Fig 1.
An NMR spectrum and the native three-dimensional structure of GroES.
(A) An HSQC spectrum of 15N-labeled GroES in 90% H2O/10% D2O at pH 6.5 and 25°C; and the backbone structures of heptameric GroES (B) and the GroES monomer (C); the flexible mobile loop (residues 17–34), Ala97 and Asn51 are shown in red. In (C), three residues (Ile25, Val26 and Leu27) are shown in a space-filling model. The figures in (B) and (C) were prepared using the GroES portion of the GroEL/GroES/ADP complex (PDB code: 1AON), and drawn by PyMOL (DeLano Scientific).
Fig 2.
HSQC spectra of 15N-labeled GroES as a function of γ (= [L]t/[P]t).
The spectra were measured in the presence of 3.0 mM ADP at pH 7.5 and 25°C. [P]t (= 21.4 μM) was kept constant.
Fig 3.
Apparent NMR titration curves of the 11 residues (Glu18, Lys20, Ser21, Ala22, Gly23, Gly24, Ile25, Leu27, Thr28, Gly29 and Asn51) of 15N-labeled GroES titrated with SR1.
The titration was carried out in the presence of 3.0 mM ADP at pH 7.5 and 25°C. [P]t (= 21.4 μM) was kept constant.
Fig 4.
Binding isotherms of 15N-labeled GroES titrated with SR1 at three different temperatures.
The experiments were carried out in the presence of 3.0 mM ADP at pH 7.5, at 20°C (A), 25°C (B) and 30°C (C). [P]t (= 21.4 μM) was kept constant. The solid lines are the theoretical curves best fit to the experimental data using Eq 6.
Table 1.
The Kb values between GroES and SR1.
Fig 5.
The van't Hoff plot for the GroES–SR1 binding reaction in the presence of 3.0 mM ADP.
The solid line indicates the theoretically best fit to the following equation: ln Kb = a + b(1/T), where a and b are constant, fitting parameters. The best-fit parameter values obtained by the least-squares analysis were a = 26.3±1.2 and b = (−4.23±0.58)×103 K. The ΔH and ΔS for the GroES–SR1 binding are given by: ΔH = −bR, and ΔS = aR, where R is the gas constant.
Fig 6.
A binding isotherm measured by the cross-peak intensities of 15N-labeled GroES titrated with SR1 in the presence of 3 mM ATP.
The titration was carried out at pH 7.5 and 25°C. [P]t (= 21.4 μM) was kept constant. The solid line is the theoretical curve best fit to the experimental data using Eq 6.