scholarly journals Multi-Step Concanavalin A Phase Separation and Early-Stage Nucleation Monitored Via Dynamic and Depolarized Light Scattering

Crystals ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 620 ◽  
Author(s):  
Hévila Brognaro ◽  
Sven Falke ◽  
Celestin Nzanzu Mudogo ◽  
Christian Betzel
Keyword(s):  
Phase Separation ◽  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Protein Interactions ◽  
Concanavalin A ◽  
Early Stage ◽  
Protein Crystallization ◽  
Cluster Formation ◽  
Macromolecular Network

Protein phase separation and protein liquid cluster formation have been observed and analysed in protein crystallization experiments and, in recent years, have been reported more frequently, especially in studies related to membraneless organelles and protein cluster formation in cells. A detailed understanding about the phase separation process preceding liquid dense cluster formation will elucidate what has, so far, been poorly understood—despite intracellular crowding and phase separation being very common processes—and will also provide more insights into the early events of in vitro protein crystallization. In this context, the phase separation and crystallization kinetics of concanavalin A were analysed in detail, which applies simultaneous dynamic light scattering and depolarized dynamic light scattering to obtain insights into metastable intermediate states between the soluble phase and the crystalline form. A multi-step mechanism was identified for ConA phase separation, according to the resultant ACF decay, acquired after an increase in the concentration of the crowding agent until a metastable ConA gel intermediate between the soluble and final crystalline phases was observed. The obtained results also revealed that ConA is trapped in a macromolecular network due to short-range intermolecular protein interactions and is unable to transform back into a non-ergodic solution.

Phase separation and toxicity of C9orf72 poly(PR) depends on alternate distribution of arginine

2021 ◽  
Vol 220 (11) ◽  
Author(s):  
Chen Chen ◽  
Yoshiaki Yamanaka ◽  
Koji Ueda ◽  
Peiying Li ◽  
Tamami Miyagi ◽  
...  
Keyword(s):  
Phase Separation ◽  
Charge Distribution ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
C9orf72 Gene ◽  
And Diffusion ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat ◽  
Liquid Liquid Phase Separation

Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

scholarly journals Applicability and Limitations in the Characterization of Poly-Dispersed Engineered Nanomaterials in Cell Media by Dynamic Light Scattering (DLS)

Materials ◽  
2019 ◽  
Vol 12 (23) ◽  
pp. 3833 ◽  
Author(s):  
Arianna Marucco ◽  
Elisabetta Aldieri ◽  
Riccardo Leinardi ◽  
Enrico Bergamaschi ◽  
Chiara Riganti ◽  
...  
Keyword(s):  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Culture Media ◽  
Raw 264.7 ◽  
Pre Treatment ◽  
The Stability ◽  
Definition Of ◽  
Cellular Tests

The dispersion protocol used to administer nanomaterials (NMs) in in vitro cellular tests might affect their toxicity. For this reason, several dispersion procedures have been proposed to harmonize the toxicological methods, allowing for the comparison of the data that were obtained by different laboratories. At the same time, several techniques and methods are available to monitor the identity of the NMs in the cell media. However, while the characterization of suspensions of engineered NMs having narrow size distribution may be easily performed, the description of aggregated NMs forming polydispersions is still challenging. In the present study, sub-micrometric/nanometric TiO2, SiO2, and CeO2 were dispersed in cell media by using two different dispersion protocols, with and without albumin (0.5%) and with different sonication procedures. Dynamic Light Scattering (DLS) was used to characterize NMs in stock solutions and culture media. Pitfalls that affect DLS measurements were identified and, guidance on a critical analysis of the results provided. The NMs were then tested for their cytotoxicity (LDH leakage) toward murine macrophages (RAW 264.7) and PMA-activated human monocytes (THP-1). As markers of pro-inflammatory response, nitric oxide (NO) and cytokine IL-1β production were measured on RAW 264.7 and THP-1 cells, respectively. The pre-treatment with albumin added to a strong sonication treatment increases the stability and homogeneity of the suspensions of nanometric samples, but not of the submicrometric-samples. Nevertheless, while TiO2 and CeO2 were non-cytotoxic in any conditions, differences in cytotoxicity, NO, and IL-1β releases were found for the SiO2, depending upon the protocol. Overall, the results suggest that there is no one-fits-all method valid for all NMs, since each class of NMs respond differently. The definition of validated procedures and parameters for the selection of the most appropriate method of dispersion for each class of NM appears to be a more efficacious strategy for the harmonization of the dispersion protocols.

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