Abstract
We studied the interaction between GroES and a single-ring mutant (SR1) of GroEL by the NMR titration of 15N-labeled GroES with SR1 at three different temperatures (20, 25 and 30°C) in the presence of 3 mM ADP in 100 mM KCl and 10 mM MgCl2 at pH 7.5. We used SR1 instead of wild-type double-ring GroEL to precisely control the stoichiometry of the GroES binding to be 1:1 ([SR1]:[GroES]). Native heptameric GroES was very flexible, showing well resolved cross peaks of the residues in a mobile loop segment (residue 17–34) and at the top of a roof hairpin (Asn51) in the heteronuclear single quantum coherence spectra. The binding of SR1 to GroES caused the cross peaks to disappear simultaneously, and hence it occurred in a single-step cooperative manner with significant immobilization of the whole GroES structure. The binding was thus entropic with a positive entropy change (219 J/mol/K) and a positive enthalpy change (35 kJ/mol), and the binding constant was estimated at 1.9×105 M−1 at 25°C. The NMR titration in 3 mM ATP also indicated that the binding constant between GroES and SR1 increased more than tenfold as compared with the binding constant in 3 mM ADP. These results will be discussed in relation to the structure and mechanisms of the chaperonin GroEL/GroES complex.
Highlights
We carried out NMR titrations of 15N-labeled GroES with SR1 in the presence of 3.0 mM ADP at three different temperatures, 20, 25 and 30 ̊C, at pH 7.5, and the 1H–15N heteronuclear single quantum coherence (HSQC) spectra of GroES at different molar ratios (γ) of SR1 to GroES, γ (= [L]t/[P]t) = 0, 0.2, 0.5, 1.0, 1.5 and 2.0, at 25 ̊C are shown in Fig 2, where [L]t and [P]t represent the total molar concentration of the ligand added to the GroES solution and the total molar concentration of the protein after addition of SR1, respectively, and [P]t was set constant (21.4 μM) throughout
Θ is represented by the volume ratio as Θ = (Vt − V)/Vt, where V is the volume of a cross peak and Vt is the initial volume of the cross peak in the absence of SR1
Native heptameric GroES is a highly flexible protein, and the amide proton signals of the flexible mobile loop and Asn51 at the top of the roof hairpin loop and Ala97 at the C terminus were well resolved in the 1H–15N HSQC spectrum (Fig 1(A))
Summary
Chaperonin complex formation of the Institute for Molecular Science, and by the Okazaki ORION project of the Okazaki Institute for Integrative Bioscience. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
