Differential effects of hydrophobic core packing residues for thermodynamic and mechanical stability of a hyperthermophilic protein.

Proteins from organisms which have adapted to environmental extremes provide excellent model systems to determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on hyperthermophilic protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal β-strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (∆GD-N) of the mutated proteins and show there is a more significant reduction for TmCSP L40A (∆∆G = 63%) than TmCSP V62A (∆∆G = 47%) as might be expected, based on the relative reduction in the size of the side chain. By contrast SMFS measures the mechanical stability (∆G*) and shows a greater reduction for TmCSP V62A (∆∆G* = 8.4%) than TmCSP L40A (∆∆G* = 2.5%). While the impact on mechanical stability is subtle, the results demonstrate the power of tuning non-covalent interactions to modulate the mechanical stability of a protein. Such understanding and control provides the opportunity to design proteins with optimized thermodynamic and mechanical properties.