Pressure- and temperature-induced unfolding studies: thermodynamics of core hydrophobicity and packing of ribonuclease A.
In this work we demonstrate that heat and pressure induce only slightly different energetic changes in the unfolded state of RNase A. Using pressure and temperature as denaturants on a significant number of variants, and by determining the free energy of unfolding at different temperatures, we estimated the stability of variants unable to complete the unfolding transition owing to the experimental conditions required for pressure experiments. The overall set of results allowed us to map the contributions to stability of the hydrophobic core residues of RNase A, with the positions most critical for stability being V54, V57, I106 and V108. We also show that the stability differences can be attributed to both hydrophobic interactions and packing density with an equivalent energetic magnitude. The main hydrophobic core of RNase A is tightly packed, as shown by the small-to-large and isosteric substitutions. In addition, we found that large changes in the number of methylene groups have non-additive positive stability interaction energies that are consistent with exquisite tight core packing and rearrangements of van der Waals' interactions in the protein interior, even after drastic deleterious substitutions.
Font
Josep
J
Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi s/n, E-17071 Girona, Spain.
Benito
Antoni
A
Torrent
Joan
J
Lange
Reinhard
R
Ribó
Marc
M
Vilanova
Maria
M
D004795:Enzyme Stability; D057927:Hydrophobic and Hydrophilic Interactions; D008697:Methane / Q000737:chemistry; D011312:Pressure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics
D012259:Ribonuclease, Pancreatic; D008697:Methane