Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped-flow circular dichroism.
Kinetics of refolding and unfolding of staphylococcal nuclease and its six mutants, each carrying single or double amino acid substitutions, are studied by stopped-flow circular dichroism measurements. A transient kinetic intermediate formed within 10 ms after refolding starts possesses a substantial part of the N-domain core beta-structure, whereas helices are formed at the later stages. The structure of the kinetic intermediate is less organized than the structure that is known to be formed by a nuclease 1-136 fragment. Only the refolding kinetics are affected by the mutations in all the mutants except two in which the mutations have changed the native structure. From this result and also from the locations of the mutation sites, the major N-terminal domain of the nuclease in the transition state of folding has a structure nearly identical to the native one. On the other hand, the minor C-terminal domain has previously been shown to be still disorganized in the transition state. The effects of the amino acid substitutions on the stability of the native and the transition states are in good agreement with the changes in the hydration free energy, expected for the corresponding amino acid replacements in the unfolded polypeptide. Since side chains of all the mutated residues are not accessible to solvent in the native structure, the result suggests that it is the unfolded state that is mainly affected by the mutations.
Kalnin
N N
NN
Department of Physics, School of Science, University of Tokyo, Japan.
Kuwajima
K
K
D002942:Circular Dichroism; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D014508:Urea / Q000494:pharmacology
D011994:Recombinant Proteins; D014508:Urea; D008836:Micrococcal Nuclease