Hays Rye

Associate Professor | Biochemistry & Biophysics

molecular chaperones, protein folding, protein quality control, disaggregation, membrane dynamics, membrane fission, nanoparticle dynamics, single molecule fluorescence

Office:BICH / 239A
Email:haysrye@tamu.edu
Phone:979-862-1123

The Rye lab is broadly interested in understanding how cells control the assembly and disassembly of important biological nanoparticles like protein aggregates and membrane transport vesicles. Specialized molecular machines powered by the energy stored in ATP or GTP are required for these activities and regulate many aspects of basic cellular biology. Our goal is to develop detailed mechanistic insights into these molecular machines and understand why their dysfunction leads to the wide range of diseases that spring from aberrant protein assembly and membrane dynamics.

Development of single particle analysis methods

Burst analysis spectroscopy (BAS) measures the population-resolved kinetics of biological nanoparticle assembly and disassembly. Fluorescent particles are advectively flowed through one or more focused laser beams.
See PNAS 105, 14400–14405 (2008).

The formation and disassembly of macromolecular particles are ubiquitous and essential features of all living organisms. Additionally, diseases are often associated with biologically active nanoparticles, such as the formation of toxic protein aggregates in diseases of protein misfolding and the growth of infectious viral particles. Simultaneously, the heterogeneous and dynamic nature of biologically active particles can make them exceedingly challenging to study. To tackle this problem, we are developing new techniques, e.g., burst analysis spectroscopy (BAS). We combine these methods with other single-molecule and single-particle approaches to uncover unique nanoparticle dynamics in cell biology.

D. Shoup, A. Roth, R. Thapa, J. Puchalla, H. S. Rye, Development and applications of multi-color burst analysis spectroscopy. Biophys. J. (2020, under revision).

Protein disaggregation and molecular chaperones

Disassembly of fluorescent clathrin coats by a yeast Hsc70 molecular chaperone system consisting of Ssa1 and Swa2. Analysis of the decay behavior of the large fluorescent bursts reveals that coat disassembly is highly cooperative.
See JBC 288, 26721–26730 (2013)

The misfolding and aggregation of essential cellular proteins is a fundamental problem for all living organisms. Aggregation of even non-essential proteins can lead to debilitating diseases like type II diabetes, Alzheimer’s, Huntington’s, and Parkinson’s. Importantly, protein folding and aggregation are heavily influenced by the cellular protein quality control machinery, involving networks of molecular chaperones. Precisely how different chaperone systems cooperate in dismantling and reactivating aggregated proteins, and how molecular chaperone action affects disease progression, is not well understood. We use a variety of approaches, including novel single-particle assays like BAS, to study the disaggregase systems of bacteria and yeast. Our goal is to develop a mechanistic understanding of how different molecular chaperone networks recognize and dismantle protein aggregates.

D. Shoup, A. Roth, J. Puchalla, H. S. Rye, The impact of hidden aggregate structure on molecular chaperone disaggregation revealed by single particle burst analysis. In preparation.

Intracellular protein folding

The bacterial GroEL chaperonin pulls non-native substrate proteins into its central cavity and, in the process, partially unfolds them. The cryoEM structure of non-native substrate protein, PepQ, was solved in collaboration with the laboratory of Dr. Junjie Zhang. When the C-terminal tails at the base of the cavity are intact, the PepQ protein is drawn toward the base of the cavity and its internal density decreases.
See Nat. Commun. 8 (2017).

Once disentangled from an aggregate, or freshly synthesized from the ribosome, many proteins cannot fold autonomously to their native conformation. The most recalcitrant class of protein requires the assistance of large, barrel-shaped molecular chaperones known as chaperonins. Exactly how chaperonins, like the GroELS system of bacteria, accelerate protein folding remains controversial. We have shown that stimulated folding requires a direct and active alteration of the folding landscape of a single polypeptide by GroELS. Using various biophysical and structural approaches, we are now trying to unravel how this active enhancement mechanism works.

M. Naqvi, M. J. Avellandeda, A. Roth, E. Koers, V. Sunderlikova, G. Kramer, H. S. Rye. S. J. Tans, GroEL-mediated acceleration of protein folding by enhanced collapse, Nat. Commun. (2020, in review).

Membrane vesicle formation

Burst analysis spectroscopy (BAS) measures the population-resolved kinetics of biological nanoparticle assembly and disassembly. Fluorescent particles are advectively flowed through one or more focused laser beams.
See PNAS 105, 14400–14405 (2008).

Membrane-enclosed transport carriers, such as vesicles and short tubules, sort biological molecules between stations in the cell in a dynamic process fundamental to the physiology of organisms. While much is known about the protein coats that sculpt membranes into vesicles, the mechanism by which vesicles are released, the so-called membrane fission reaction, remains poorly understood. We are using the release of transport carriers from the recycling endosome of C. elegans and from the plasma membrane of yeast as model systems for examining membrane fission mechanisms. We have developed cell-free, single-particle fission assays based on BAS and are then using it to understand how nucleotide hydrolysis is coupled to membrane binding, rearrangement, and fission.

A. Brooks, D. Shoup, L. Kustigian, J. Puchalla, C. M. Carr, H. S. Rye, Single particle fluorescence burst analysis of Epsin induced membrane fission. PloS One 10 (2015).