Energetics of complementary side-chain packing in a protein hydrophobic core.
The energetics of complementary packing of nonpolar side chains in the hydrophobic core of a protein were analyzed by protein engineering experiments. We have made the mutations Ile----Val, Ile----Ala, and Leu----Ala in a region of the small bacterial ribonuclease barnase where the major alpha-helix packs onto the central beta-sheet. The destabilization resulting from the creation of cavities was determined by measuring the decrease in free energy of folding from reversible denaturation induced by urea, guanidinium chloride, or heat. The different methods give consistent and reproducible results. The loss in free energy of folding for the mutant proteins is 1.0-1.6 kcal/mol per methylene group removed. This exceeds by severalfold the values obtained from model experiments of the partitioning of relevant side chains between aqueous and nonpolar solvents. Much of this discrepancy arises because two surfaces are buried when a protein folds--both the amino acid side chain in question and the portions of the protein into which it packs. These experiments directly demonstrate that the interior packing of a protein is crucial in stabilizing its three-dimensional structure: the conversion of leucine or isoleucine to alanine in the hydrophobic core loses half the net free energy of folding of barnase with a concomitant decrease in yield of the expressed recombinant protein.
Kellis
J T
JT
Department of Chemistry, University of Cambridge, U.K.
Nyberg
K
K
Fersht
A R
AR
D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008433:Mathematics; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012260:Ribonucleases / Q000096:biosynthesis* / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D000596:Amino Acids; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease