@article {155540, title = {Ionic Liquid-Induced Modulation of Ubiquitin Stability: The Dominant Role of Hydrophobic Interactions.}, journal = {Langmuir}, volume = {41}, year = {2025}, month = {2025 Mar 11}, pages = {5823-5837}, abstract = {

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 β-grasp structure. We found that IL-induced destabilization follows a consistent order with previous findings: [BMIM] \> [BMPyr] \> [EMIM] for cations and [BF] \> [MeSO] \> [Cl] for anions. 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][BF]; however, site-directed mutagenesis of these residues showed minimal changes in destabilization, 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 lower the unfolding barrier without altering the transition state structure, offering insights into protein folding dynamics. ILs appear to modulate the stability landscape of proteins by energetically and kinetically favoring 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.

}, keywords = {Hydrogen-Ion Concentration, Hydrophobic and Hydrophilic Interactions, Imidazoles, Ionic Liquids, Protein Stability, Thermodynamics, Ubiquitin}, issn = {1520-5827}, doi = {10.1021/acs.langmuir.4c03886}, url = {https://pubmed.ncbi.nlm.nih.gov/40065704/}, author = {Shrivastava, Aditya and Kamma, Harika and Das, Ranabir and Ainavarapu, Sri Rama Koti} } @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 {28252, title = {The G126V Mutation in the Mouse Prion Protein Hinders Nucleation-Dependent Fibril Formation by Slowing Initial Fibril Growth and by Increasing the Critical Concentration.}, journal = {Biochemistry}, volume = {56}, year = {2017}, month = {2017 11 07}, pages = {5931-5942}, abstract = {

The middle disordered hydrophobic region of the prion protein plays a critical role in conformational conversion of the protein, with pathogenic as well as protective mutations being localized to this region. In particular, it has been shown that the G127V mutation in this region of the human prion protein (huPrP) is protective against the spread of prion disease, but the mechanism of protection remains unknown. In this study, quantitative analyses of the kinetics of fibril formation by wild-type mouse prion protein (moPrP) and G126V moPrP (equivalent to G127V huPrP) reveal important differences: the critical concentration is higher, the lag phase is longer, and the initial effective rate constant of fibril growth is slower for the mutant variant. The study offers a simple biophysical explanation for why the G127V mutation in huPrP would be protective in humans: the \~{}5-fold increase in critical concentration caused by the mutation likely results in the critical concentration (below which fibril formation cannot occur) being higher that the concentration of the protein present in and on cells in vivo.

}, keywords = {Animals, Humans, Hydrophobic and Hydrophilic Interactions, Kinetics, Mice, Models, Theoretical, Mutation, Missense, Polymerization, Prion Proteins, PrP 27-30 Protein}, issn = {1520-4995}, doi = {10.1021/acs.biochem.7b00894}, author = {Sabareesan, Ambadi Thody and Udgaonkar, Jayant B} } @article {1027, title = {Rise of the Helix from a Collapsed Globule during the Folding of Monellin.}, journal = {Biochemistry}, volume = {54}, year = {2015}, month = {2015 Sep 1}, pages = {5356-65}, abstract = {

Early kinetic intermediates observed during the folding of many proteins are invariably compact and appear to possess some secondary structure. Consequently, it has been difficult to understand whether compaction drives secondary structure formation or secondary structure formation facilitates compaction during folding. In this study of the folding of single-chain monellin, it is shown that a kinetic molten globule (MG) is populated at 2 ms of folding. Far-UV circular dichroism (CD) measurements show that the kinetic MG is devoid of any helical structure even under the most stabilizing folding conditions. Multisite fluorescence resonance energy transfer (FRET) measurements show that the kinetic MG is compact with different segments having contracted to different extents. It is shown that the sequence segment that goes on to form the sole helix in the native protein is fully collapsed in the kinetic MG. This segment expands to accommodate the helix as the kinetic MG folds further to the native state, while other segments of the protein contract. Helix formation starting from the kinetic MG is shown to occur in multiple kinetic steps, whether measured by far-UV CD or by FRET.

}, keywords = {Amino Acid Substitution, Circular Dichroism, Cysteine, Fluorescence Resonance Energy Transfer, Hydrophobic and Hydrophilic Interactions, Kinetics, Models, Molecular, Mutagenesis, Site-Directed, Plant Proteins, Protein Conformation, Protein Folding, Protein Stability, Protein Structure, Secondary, Spectrometry, Fluorescence}, issn = {1520-4995}, doi = {10.1021/acs.biochem.5b00730}, author = {Goluguri, Rama Reddy and Udgaonkar, Jayant B} }