Macromolecular Crowding-Induced Unusual Liquid–Liquid Phase Separation of Human Serum Albumin via Soft Protein–Protein Interactions

Patel, Chinmaya Kumar; Singh, Shivendra; Saini, Bhawna; Mukherjee, Tushar Kanti

doi:10.1021/acs.jpclett.2c00307

Cited by 19 publications

(85 citation statements)

References 53 publications

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“…Moreover, HRP and GOx droplets are stable in the temperature range of 4-80 ℃; however, inhibited completely at 90 ℃ (Figures 4B and S18), suggesting an upper critical solution temperature (UCST) profile. 34 The observed UCST profiles indicate a dominant role of enthalpically driven intermolecular interactions over the entropy of mixing as described previously by the Flory-Huggins theory. 46,47 Next, we tested the feasibility of LLPS in the presence of neutral salt, sodium chloride (NaCl), chaotropic salt, sodium thiocyanate (NaSCN), and an aliphatic alcohol, 1,6-hexanediol.…”

Section: Resultssupporting

confidence: 74%

“…Moreover, HRP and GOx droplets are stable in the temperature range of 4–80 °C; however, inhibited completely at 90 °C (Figures 4B and S18), suggesting an upper critical solution temperature (UCST) profile. 34 The observed UCST profiles indicate a dominant role of enthalpically driven intermolecular interactions over the entropy of mixing as described previously by the Flory–Huggins theory. 46,47…”

Section: Resultssupporting

confidence: 73%

“…30,31,34 In contrast, droplets remain intact even at high concentrations of 1,6-hexanediol (6%) and NaSCN (1 M) (Figures 4D,E, and S20), suggesting an insignificant role of hydrophobic interactions. 30,34 Taken together, our findings illustrate that droplet formation is primarily driven by multivalent electrostatic intermolecular interactions between short patches of oppositely charged polypeptide chains. On the basis of these findings, we present a mechanistic model where LLPS of enzymes via droplet formation under a crowded milieu amplifies the catalytic efficiencies of metabolic reactions way in our group to explore this phenomenon for other enzymes in a crowded milieu.…”

Section: Resultsmentioning

confidence: 99%

“…Droplet formation of both the enzymes in the presence of PEG is completely inhibited in the presence of 3 M NaCl (Figures 4C and S19), indicating the key role of strong electrostatic intermolecular interactions behind the LLPS of both the enzymes. 30,31,34 In contrast, droplets remain intact even at high concentrations of 1,6-hexanediol (6%) and NaSCN (1 M) (Figures 4D,E, and S20), suggesting an insignificant role of hydrophobic interactions. 30,34 Taken together, our findings illustrate that droplet formation is primarily driven by multivalent electrostatic intermolecular interactions between short patches of oppositely charged polypeptide chains.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Biomolecular Condensate Regulates Enzymatic Activity under Crowded Milieu: Synchronization of Liquid-Liquid Phase separation and Enzymatic Transformation

Saini

Mukherjee

2022

Preprint

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Cellular crowding plays a key role in regulating the enzymatic reactivity, which is challenging to realize in the dilute phase. However, the molecular interpretation behind this enhanced reactivity is poorly understood. Herein, using horseradish peroxidase (HRP) and glucose oxidase (GOx) cascade pair, we demonstrate for the first time that macromolecular crowding induces liquid-liquid phase separation via liquid-like droplet formation and thereby increases the intrinsic catalytic efficiencies of HRP and GOx. Our findings reveal unprecedented enhancement (18-338-fold) in the catalytic efficiency within the droplet phase relative to the bulk aqueous phase. The present discovery highlights the active role of liquid-like membraneless condensates in regulating various complex metabolic reactions under the crowded cellular environment.

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Section: Resultssupporting

confidence: 74%

Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Biomolecular Condensate Regulates Enzymatic Activity under Crowded Milieu: Synchronization of Liquid-Liquid Phase separation and Enzymatic Transformation

Saini

Mukherjee

2022

Preprint

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Macromolecular Crowding Promotes Re-entrant Liquid–Liquid Phase Separation of Human Serum Transferrin and Prevents Surface-Induced Fibrillation

Patel,

Rani,

Kumar

et al. 2023

Biomacromolecules

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Protein aggregation and inactivation upon surface immobilization are major limiting factors for analytical applications in biotechnology-related fields. Protein immobilization on solid surfaces often requires multi-step surface passivation, which is time-consuming and inefficient. Herein, we have discovered that biomolecular condensates of biologically active human serum transferrin (Tf) can effectively prevent surface-induced fibrillation and preserve the native-like conformation of phase-separated Tf over a period of 30 days. It has been observed that macromolecular crowding promotes homotypic liquid–liquid phase separation (LLPS) of Tf through enthalpically driven multivalent hydrophobic interactions possibly via the involvement of its low-complexity domain (residues 3–20) containing hydrophobic amino acids. The present LLPS of Tf is a rare example of salt-mediated re-entrant phase separation in a broad range of salt concentrations (0–3 M) solely via the involvement of hydrophobic interactions. Notably, no liquid-to-solid-like phase transition has been observed over a period of 30 days, suggesting the intact conformational integrity of phase-separated Tf, as revealed from single droplet Raman, circular dichroism, and Fourier transform infrared spectroscopy measurements. More importantly, we discovered that the phase-separated condensates of Tf completely inhibit the surface-induced fibrillation of Tf, illustrating the protective role of these liquid-like condensates against denaturation and aggregation of biomolecules. The cell mimicking compact aqueous compartments of biomolecular condensates with a substantial amount of interfacial water preserve the structure and functionality of Tf. Our present study highlights an important functional aspect of biologically active protein condensates and may have wide-ranging implications in cell physiology and biotechnological applications.

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Kinetic Study of the Esterase-like Activity of Albumin following Condensation by Macromolecular Crowding

Lamy,

Bullier-Marchandin,

Pointel

et al. 2024

Biomacromolecules

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The ability of bovine serum albumin (BSA) to form condensates in crowded environments has been discovered only recently. Effects of this condensed state on the secondary structure of the protein have already been unraveled as some aging aspects, but the pseudoenzymatic behavior of condensed BSA has never been reported yet. This article investigates the kinetic profile of para-nitrophenol acetate hydrolysis by BSA in its condensed state with poly(ethylene) glycol (PEG) as the crowding agent. Furthermore, the initial BSA concentration was varied between 0.25 and 1 mM which allowed us to modify the size distribution, the volume fraction, and the partition coefficient (varying from 136 to 180). Hence, the amount of BSA originally added was a simple way to modulate the size and density of the condensates. Compared with dilute BSA, the initial velocity (v i ) with condensates was dramatically reduced. From the Michaelis−Menten fits, the extracted Michaelis constant K m and the maximum velocity V max decreased in control samples without condensates when the BSA concentration increased, which was attributed to BSA self-oligomerization. In samples containing condensates, the observed v i was interpreted as an effect of diluted BSA remaining in the supernatants and from the condensates. In supernatants, the crowding effect of PEG increased the k cat and catalytic efficiency. Last, V max was proportional to the volume fraction of the condensates, which could be controlled by varying its initial concentration. Hence, the major significance of this article is the control of the size and volume fraction of albumin condensates, along with their kinetic profile using liquid−liquid phase separation.

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Macromolecular Crowding-Induced Unusual Liquid–Liquid Phase Separation of Human Serum Albumin via Soft Protein–Protein Interactions

Cited by 19 publications

References 53 publications

Biomolecular Condensate Regulates Enzymatic Activity under Crowded Milieu: Synchronization of Liquid-Liquid Phase separation and Enzymatic Transformation

Biomolecular Condensate Regulates Enzymatic Activity under Crowded Milieu: Synchronization of Liquid-Liquid Phase separation and Enzymatic Transformation

Macromolecular Crowding Promotes Re-entrant Liquid–Liquid Phase Separation of Human Serum Transferrin and Prevents Surface-Induced Fibrillation

Kinetic Study of the Esterase-like Activity of Albumin following Condensation by Macromolecular Crowding

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