Biophysics

Biomolecular condensates are macromolecular assemblies constituted of proteins that possess intrinsically disordered regions and RNA-binding ability together with nucleic acids. These compartments formed via liquid-liquid phase separation (LLPS) provide spatiotemporal control of crucial cellular processes such as RNA metabolism. The liquid-like state is dynamic and reversible, containing highly diffusible molecules, whereas gel, glass, and solid phases might not be reversible due to the strong intermolecular crosslinks. Neurodegeneration-associated proteins such as the prion protein (PrP) and Tau form liquid-like condensates that transition to gel- or solid-like structures upon genetic mutations and/or persistent cellular stress. Mounting evidence suggests that progression to a less dynamic state underlies the formation of neurotoxic aggregates. Understanding the dynamics of proteins and biomolecules in condensates by measuring their movement in different timescales is indispensable to characterize their material state and assess the kinetics of LLPS. Herein, we describe protein expression in E. coli and purification of full-length mouse recombinant PrP, our in vitro experimental system. Then, we describe a systematic method to analyze the dynamics of protein condensates by X-ray photon correlation spectroscopy (XPCS). We also present fluorescence recovery after photobleaching (FRAP)-optimized protocols to characterize condensates, including in cells. Next, we detail strategies for using fluorescence microscopy to give insights into the folding state of proteins in condensates. Phase-separated systems display non-equilibrium behavior with length scales ranging from nanometers to microns and timescales from microseconds to minutes. XPCS experiments provide unique insights into biomolecular dynamics and condensate fluidity. Using the combination of the three strategies detailed herein enables robust characterization of the biophysical properties and the nature of protein phase-separated states.

Cytochrome P450 reductase (CPR) is a multi-domain protein that acts as a redox partner of cytochrome P450s. The CPR contains a flavin adenine dinucleotide (FAD)–binding domain, a flavin mononucleotide (FMN)-binding domain, and a connecting domain. To achieve catalytic events, the FMN-binding domain needs to move relative to the FAD-binding domain, and this high flexibility complicates structural determination in high-resolution by X-ray crystallography. Here, we demonstrate a seeding technique of sorghum CPR crystals for resolution improvement, which can be applied to other poorly diffracting protein crystals. Protein expression is completed using an E. coli cell line with a high protein yield and purified using chromatography techniques. Crystals are screened using an automated 96-well plating robot. Poorly diffracting crystals are originally grown using a hanging drop method from successful trials observed in sitting drops. A macro seeding technique is applied by transferring crystal clusters to fresh conditions without nucleation to increase crystal size. Prior to diffraction, a dehydration technique is applied by serial transfer to higher precipitant concentrations. Thus, an increase in resolution by 7 Å is achieved by limiting the inopportune effects of the flexibility inherent to the domains of CPR, and secondary structures of SbCPR2c are observed.

Graphical abstract:

Cancer cells often overexpress glutaminase enzymes, in particular glutaminase C (GAC). GAC resides in the mitochondria and catalyzes the hydrolysis of glutamine to glutamate. High levels of GAC have been observed in aggressive cancers and the inhibition of its enzymatic activity has been shown to reduce their growth and survival. Numerous GAC inhibitors have been reported, the most heavily investigated being a class of compounds derived from the small molecule BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide). X-ray structure determination under cryo-cooled conditions showed that the binding contacts for the different inhibitors were largely conserved despite their varying potencies. However, using the emerging technique serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors. Here, we describe a step-by-step protocol for crystal handling, data collection, and data processing of GAC in complex with allosteric inhibitors using serial room temperature crystallography.

Graphical abstract:

Workflow for serial room temperature crystallography. Diagram showing the processing and scaling routine for crystals analyzed using serial room temperature crystallography.

The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 binds angiotensin converting enzyme-2 (ACE-2) on the surface of epithelial cells, leading to fusion, and entry of the virus into the cell. This interaction can be blocked by the binding of llama-derived nanobodies (VHHs) to the RBD, leading to virus neutralisation. Structural analysis of VHH-RBD complexes by X-ray crystallography enables VHH epitopes to be precisely mapped, and the effect of variant mutations to be interpreted and predicted. Key to this is a protocol for the reproducible production and crystallization of the VHH-RBD complexes. Based on our experience, we describe a workflow for expressing and purifying the proteins, and the screening conditions for generating diffraction quality crystals of VHH-RBD complexes. Production and crystallization of protein complexes takes approximately twelve days, from construction of vectors to harvesting and freezing crystals for data collection.