Pathway and stability of protein folding.
We describe an experimental approach to the problem of protein folding and stability which measures interaction energies and maps structures of intermediates and transition states during the folding pathway. The strategy is based on two steps. First, protein engineering is used to remove interactions that stabilize defined positions in barnase, the RNAse from Bacillus amyloliquefaciens. The consequent changes in stability are measured from the changes in free energy of unfolding of the protein. Second, each mutation is used as a probe of the structure around the wild-type side chain during the folding process. Kinetic measurements are made on the folding and unfolding of wild-type and mutant proteins. The kinetic and thermodynamic data are combined and analysed to show the role of individual side chains in the stabilization of the folded, transition and intermediate states of the protein. The protein engineering experiments are corroborated by nuclear magnetic resonance studies of hydrogen exchange during the folding process. Folding is a multiphasic process in which alpha-helices and beta-sheet are formed relatively early. Formation of the hydrophobic core by docking helix and sheet is (partly) rate determining. The final steps involve the forming of loops and the capping of the N-termini of helices.
Fersht
A R
AR
Department of Chemistry, University of Cambridge, U.K.
Bycroft
M
M
Horovitz
A
A
Kellis
J T
JT
Matouschek
A
A
Serrano
L
L
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins; D001665:Binding Sites; D004795:Enzyme Stability; D008961:Models, Structural; D011487:Protein Conformation; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D011506:Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D014448:Tyrosine-tRNA Ligase