Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts” (PNAS). Published online March 4, 2014.