Browsing by Subject "intrinsically disordered protein"

Phase separation of macromolecules is a critical phenomenon for the human condition. This phenomenon has also been exploited for biotechnological development to improve human morbidity and mortality. However, there is still much more to learn regarding how this behavior is encoded within a protein sequence. Thus, this thesis seeks to 1) further explore the sequence space to understand how phase separation is encoded, with an emphasis on polypeptides with upper critical solution temperature (UCST) transitions and 2) use this phase separation to control availability of macromolecules at various length scales.

Using traditional molecular biology techniques, we will recombinantly express and purify a large number of polypeptides with variable sequence composition and sequence architecture. Then, using traditional polymer science and material science techniques combined with microscopic techniques that span the macro-scale and nano-scale, we will characterize their phase separation behavior and the interaction of these materials with biological systems.

We developed a practical mutation strategy that allows for complete control of the UCST binodal line in physiologic conditions that is useful for de novo design of artificial IDPs with UCST phase behavior. We evaluated the interaction of these polypeptides and their phase separation in the presence of bacterial, eukaryotic cells and in mice demonstrating how this binodal line fused to biological active partners can control biologic functions.

In bacteria, we made artificial phase separated puncta, akin to naturally occurring phase separated droplets, that have non-canonical function, demonstrating how primary features of the polypeptide chain affect enzymatic function. We created block co-polypeptides comprised of UCST and LCST protein sequences that exhibit remarkably tunable and robust nanoscale self-assembly into spherical micelles, worm-like micelles and vesicular structures capable of displaying large targeting domains on their surface. In the presence of eukaryotic cells, these nanomaterials can dramatically increase polypeptide uptake, increasing the avidity of the targeting molecule by over 1000-fold.

Finally, we demonstrated that phase separated polypeptides can sequester an active peptide GLP-1 from systemic circulation, controlling the peptide’s bioactivity through control of the phase diagram. Taken together, we demonstrate the universal power of the phase diagram, across many length scales, where the transducing agent for controlling biological activity is an engineered, repetitive polypeptide sequence.