On the utility of microfluidic systems to study protein interactions: advantages, challenges, and applications

Watkin, Serena A.J.; Bennie, Rachel Z; Gilkes, J.M.; Nock, Volker; Pearce, F. Grant; Dobson, Renwick C. J.

doi:10.1007/s00249-022-01626-9

Cited by 6 publications

(5 citation statements)

References 86 publications

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“…To further evaluate stabilizing or destabilizing effects of amino acid substitutions on the interaction between LC3B and phosphorylated optineurin, we established an approach based on Microfluidic Diffusional Sizing (MDS) 48,49,102 to study the binding affinity between GST-tagged LC3B and different peptide variants corresponding to the 169-185 residues of the full-length OPTN. We have purchased FITC-labeled peptides and titrated them with increasing concentrations of GST-LC3B to follow changes in hydrodynamic radius that could be fitted to obtain a Kd value for each peptide.…”

Section: Resultsmentioning

confidence: 99%

“…We here show how Alphafold-based models for interaction 39,40 , biomolecular simulations [41][42][43] and other structure-based computational approaches [44][45][46][47] , together with experimental biophysical data based on Microfluidic Diffusional Sizing 48,49 are effective to study the modulation by phosphorylation of SLiMs focusing on the OPTNLIR-LC3B complex and its disease-related mutations. In a broader context, our work provides a computational framework with in vitro experimental validation to apply to other LIRs or SLiMs regulated by phosphorylation.…”

Section: Introductionmentioning

confidence: 99%

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Decoding Phospho-Regulation and Flanking Regions in Autophagy-Associated Short Linear Motifs: A Case Study of Optineurin-LC3B Interaction

Antonescu,

Utichi,

Sora

et al. 2023

Preprint

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Short Linear Motifs (SLiMs) are pivotal in mediating interactions between intrinsically disordered proteins and their binding partners. SLiMs exhibit sequence degeneracy and undergo regulation by post-translational modifications, including phosphorylation. These modifications can either augment or attenuate the binding affinities for a SliM and its binding partner. In this study, we set out to integrate biomolecular simulations, in silico high-throughput mutational scans, and biophysical measurements to elucidate the structural details of phospho-regulation in a class of SLiMs crucial for autophagy, known as LC3 interacting regions (LIRs). As a case study, we investigated the interaction between optineurin and LC3B, a system extensively characterized by experiments. We confirmed the robustness of our computational approach through the comparison with the available experimental data. Furthermore, we unveiled the previously unexplored role of the N-terminal flanking region upstream of the LIR core motif. Our study offers an atom-level perspective on the structural mechanisms and conformational alterations induced by phosphorylation in optineurin and LC3B recognition. Additionally, we assessed the impact of disease-related mutations on optineurin, accounting for different functional features.Notably, we established an approach based on Microfluidic Diffusional Sizing to investigate the binding affinity of SLiMs to target proteins, enabling precise measurements of the dissociation constant for a selection of variants from the in silico mutational screening. Overall, our work provides a versatile toolkit to characterize other LIR-containing proteins and their modulation by phosphorylation or other phospho-regulated SLiMs, thereby advancing the understanding of important cellular processes.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Decoding Phospho-Regulation and Flanking Regions in Autophagy-Associated Short Linear Motifs: A Case Study of Optineurin-LC3B Interaction

Antonescu,

Utichi,

Sora

et al. 2023

Preprint

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“…Future research must focus on strategies to enhance throughput while maintaining the precision of microfluidic techniques. [51][52][53]…”

Section: Scalability Challengesmentioning

confidence: 99%

“…The intricate nature of microfluidic devices and the need for specialized equipment limit the scalability of nanogel synthesis. Future research must focus on strategies to enhance throughput while maintaining the precision of microfluidic techniques 51–53 …”

Section: Microfluidics: a Platform For Precision Engineeringmentioning

confidence: 99%

Controlled polymerization in microfluidics: Advancement in nanogel synthesis

Pandey,

Sharma,

Sandhu

et al. 2023

SPE Polymers

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This comprehensive review provides a detailed examination of controlled polymerization in the development of nanogels‐based microfluidic devices. Here, we have explored the integration of atom transfer radical polymerization (ATRP) and reversible addition‐fragmentation chain transfer (RAFT) techniques within microfluidic platforms for the synthesis of nanogels. The synergistic combination of ATRP and RAFT with microfluidics has emerged as a powerful tool for precise control over polymerization reactions, enabling the fabrication of well‐defined, multifunctional nanogels with tailored properties. The review begins with a thorough introduction to the principles of ATRP and RAFT, highlighting their respective advantages in controlled radical polymerization. Subsequently, it elucidates the principles of microfluidics and its profound impact on polymerization processes. The merging of these techniques enables precise control over reaction kinetics, monomer conversions, and molecular weight distributions, facilitating the synthesis of nanogels with unprecedented precision and reproducibility. Furthermore, this review delves into the chemical mechanisms underlying ATRP and RAFT reactions in microfluidic environments, emphasizing the role of various initiators, catalysts, and chain transfer agents. Special attention is given to the impact of microscale flow conditions on reaction kinetics and polymer structure. In addition, the review discusses emerging trends in the field, such as the incorporation of novel monomers and the exploration of environmentally benign reaction conditions.Highlights The controlled polymerization is the mechanism by which we can obtain tailormade material. The nanogel synthesis must ensure the gelation to be in confined dimensions here microfluidics plays key role.

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“…Here, we provide a detailed description of the phase behavior of a polyelectrolytic ELP in different solution conditions and strive to interpret the results through a post-reductionist lens. , This perspective acknowledges the full complexity of weak surface interactions, spatiotemporal partitioning, and solution environment in examining protein function. , Dynamic light scattering (DLS) serves as our primary technique, surpassing turbidity measurements to offer valuable physical insights into protein dynamics, assembly, and microscale biomolecular condensation . The intricate interplay of influences on ELP LCST behavior is ultimately distilled in the rapid and accurate quantification of microscopic diffusion.…”

Section: Introductionmentioning

confidence: 99%

Understanding the Phase Behavior of a Multistimuli-Responsive Elastin-like Polymer: Insights from Dynamic Light Scattering Analysis

Swanson,

Arnold,

Curley

et al. 2024

J. Phys. Chem. B

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Elastin-like polymers are a class of stimuliresponsive protein polymers that hold immense promise in applications such as drug delivery, hydrogels, and biosensors. Yet, understanding the intricate interplay of factors influencing their stimuli-responsive behavior remains a challenging frontier. Using temperature-controlled dynamic light scattering and zeta potential measurements, we investigate the interactions between buffer, pH, salt, water, and protein using an elastin-like polymer containing ionizable lysine residues. We observed the elevation of transition temperature in the presence of the common buffering agent HEPES at low concentrations, suggesting a "salting-in" effect of HEPES as a cosolute through weak association with the protein.Our findings motivate a more comprehensive investigation of the influence of buffer and other cosolute molecules on elastin-like polymer behavior.

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On the utility of microfluidic systems to study protein interactions: advantages, challenges, and applications

Cited by 6 publications

References 86 publications

Decoding Phospho-Regulation and Flanking Regions in Autophagy-Associated Short Linear Motifs: A Case Study of Optineurin-LC3B Interaction

Decoding Phospho-Regulation and Flanking Regions in Autophagy-Associated Short Linear Motifs: A Case Study of Optineurin-LC3B Interaction

Controlled polymerization in microfluidics: Advancement in nanogel synthesis

Understanding the Phase Behavior of a Multistimuli-Responsive Elastin-like Polymer: Insights from Dynamic Light Scattering Analysis

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