Disordered Proteins as Biotech Building Blocks

Intrinsically disordered proteins can be used to easily create microparticles with complex architectures.

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Intrinsically disordered proteins (IDPs) are a class of proteins that are rapidly turning upside down the dogmatic idea that structure encodes function in proteins1,2.  Proteins that were previously ignored because they had large structurally disordered regions are now emerging as important contributors to a myriad of functions ranging from regulation of stress response to structural materials in organisms.  A unique property of many IDPs is their ability to phase separate in response to stimuli such as concentration, pH, and temperature3-5. While IDPs are a hot topic in cellular biology, in many ways the field of genetically engineered protein materials that we come from has been unwittingly working on —and designing at the sequence level— artificial IDPs for decades6,7. Silk, resilin, and elastin, for example, all depend on intrinsically disordered regions for their attractive material properties, and years of materials design and engineering in this field has led to an understanding of how to use structural disorder to design phase behavior from the ground up.

Examples of microparticle architectures achievable using intrinsically disordered proteins and simple phase behavior.

Using these principles, in 2018 our team published a paper on a new type of protein material, a “partially-ordered polymer”, that is capable of reversibly phase separating into a solid scaffold with two unique properties8: first, upon undergoing its phase transition, rather than forming a featureless liquid phase that is immiscible in water, these polymers phase separate into a solid material with a beautiful fractal-like network of interconnected pores. Second, the temperature at which these features form upon heating is higher than the temperature required to dissolve them upon cooling— this is a form of thermal hysteresis. Both features are very difficult —if not impossible—to predict or program into a material a priori, and we were hence excited that we had serendipitously stumbled upon a material where both features could be rationally tuned at the sequence level.  A consistent comment we would get from our colleagues was: “Really cool! But what are these properties good for?”  Our arguments “it allows us to probe solubility dynamics in IDPs related to certain diseases” or “the thermodynamic driving force behind hysteresis is the key to scaffold formation” were accepted but did not gain any true traction. We knew at the end of the day, that we had to build something new with it.

This manuscript is, in many ways, our response, a demonstration that a new material with new properties can have new applications.  By borrowing a key concept from cell biology, that IDPs are used to create spatially segregated regions within a cell —membraneless organelles, we set out to apply that same concept to the development of microparticles with internal architectures that were previously not possible to create. These new microparticles should, we hope, lead to new applications in biotechnology, biomaterials, and drug delivery.

 

1            Babu, M. M. The contribution of intrinsically disordered regions to protein function, cellular complexity, and human disease. Biochem Soc Trans 44, 1185-1200, doi:10.1042/BST20160172 (2016).

2            van der Lee, R. et al. Classification of intrinsically disordered regions and proteins. Chem Rev 114, 6589-6631, doi:10.1021/cr400525m (2014).

3            Boeynaems, S. et al. Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties. Proc Natl Acad Sci U S A 116, 7889-7898, doi:10.1073/pnas.1821038116 (2019).

4            Pak, C. W. et al. Sequence determinants of intracellular phase separation by complex coacervation of a disordered protein. Mol Cell 63, 72-85, doi:10.1016/j.molcel.2016.05.042 (2016).

5            Quiroz, F. G. & Chilkoti, A. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. Nat Mater 14, 1164-1171, doi:10.1038/nmat4418 (2015).

6            Dzuricky, M., Roberts, S. & Chilkoti, A. Convergence of artificial protein polymers and intrinsically disordered proteins. Biochemistry 57, 2405-2414, doi:10.1021/acs.biochem.8b00056 (2018).

7            Roberts, S., Dzuricky, M. & Chilkoti, A. Elastin-like polypeptides as models of intrinsically disordered proteins. FEBS Lett 589, 2477-2486, doi:10.1016/j.febslet.2015.08.029 (2015).

8        Roberts, S. et al. Injectable tissue integrating networks from recombinant polypeptides with tunable order. Nat Mater 17(12):1154–1163. doi:10.1038/s41563-018-0182-6 (2018).

Stefan Roberts

Research Scientist, Duke University

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