Sac7d is a chromatin protein from the hyperthermophile Sulfolobus acidocaldarius that severely kinks duplex DNA with negligible change in protein structure. In previous work, the overall stability of Sac7d has been well-characterized with a global analysis of the linkage of folding, protonation, and anion binding. We extend that work here with NMR measurements of global stability as well as the distribution of stability and flexibility in the solution structure. Native state amide hydrogen exchange has been used to identify the most-protected core amide protons which exchange through global unfolding. The pH and temperature dependence of stability defined by native state exchange is in excellent agreement with the free energy surface determined by a linkage analysis of the dependence of folding on pH, salt, and temperature. These results confirm that the deltaC(P) obtained from a Kirchhoff analysis of DSC data (i.e., deltaH vs Tm) is incorrect, and an accurate description of the protein stability curve for Sac7d requires a measure of the thermodynamic contributions of protonation and anion binding. Amide hydrogen exchange, along with generalized order parameters determined by 15N relaxation data, demonstrates considerable variation in stability throughout the structure with some of the least stable regions occurring at the N- and C-termini. The most stable and inflexible region of the backbone occurs primarily in the DNA-binding beta-sheet which is responsible for bending DNA. Study holds ProTherm entries: 19584, 19585, 19586, 19587, 19588, 19589, 19590, 19591, 19592 Extra Details: Experiments were carried out on the amide group of W24 hyperthermophile; flexibility; anion binding; DNA-binding
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:51 p.m.
|Number of data points||9|
|Proteins||DNA-binding protein 7d ; DNA-binding protein 7d|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence A:MVKVKFKYKGEEKEVDTSKIKKVWRVGKMVSFTYDDNGKTGRGAVSEKDAPKELLDMLARAEREKK/B:GCGATCGC/C:GCGATCGC|