Sengupta Rituparna, Capp Michael W, Shkel Irina A, Record M Thomas
Program in Biophysics, University of Wisconsin-Madison, Madison, WI 53706, USA.
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Nucleic Acids Res. 2017 Dec 15;45(22):12671-12680. doi: 10.1093/nar/gkx862.
Significant, otherwise-unavailable information about mechanisms and transition states (TS) of protein folding and binding is obtained from solute effects on rate constants. Here we characterize TS for lac repressor(R)-lac operator(O) binding by analyzing effects of RO-stabilizing and RO-destabilizing solutes on association (ka) and dissociation (kd) rate constants. RO-destabilizing solutes (urea, KCl) reduce ka comparably (urea) or more than (KCl) they increase kd, demonstrating that they destabilize TS relative to reactants and RO, and that TS exhibits most of the Coulombic interactions between R and O. Strikingly, three solutes which stabilize RO by favoring burial/dehydration of amide oxygens and anionic phosphate oxygens all reduce kd without affecting ka significantly. The lack of stabilization of TS by these solutes indicates that O phosphates remain hydrated in TS and that TS preferentially buries aromatic carbons and amide nitrogens while leaving amide oxygens exposed. In our proposed mechanism, DNA-binding-domains (DBD) of R insert in major grooves of O pre-TS, forming most Coulombic interactions of RO and burying aromatic carbons. Nucleation of hinge helices creates TS, burying sidechain amide nitrogens. Post-TS, hinge helices assemble and the DBD-hinge helix-O-DNA module docks on core repressor, partially dehydrating phosphate oxygens and tightening all interfaces to form RO.
通过溶质对速率常数的影响，可以获得有关蛋白质折叠和结合机制及过渡态（TS）的重要且其他方式难以获得的信息。在此，我们通过分析稳定RO和破坏RO的溶质对结合速率常数（ka）和解离速率常数（kd）的影响，来表征乳糖阻遏物（R）-乳糖操纵子（O）结合的过渡态。破坏RO的溶质（尿素、KCl）降低ka的程度相当（尿素）或大于（KCl）它们增加kd的程度，这表明相对于反应物和RO，它们使过渡态不稳定，并且过渡态呈现出R与O之间的大部分库仑相互作用。引人注目的是，三种通过促进酰胺氧和阴离子磷酸氧的埋藏/脱水来稳定RO的溶质均降低kd，而对ka没有显著影响。这些溶质对过渡态缺乏稳定作用，表明O磷酸盐在过渡态中保持水合状态，并且过渡态优先埋藏芳香族碳和酰胺氮，同时使酰胺氧暴露在外。在我们提出的机制中，R的DNA结合结构域（DBD）在过渡态前插入O的大沟中，形成RO的大部分库仑相互作用并埋藏芳香族碳。铰链螺旋的成核产生过渡态，埋藏侧链酰胺氮。过渡态后，铰链螺旋组装，DBD-铰链螺旋-O-DNA模块停靠在核心阻遏物上，部分脱水磷酸氧并收紧所有界面以形成RO。
