Biology subjects

Mukherjee, T. K.

Publications and source records attributed to Mukherjee, T. K..

3 recordsLinked to original sources

bioRxiv2024

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

The presence of Ca2+ ions is known to facilitates the biocatalytic 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 -chymotrypsin under physiological conditions. We discovered that macromolecular crowding promotes spontaneous homotypic condensation of native trypsin via liquid-liquid phase separation to yield membraneless condensates/droplets in a broad range of concentrations, pH, and temperature. These condensates are stabilized by multivalent hydrophobic interactions between short patches of hydrophobic residues. Importantly, no liquid-to-solid-like phase transition has been observed over a period of 14 days, indicating the structural intrigrity of phase-separated trypsin within the droplets. Structural insights revealed minimal conformational perturbation of trypsin upon phase separation. Interestingly, we found that Ca2+ binding in the calcium binding loop reversibly regulates the biomolecular condensation of trypsin and -chymotrypsin. While Ca2+-bound trypsin are ineffective to undergo LLPS to form condensate, its removal facilitates condensation under similar experimental conditions. More importantly, we show that biomolecular condensation effectively prevents autolysis of trypsin at physiological conditions and preserve its native-like esterase activity over a period of 14 days, whereas free trypsin loses 86% of its initial activity. In addition, it has 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. Our findings indicate that biomolecular condensates of trypsin and trypsin-like serine proteases act as storage media to prevent autolysis and premature activation, and at the same time preserve their native-like active conformations. The present study highlights an important physiological aspect of biomolecular condensates of trypsin-like serine proteases by which cells can spatio-temporally regulate their biocatalytic efficacy via Ca2+-signalling.

biochemistry↗

bioRxiv2023

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

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 (residue 3-20) containing hydrophobic amino acids. The present LLPS of Tf is a rare example of salt-mediated reentrant 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 aqueous compartments of biomolecular condensates with a substantial amount of interfacial water preserve the structure and functionality of biomolecules. 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.

biochemistry↗

bioRxiv2022

Liquid-Liquid Phase Separation Regulates Enzymatic Activity

Cellular crowding plays a key role in regulating the enzymatic reactivity in physiological conditions, which is challenging to realize in the dilute phase. Enzymes drive a wide range of complex metabolic reactions with high efficiency and selectivity under extremely heterogeneous and crowded cellular environments. However, the molecular interpretation behind the enhanced enzymatic reactivity under a crowded milieu 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 (LLPS) via the formation of liquid-like condensates/droplets and thereby increases the intrinsic catalytic efficiencies of HRP and GOx. Both these enzymes undergo crowding induced homotypic LLPS via enthalpically driven multivalent electrostatic as well as hydrophobic interactions. Using a set of kinetic and microscopic experiments, we show that precise synchronization of spontaneous LLPS and enzymatic transformations is key to realize the enhanced enzymatic activity under the crowded environments. Our findings reveal an unprecedented enhancement (91-205-fold) in the catalytic efficiency (kcat/Km) of HRP at pH 4.0 within the droplet phase relative to that in the bulk aqueous phase in the presence of different crowders. In addition, we have shown that other enzymes also undergo spontaneous LLPS under macromolecular crowding, signifying the generality of this phenomenon under the crowded environments. More importantly, coalescence driven highly regulated GOx/HRP cascade reactions within the fused droplets have been demonstrated with enhanced activity and specificity under the crowded environments. The present discovery highlights the active role of membraneless condensates in regulating the enzymatic efficacy for complex metabolic reactions under the crowded cellular environments and may find significant importance in the field of biocatalysis.

biochemistry↗