Overview

The conventional view that proteins form globular structures has dramatically changed with the discovery of intrinsically disordered proteins (IDPs), or proteins that persist as polymers in solution. This conformational plasticity of IDPs allows them to behave collectively, resulting in emergent phenomena ranging from polyelectrolyte brushes that sterically stabilize biological surfaces (see image, right) to the recent, exciting discovery of IDP-rich membrane-less organelles. However, that very conformational plasticity enables IDPs to form pathogenic aggregates in nearly every major neurodegenerative disease.  From a soft matter and public health perspective, the importance of IDPs demands quantitative characterization beyond traditional biological methods.

Techniques used in the Chung Lab

We are a molecular biophysics laboratory. The Chung Lab expresses and purifies IDPs (that reflect healthy/diseased states) and biophysically characterize them in cell free environments that replicate a variety of cellular conditions. Some of the advanced tools we use include:

1. Expressing proteins with canonical and non-canonical amino acids: While the bacterial expression of recombinant proteins has elucidated their many biophysical properties, post-translational modifications allow for reprogramming of protein function after cellular translation (especially in disease). Using recently-developed Genetic Code Expansion, we incorporate post-translational modifications into IDPs to try to understand their new function.

2. Advanced X-ray scattering and spectroscopy techniques: The Chung lab has been at the forefront of adapting advanced X-ray synchrotron techniques for use with biological assemblies. We collaborate with experimental beamlines at Stanford Synchrotron Radiation Lightsource and the Advanced Photon Source to characterize biological assemblies with unprecedented spatial and temporal fidelity. We also have an in-house Xenocs Xeuss 3.0 Small-Angle X-ray Scattering instrument for preliminary measurements.

3. High-throughput screening: Combining robotics and fluorescence measurements, we characterize IDPs in a variety of cellular conditions in order to determine their physiological function and diseased dysfunction.

Unlocking the effect of phosphorylation on fibrils found in Alzheimer’s

Although the presence of neurofibrillary tangles is increasingly linked to the etiology of Alzheimer’s disease (AD), the inability to recreate tangles in the laboratory precludes efforts to understand how they form or even to investigate compounds that may target them. Neurofibrillary tangles consist of paired helical filaments (PHFs) that are, in turn, made up of hyperphosphorylated full-length tau. Efforts to reconstitute in vitro PHFs often lack structural features of PHFs from AD brains, such as the presence of a “fuzzy coat”, which may be a more significant therapeutic target. Our lab will reconstitute AD-like PHFs by using genetic code expansion and high-throughput screening to incorporate phosphorylated full-length tau into PHFs. Then, we will use high-resolution cryo-EM to confirm their fidelity to PHFs from AD brains. With this new in vitro platform, we will investigate the proteopathic effects of the PHF “fuzzy coat” at a scale previously impossible.  In the future, this platform will provide the researchers at USC and the scientific community-at-large the ability to screen a wide variety of therapeutics targeting structures that are otherwise only found in PHFs from AD brains without being limited by sample constraints. 

Funding sources: Daniel J. Epstein Breakthrough Alzheimer’s Research Fund

Reprogramming bulk and transport properties of protein liquid droplets

Perhaps the most intriguing aspect in looking to structures within the cell to inspire the next generation of novel materials is not their bulk structural properties but the cellular ability to “reprogram” these structures. Much as reprogramming software allows it to perform different tasks, reprogramming materials allows them to take on novel functions after production. This is especially evident in select proteins that phase separate into protein-rich and protein-dilute liquid phases, much as oil added to water will separate into an oil-rich liquid phase. This phase separation has been shown to be controlled by phosphorylation, or the addition of a charged phosphate group to the protein. Not all phosphorylations are created equal; phosphorylation at certain sites within the protein will eliminate the ability to phase separate while others still can control the transport of molecules in-and-out of the protein-rich liquid phase. We will use genetic code expansion to manufacture proteins with specific phosphorylation code and create a framework for the design and use of similar reprogrammable biomaterials.

Funding Sources: National Science Foundation