Scientists from St. Jude Children’s Research Hospital and Washington University in St. Louis have established a model for predicting how proteins phase separate. The stickers-and-spacers model is a quantitative framework, applicable to many proteins, to predict phase separation. The findings were reported today in Science.
Liquid-liquid phase separation (LLPS) is a biophysical process that underlies many fundamental biological activities in cells. In particular, cells sort and separate proteins and other components through phase separation. Membraneless organelles are cellular bodies without membranes; many of these behave like liquid droplets that are formed through phase separation. When phase separation goes awry, it can also contribute to neurological diseases and cancer.
Interactions among intrinsically disordered proteins, notable for their lack of structure, can drive LLPS.
Scientists have not understood the interacting regions or “stickers” in the intrinsically disordered proteins that drive LLPS. The researchers wanted to identify these stickers and thus predict phase separation.
“Predicting phase separation quantitatively is important because this process is not only the underlying mechanism for the formation of membraneless organelles, but it is involved in many other cellular processes,” said co-senior author Tanja Mittag, Ph.D., of the St. Jude Department of Structural Biology. “Dysregulation of phase separation is also involved in disease, for example, when phase-separating, disordered domains are translocated in cancers and give rise to oncogenic fusion proteins.”
This work is part of the St. Jude Research Collaborative on Membraneless Organelles. Through the collaborative, investigators at St. Jude and Washington University conducted research that required the expertise of scientists at both institutions, streamlining and speeding up progress in this field.
The team identified stickers, regions in the disordered protein that drive its compaction. That makes the stickers critical to the interactions that lead to phase separation. Knowing the identity of the stickers and the extent of compaction allowed the team to determine the strength of the protein interactions. This was a key step to predict phase separation quantitatively.
This work used nuclear magnetic resonance spectroscopy and small-angle X-ray scattering to identify the stickers on a protein called hnRNAP1. The stickers are aromatic residues in the protein chain. Results showed that these stickers are sufficient to explain phase behavior. The team also found that the patterning of the stickers (how they are arranged in the sequence and interspersed by “spacers”) is necessary for functional LLPS to occur. The team learned that clustering of stickers in the sequence results in the formation of solid aggregates rather than liquid droplets.
These findings led to the creation of a sticker-and-spacers model for predicting phase separation. This model can be applied to other proteins, thus creating a tool that can be widely used to predict phase separation.
“We hope the stickers-and-spacers model will have value for facilitating research into and understanding of protein phase separation,” said co-senior author Rohit Pappu, Ph.D., of the Washington University McKelvey School of Engineering. “The Research Collaborative propelled this effort by bringing the force of both laboratories together to tackle this question.”
The study’s co-first authors are Erik Martin and Ivan Peran of St. Jude, and Alex Holehouse of Washington University. Additional authors on the paper are Anne Bremer and Christy Grace of St. Jude and Mina Farag, J. Jeremias Incicco and Andrea Soranno of Washington University.
The research was funded by St. Jude Children’s Research Hospital Research Collaborative on Membraneless Organelles in Health and Disease; The U.S. National Science Foundation; the Human Frontier Science Program; the American Federation for Aging Research; and ALSAC, the fundraising and awareness organization of St. Jude.
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