title: Effect of single amino acid replacements on the folding and stability of dihydrofolate reductase from Escherichia coli.
abstract: The role of the secondary structure in the folding mechanism of dihydrofolate reductase from Escherichia coli was probed by studying the effects of amino acid replacements in two alpha helices and two strands of the central beta sheet on the folding and stability. The effects on stability could be qualitatively understood in terms of the X-ray structure for the wild-type protein by invoking electrostatic, hydrophobic, or hydrogen-bonding interactions. Kinetic studies focused on the two slow reactions that are thought to reflect the unfolding/refolding of two stable native conformers to/from their respective folding intermediates [Touchette, N. A., Perry, K. M., & Matthews, C. R. (1986) Biochemistry 25, 5445-5452]. Replacements at three different positions in helix alpha B selectively alter the relaxation time for unfolding while a single replacement in helix alpha C selectively alters the relaxation time for refolding. This behavior is characteristic of mutations that change the stability of the protein but do not affect the rate-limiting step. In striking contrast, replacements in strands beta F and beta G can affect both unfolding and refolding relaxation times. This behavior shows that these mutations alter the rate-limiting step in these native-to-intermediate folding reactions. It is proposed that the intermediates have an incorrectly formed beta sheet whose maturation to the structure found in the native conformation is one of the slow steps in folding.
authors.forename: K M
authors.initials: KM
authors.lastname: Perry
authors.forename: J J
authors.initials: JJ
authors.lastname: Onuffer
authors.forename: N A
authors.initials: NA
authors.lastname: Touchette
authors.forename: C S
authors.initials: CS
authors.lastname: Herndon
authors.forename: M S
authors.initials: MS
authors.lastname: Gittelman
authors.forename: C R
authors.initials: CR
authors.lastname: Matthews
authors.forename: J T
authors.initials: JT
authors.lastname: Chen
authors.forename: R J
authors.initials: RJ
authors.lastname: Mayer
authors.forename: K
authors.initials: K
authors.lastname: Taira
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D000596:Amino Acids; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase