@article {161980, title = {Structure-guided engineering of protein stability through core hydrophobicity.}, journal = {Protein Sci}, volume = {34}, year = {2025}, month = {2025 Dec}, pages = {e70360}, abstract = {

Engineering thermostable proteins is advantageous for industrial and biomedical applications, where improved thermal stability can enhance conformational integrity, prolong functional half-life, and increase catalytic efficiency at elevated temperatures. We explored modifying the length of buried hydrocarbon chains to improve protein thermal stability. By optimizing the hydrophobic core through targeted amino acid substitutions, we aimed to minimize internal voids and improve core packing. To achieve this, we developed an algorithm that replaces buried hydrophobic residues with longer or bulkier hydrophobic side chains. The algorithm calculates the free energy of unfolding (ΔG) for each substitution, selecting only significantly stabilizing configurations. Functionally important residues and contact networks were excluded from mutation to preserve protein function. We applied the method to several proteins from the beta-grasp fold family. For experimental validation, we chose NEDD8, a beta-grasp protein with poor solubility and low thermal stability. Two subtle substitutions predicted by our algorithm increased NEDD8{\textquoteright}s thermal stability by 1.7 kcal/mol and raised its melting point by 17{\textdegree}C. MD simulations and NMR spectroscopy revealed reduced conformational fluctuations and increased stabilizing interactions, such as hydrogen bonding and electrostatic contacts. Functional assays confirmed that the substitutions did not perturb NEDD8{\textquoteright}s global fold or interactions with cofactors and enzymes. These results highlight the effectiveness of tuning buried hydrophobic residues to enhance protein stability without compromising function. This strategy could serve as a general framework for designing robust therapeutic proteins and enzymes for industrial or biomedical applications.

}, keywords = {Algorithms, Amino Acid Substitution, Hydrophobic and Hydrophilic Interactions, Molecular Dynamics Simulation, NEDD8 Protein, Protein Conformation, Protein Engineering, Protein Stability, Thermodynamics}, issn = {1469-896X}, doi = {10.1002/pro.70360}, author = {Ravichandran, Aravind and Puri, Anindita and Bhate, Suhas H and Habibullah, Batul Ismail and Singh, Gajendra and Das, Ranabir} } @article {78107, title = {Monitoring protein ubiquitination and SUMOylation in real-time by NMR.}, journal = {Chem Commun (Camb)}, volume = {56}, year = {2020}, month = {2020 Jun 18}, pages = {6735-6738}, abstract = {

The covalent conjugation of ubiquitin (Ub), known as ubiquitination, is a multi-step reaction involving multiple enzymes. We report a real-time, tag-free method to monitor protein ubiquitination by NMR spectroscopy under physiological conditions. The approach is also applicable for monitoring other ubiquitin-like modifications, and the disassembly of Ub polymers.

}, issn = {1364-548X}, doi = {10.1039/d0cc02252g}, author = {Habibullah, Batul Ismail and Tripathi, Vasvi and Surana, Parag and Das, Ranabir} } @article {69319, title = {Deamidation disrupts native and transient contacts to weaken the interaction between UBC13 and RING-finger E3 ligases.}, journal = {Elife}, volume = {8}, year = {2019}, month = {2019 Oct 22}, abstract = {

The deamidase OspI from enteric bacteria deamidates a glutamine residue in the host ubiquitin-conjugating enzyme UBC13 and converts it to glutamate (Q100E). Consequently, its polyubiquitination activity in complex with the RING-finger ubiquitin ligase TRAF6 and the downstream NF-κB inflammatory response is silenced. The precise role of deamidation in silencing the UBC13/TRAF6 complex is unknown. We report that deamidation inhibits the interaction between UBC13 and TRAF6 RING-domain (TRAF6) by perturbing both the native and transient interactions. Deamidation creates a new intramolecular salt-bridge in UBC13 that competes with a critical intermolecular salt-bridge at the native UBC13/TRAF6 interface. Moreover, the salt-bridge competition prevents transient interactions necessary to form a typical UBC13/RING complex. Repulsion between E100 and the negatively charged surface of RING also prevents transient interactions in the UBC13/RING complex. Our findings highlight a mechanism wherein a post-translational modification perturbs the conformation and stability of transient complexes to inhibit protein-protein association.

}, issn = {2050-084X}, doi = {10.7554/eLife.49223}, author = {Mohanty, Priyesh and Agrata, Rashmi and Habibullah, Batul Ismail and G S, Arun and Das, Ranabir} }