Single-molecule dynamics in solution with anti-Brownian trapping

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications & trials

Abstract

We have made progress on the following two areas during the past year a) phase-separating pyrenoid proteins form complexes in the dilute phase. Recently, liquid-liquid phase separation was found to drive the assembly of many cellular compartments that lack membranes (also referred to as biomolecular condensates) and became an emergent new paradigm in cellular biology. While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play important roles in the stability, dynamics, and regulation of condensates. b) Single-molecule dissection of gRNA conformation during Cas9 holoenzyme assembly. Biomolecules carry out their function by cycling through a series of functional states. To better understand the structural-functional relations, it is of tremendous interest to probe structure at sequential stages of the functional states. We recently used the ABEL-FRET platform to probe the 3-end structure of CRISPR RNA (crRNA) at the single-molecule level as it assembles into the Cas9 holoenzyme. For every molecule, its assembly state is unambiguously determined using hydrodynamic profiling and its 3-end structure is probed by a pair of strategically placed FRET dyes. Strikingly, we discovered structural heterogeneity and dynamics at every stage of the assembly pathway that is, crRNA, guide RNA (gRNA, or crRNA-tracrRNA hybrid), Cas9-gRNA complex and Cas9-gRNA bound with substrate DNA, highlighting the importance of RNA structural diversity. Current work focuses on using RNA structural prediction tools to generate structural models consistent with single-molecule FRET measurements and devising plausible pathways of Cas9 holoenzyme assembly. This work could potentially shed light on fundamental biophysical principles of Cas9-gRNA recognition c) Towards a single-molecule view of SARS-COV-2 main protease activity. The main protease of SARS-COV-2 (MPro) is indispensable for the coronavirus replication and propagation. MPro exists as a homodimer and is responsible for most maturation cleavage events within the precursor polyprotein. Due to its vital roles, MPro has been a prominent drug target. For example, Paxlovid (PF-7321332) from Pfizer is an active site inhibitor of MPro. To better understand the enzymatic properties of MPro and provide mechanistic insights of how the drug disrupts the enzymatic cycle of the protease, we aim to develop a new single-molecule assay to watch individual MPro enzymes process their substrates, one molecule at a time. In the first 6 months of this project, we have established a protocol to fluorescently label Mpro proteins for single-molecule observations. We have also successfully monitored the dimer-monomer equilibrium at the single-molecule level which validated the activity of the labeled enzyme. These progress set the stage to future single-molecule enzymology studies of MPro.