title: Thermodynamic effects of multiple protein conformations on stability and DNA binding.
abstract: The side-chain conformations of amino acids in the hydrophobic core are important for protein folding and function. A previous NMR study has shown that a mutant protein of transcriptional activator c-Myb, I155L/I181L R3, has multiple conformations and increased fluctuation in comparison with the wild type. To elucidate the quantitative correlation of structural fluctuation with stability and function, we analyzed the thermodynamic effects of I155L and I181L mutations, using R2R3 that encompasses the minimum specific DNA-binding region. Circular dichroism and differential scanning calorimetry measurements showed that the mutation of I155L had little effect on stability, while the I181L mutation significantly destabilized the protein. It is noteworthy that the decreased stability resulting from the I181L mutation was mainly due to decreased enthalpy change, which is partially compensated by decreased entropy change. Isothermal titration calorimetry measurements showed that the specific DNA-binding affinity was decreased owing to the I181L mutation, which was due to decreased binding entropy change. Entropy in the folded state, which corresponds to the DNA-free state, increases due to the I181L mutation because of the increased conformational fluctuation observed in I155L/I181L mutant of R2R3 by CLEANEX-PM NMR analysis, which in turn results in decreased folding entropy and DNA-binding entropy changes.
authors.affiliation: Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Inaba
authors.forename: Harumi
authors.initials: H
authors.lastname: Fukada
authors.forename: Takahisa
authors.initials: T
authors.lastname: Ikegami
authors.forename: Masayuki
authors.initials: M
authors.lastname: Oda
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure*; D004735:Energy Transfer; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D020598:Proto-Oncogene Proteins c-myb / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D020598:Proto-Oncogene Proteins c-myb; D004247:DNA
keywords: Binding function; Conformational entropy; Protein fluctuation; Protein folding; Thermodynamics