While molecular probes have been developed that distinguish between properly folded and misfolded proteins, they have been regarded with suspicion. By binding with a properly folded protein, such probe effectively stabilizes it. And, if many copies of the probe stabilize a population of well-folded proteins, measurement of the proportion of properly folded to misfolded proteins could be skewed. That is, the probe could produce measurements that overstate the fraction of properly folded proteins.
Now it appears that molecular probes could become more reliable, thanks to a new technique developed by scientists at The Scripps Research Institute (TSRI). These scientists combined folding probes with cell lysis and ATP depletion. With this approach, chaperones in the cell hold onto the unfolded proteome, preventing its folding and providing a snapshot to the folded protein-of-interest population while minimizing the overrepresentation of proteins in the folded state.
The new technique could enjoy wide use, given that many researchers are interested in the role of protein misfolding in causing tissue damage. Disorders that feature excessive protein misfolding afflict millions of people worldwide and include Alzheimer’s and Parkinson’s diseases, the systemic amyloidoses, and prion (“mad cow-type”) infections, as well as common enzyme deficiencies.
The TSRI scientists described their work March 3 in the Proceedings of the National Academy of Sciences, in an article entitled “Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts.” In this article, the authors explained how they developed small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest.
The probes, added the authors, enable fluorescence-based quantification of the folded and functional fraction in cell lysate at a time point of interest. “Importantly,” the authors emphasized, “these probes minimally perturb a protein’s folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis.”
“This new probe technology should lead to a better understanding of how to fold misfolding-prone proteins in cells,” said Jeffery W. Kelly, chair of TSRI’s Department of Molecular and Experimental Medicine, Lita Annenberg Hazen Professor of Chemistry, and member of the Skaggs Institute for Chemical Biology at TSRI. “The ability to quantify protein folding in a cell using this simple fluorescence-based technology should speed the development of new therapies.”
One of the most important applications of new probes like these will be for the rapid, high-throughput screening of very large drug compound libraries to identify drug candidates that prevent protein misfolding by improving the quality of cellular folding. “Using these probes to quantify the concentration of a functional, folded protein-of-interest, we can screen for compounds that boost this concentration,” said research associate Xin Zhang, who conceived and designed the study with Kelly.
In their study, the researchers cleared another hurdle for the use of probes in high-throughput screens with the design of a probe whose fluorescent beacon isn’t lit all the time, but only turns on when it reacts with the folded protein-of-interest. “That fluorescent signal quickly shows you the concentration of the folded, functional protein that was in the cell at the time of lysis,” Zhang said. “There is no need for the time-consuming removal of fluorescence probes that aren’t bound to targets or separation of the probe–protein-of-interest conjugate.”
