@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 {22020, title = {Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.}, journal = {Biochemistry}, volume = {56}, year = {2017}, month = {2017 Aug 08}, pages = {4053-4063}, abstract = {

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

}, keywords = {Amino Acid Substitution, Deuterium Exchange Measurement, Energy Transfer, Guanidine, Indicators and Reagents, Kinetics, Menispermaceae, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Plant Proteins, Protein Conformation, Protein Denaturation, Protein Folding, Protein Stability, Thermodynamics}, issn = {1520-4995}, doi = {10.1021/acs.biochem.7b00367}, author = {Malhotra, Pooja and Jethva, Prashant N 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} }