Deciphering the liquid–liquid phase separation induced modulation in the structure, dynamics, and enzymatic activity of an ordered protein β-lactoglobulin

Rai, Saurabh; Pramanik, Srikrishna; Mukherjee, Saptarshi

doi:10.1039/d3sc06802a

Cited by 5 publications

(3 citation statements)

References 71 publications

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“…It is interesting to note here that these superstructures observed in the electron microscopic images are reflections of their discrete nature in the solution. This was confirmed by morphological analysis of the superstructures in the solution state by the sandwiching method . Distinctly fluorescent superstructures observed through confocal fluorescence microscopy, well matched with the obtained differential interference contrast (DIC) pattern, are evident in Figures f–h and S8.…”

supporting

confidence: 75%

Assemble–Disassemble–Reassemble Dynamics in Copper Nanocluster-Based Superstructures

Agrawal,

Rai,

Mahato

et al. 2024

J. Phys. Chem. Lett.

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supporting

confidence: 75%

Assemble–Disassemble–Reassemble Dynamics in Copper Nanocluster-Based Superstructures

Agrawal,

Rai,

Mahato

et al. 2024

J. Phys. Chem. Lett.

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“…These findings authenticate that the LLPS of trypsin in the presence of inert crowders is homotypic in nature similar to other proteins. [45][46][47][48][49]54 The mean size of trypsin condensates increases progressively upon an increase in the concentrations of trypsin at a fixed concentration of PEG (10%) (Figure 1G). This observation can be explained by considering dominant protein−protein interactions as well as extensive number of coalescence/fusion events at high trypsin concentration.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Proteins often interact with neighboring proteins through various noncovalent interactions, and inert crowders are known to promote these interactions via the excluded volume effect. [45][46][47][48][49]54 These weak but multivalent protein−protein interactions often drive the phase separation of biomolecules and stabilize the phase-separated condensates. To determine the nature of the intermolecular interactions in the present system, we first studied the influence of pH and temperature on the phase separation propensity of trypsin.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca²⁺-Mediated Activation of Esterase Activity

Patel,

Mukherjee

2024

Biomacromolecules

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The presence of Ca2+ ions is known to facilitate the activity of trypsin-like serine proteases via structural stabilization against thermal denaturation and autolysis. Herein, we report a new and hidden regulatory role of Ca2+ in the catalytic pathways of trypsin and α-chymotrypsin under physiological conditions. We discovered that macromolecular crowding promotes spontaneous homotypic condensation of trypsin via liquid–liquid phase separation to yield membraneless condensates over a broad range of concentrations, pH, and temperature, which are stabilized by multivalent hydrophobic interactions. Interestingly, we found that Ca2+ binding in the calcium binding loop reversibly regulates the condensation of trypsin and α-chymotrypsin. Spontaneous condensation effectively prevents autolysis of trypsin and preserves its native-like esterase activity for a prolonged period of time. It has also been found that phase-separated trypsin responds to Ca2+-dependent activation of its esterase activity even after 14 days of storage while free trypsin failed to do so. The present study highlights an important physiological aspect by which cells can spatiotemporally regulate the biocatalytic efficacy of trypsin-like serine proteases via Ca2+-signaling.

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Mechanistic Insights into the c-MYC G-Quadruplex and Berberine Binding inside an Aqueous Two-Phase System Mimicking Biomolecular Condensates

Pradhan,

Campanile,

Sharma

et al. 2024

J. Phys. Chem. Lett.

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We investigated the binding between the c-MYC Gquadruplex (GQ) and berberine chloride (BCl) in an aqueous twophase system (ATPS) with 12.3 wt % polyethylene glycol and 5.6 wt % dextran, mimicking the highly crowded intracellular biomolecular condensates formed via liquid−liquid phase separation. We found that in the ATPS, complex formation is significantly altered, leading to an increase in affinity and a change in the stoichiometry of the complex with respect to neat buffer conditions. Thermodynamic studies reveal that binding becomes more thermodynamically favorable in the ATPS due to entropic effects, as the strong excluded volume effect inside ATPS droplets reduces the entropic penalty associated with binding. Finally, the binding affinity of BCl for the c-MYC GQ is higher than those for other DNA structures, indicating potential specific interactions. Overall, these findings will be helpful in the design of potential drugs targeting the c-MYC GQ structures in cancer-related biocondensates.

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Deciphering the liquid–liquid phase separation induced modulation in the structure, dynamics, and enzymatic activity of an ordered protein β-lactoglobulin

Abstract: Liquid–liquid phase separation (LLPS) exhibited by an ordered protein β-lactoglobulin (β-LG) under crowded environment prompts alterations in conformational distribution leading to emergence of new promiscuous (esterase-like) activity.

Cited by 5 publications

References 71 publications

Assemble–Disassemble–Reassemble Dynamics in Copper Nanocluster-Based Superstructures

Assemble–Disassemble–Reassemble Dynamics in Copper Nanocluster-Based Superstructures

Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca²⁺-Mediated Activation of Esterase Activity

Mechanistic Insights into the c-MYC G-Quadruplex and Berberine Binding inside an Aqueous Two-Phase System Mimicking Biomolecular Condensates

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Deciphering the liquid–liquid phase separation induced modulation in the structure, dynamics, and enzymatic activity of an ordered protein β-lactoglobulin

Abstract: Liquid–liquid phase separation (LLPS) exhibited by an ordered protein β-lactoglobulin (β-LG) under crowded environment prompts alterations in conformational distribution leading to emergence of new promiscuous (esterase-like) activity.

Cited by 5 publications

References 71 publications

Assemble–Disassemble–Reassemble Dynamics in Copper Nanocluster-Based Superstructures

Assemble–Disassemble–Reassemble Dynamics in Copper Nanocluster-Based Superstructures

Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca2+-Mediated Activation of Esterase Activity

Mechanistic Insights into the c-MYC G-Quadruplex and Berberine Binding inside an Aqueous Two-Phase System Mimicking Biomolecular Condensates

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Resources

About

Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca²⁺-Mediated Activation of Esterase Activity