Folding kinetics of T4 lysozyme and nine mutants at 12 degrees C.
The kinetics of unfolding and refolding of T4 lysozyme and nine of its mutants have been investigated as a function of guanidinium chloride concentration at 12 degrees C. All show simple two-state, first-order kinetics. Two types of mutants were studied: proline-alanine interchanges and substitutions at position 3 with side chains of varying hydrophobicity. Crystal structures are available for seven of the ten proteins. The effect of mutations on the folding kinetics is more pronounced and complex than on equilibrium thermodynamics. The proteins fall into two broad kinetic classes with one class rather close to the wild type. P86A is a mutant with marked changes in kinetics but only a very small change in stability. Since the 86 position is in the middle of an alpha-helix, the indications are that the helix containing an A residue is more stable in the transition state than one containing a P residue. The other mutants are more complicated, with the refolding and unfolding rates unequally affected by the mutations. On the basis of comparisons with other investigations, we conclude that the rate-determining step in the presence of guanidinium chloride is not the same as in aqueous solution and that it most likely precedes it. The indications are that we are studying the formation of a transition intermediate which is destabilized by the denaturant and which resembles the A intermediate of the framework or molten globule models for protein folding.
Chen
B L
BL
Institute of Molecular Biology, University of Oregon, Eugene 97403-1229.
Baase
W A
WA
Nicholson
H
H
Schellman
J A
JA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D012996:Solutions; D013604:T-Phages / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics
D000596:Amino Acids; D012996:Solutions; D009113:Muramidase