Biology subjects

Ainavarapu, S. R. K.

Publications and source records attributed to Ainavarapu, S. R. K..

5 recordsLinked to original sources

bioRxiv2026

Redox-modulated bacterial deubiquitinase ElaD: Target recognition and suppression of K63-linked polyubiquitin accumulation in yeast.

Bacterial deubiquitinases (DUBs) are important virulence effectors that manipulate host ubiquitin signaling during infection. ElaD, a CE-clan DUB expressed by enterohemorrhagic Escherichia coli, preferentially cleaves K63-linked ubiquitin chains, yet its effects on conserved cellular stress responses remain poorly understood. We demonstrate that ElaD exhibits redox-dependent DUB activity in vitro. In addition, we identified the molecular basis underlying the selective recognition of substrate proteins, ubiquitin and NEDD8 by ElaD. Structural and mutational analyses reveal that, beyond the conserved catalytic site, ElaD engages ubiquitin through a combination of electrostatic and hydrophobic interactions. Using Saccharomyces cerevisiae as a heterologous model system, we show that wild-type ElaD rescues the proteotoxic stress phenotype of ubp2{Delta} yeast cells, whereas specific ElaD mutants fail to confer a similar response. Furthermore, expression of ElaD suppresses oxidative stress-induced accumulation of K63-linked polyubiquitin and may perturb stress-associated translational regulation linked to K63 ubiquitin signaling. Consequently, cells expressing ElaD exhibit altered stress adaptation and diminished fitness during prolonged oxidative stress. Collectively, these findings indicate that ElaD perturbs ubiquitin-mediated stress signaling by counteracting K63-linked ubiquitination events that support adaptive cellular responses. Our study highlights how a bacterial DUB can reprogram conserved ubiquitin-dependent pathways and exploit host ubiquitin signaling networks to modulate cellular stress responses and protein homeostasis. These findings further suggest potential host targets of bacterial DUBs during infection.

biophysics↗

bioRxiv2025

Circular permutants of azurin exhibit molten globule intermediates not observed in WT azurin

Molten globule (MG) states of proteins have been described as special types of intermediate states within the energy landscape diagram of proteins. In a cell, the MG state has functional significance compared to the native or unfolded states owing to its enhanced side-chain dynamics. In this study, we re-examined the acid-denatured state of the metalloprotein azurin and also investigated whether circular permutation (CP) of azurin can lead to modulation of the energy landscape to yield intermediate or MG states. CP is a protein engineering tool that allows the rearrangement of the secondary structural elements without perturbing the overall three-dimensional protein structure. We carried out comprehensive thermal, chemical, and pH denaturation studies to examine possible MG states in azurin WT. We additionally examined two CPs of azurin, whose energy landscapes in the metal-free and metal-bound forms have been previously characterized. One of the positions of CP is near the metal-binding loop (cpN42) and the other is within the active site of the protein (cpF114). We used thermal denaturation monitored by far-UV and near-UV CD, time-resolved fluorescence anisotropy of a buried Trp, and solvent accessibility of the same Trp residue to evaluate the presence of MG states in these proteins. WT azurin shows binding with ANS at a low pH due to ionic interaction with the fluorophore at this pH. We find that though WT azurin does not adopt an MG state, CP of azurin(s) exhibited an MG state. Our findings highlight the potential of CP to modulate protein energy landscapes and consequently the physiological functions of proteins.

biophysics↗

bioRxiv2024

Ionic Liquid-Induced Modulation of Ubiquitin Stability: The Dominant Role of Hydrophobic Interactions

Despite the widespread use of imidazolium-based ionic liquids (ILs) in biotechnology, pharmaceuticals, and green chemistry, their detailed interactions with proteins, particularly affecting structural stability, remain poorly understood. This study examines the effects of ILs on ubiquitin, a thermodynamically robust protein with a {beta}-grasp structure. We found that IL-induced destabilisation follows a consistent order with previous findings: [BF4]- > [MeSO4]- > [Cl]- for anions and [BMIM]+ > [BMPyr]+ > [EMIM]+ for cations. Through pH and ionic strength-dependent studies, we observed that hydrophobic interactions predominantly influence the stability of positively charged ubiquitin, with electrostatic interactions playing a secondary role. NMR studies identified residues impacted by [BMIM][BF4]; however, mutagenesis of these residues showed minimal changes in destabilisation, suggesting a global effect. This led us to conduct a broader empirical analysis, incorporating solvent-accessible surface area evaluations, which confirmed that hydrophobic residues are the primary drivers of stability alterations in ubiquitin, with charged residues playing a minimal role. Additionally, single-molecule force spectroscopy results indicate that imidazolium ILs decrease the unfolding barrier without altering transition state structures, offering insights into protein folding dynamics. ILs appear to modulate the stability landscape of proteins by energetically and kinetically favouring the unfolded state over the folded state. These insights offer potential strategies for the selective tuning of protein stability, which could be exploited to modulate protein-protein or protein-substrate interactions in various applications of ILs.

biophysics↗

bioRxiv2023

Liquid-liquid phase separation and aggregation of a globular folded protein SUMO1

Many studies in recent years have investigated the phenomenon of liquid-liquid phase separation (LLPS) in proteins. LLPS is reported in intrinsically disordered proteins (IDPs) or in proteins with intrinsically disordered regions (IDRs) lacking a well-defined three-dimensional structure. However, the occurrence of LLPS in folded proteins, that lacks IDRs is not widely known. It is generally assumed that the compact structure and limited flexibility of folded proteins hinder their ability to establish weak and dynamic interactions crucial for LLPS. Contrary to the prevailing understanding, we present direct evidence of rapid phase separation of a globular protein, SUMO1, occurring under crowded conditions at physiological pH and room temperature. The protein molecules in the liquid droplets undergo conformational changes with time, monitored by intrinsic tryptophan fluorescence. We also demonstrate the phase transition of SUMO1 droplets from liquid to solid state with maturation, ultimately leading to aggregation. The SUMO1 aggregates contain a significant amount of beta-sheet structure but have amorphous morphology, probed by several spectroscopic techniques (Thioflavin T fluorescence, Raman Spectroscopy, and TEM). Our findings provide insights into the behaviour of SUMO1 protein in crowded environment, albeit, the underlying mechanism is not well understood and may differ from those of IDPs. Furthermore, SUMO1 protein is known to colocalize in inclusion bodies of IDPs, and therefore, LLPS of SUMO1 may have important biological implications in neurodegenerative diseases.

biophysics↗

bioRxiv2020

Rational design of protein-specific folding modifiers

Protein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a xenonucleus, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Forster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 {+/-} 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

biophysics↗