PMC

Simulation of DEER modulation depth versus subunit exchange time according to . The initial fraction of MM out of the total dimer concentration, [MM]₀/([MM]₀ + [NN]₀), is indicated at the right of each curve. A k₋₁ = 0.037 min⁻¹ was used in for the simulations.

Simulation of NMR cross peak intensity versus subunit exchange time for PRE experiments for a series of NN concentration fractions of the total dimers [NN]₀/([MM]₀ + [NN]₀) as indicated. (a) Simulation curves with PREs indicated by I_para/I_dia of 0.8. (b) I_para/I_dia of 0.5. (c) I_para/I_dia of 0.2. (d) I_para/I_dia of 0. k₋₁ of 0.037 min⁻¹ was used with for simulations.

Simulation of NMR cross peak intensity versus subunit exchange time for the PRE experiment using . (a) Simulation of NMR cross peak intensity decay from NN (I_dia, normalized to 1, magenta) versus mixing time, and growth from MN for three different PRE magnitudes indicated by I_para, which was normalized to I_dia (orange, I_para=1; wine, I_para=0.5; and royal, I_para=0). The olive line stands for the intensity of 0.5. (b) The summation of the NMR signal intensity (I_obs) from NN and MN. k₋₁ of 0.037 min⁻¹ was used in for the simulations.

PRE experiments and fitted curves for determination of k₋₁ values. (a) Average normalized intensity of 39 non-overlapped peaks from ten-repeated 2D SOFAST-HMQC data sets collected on 0.3 mM Dsy0195-S52C. 2D SOFAST-HMQC spectra of 0.3 mM ¹⁵N-Dsy0195-S52C (b), and of a 1:1 mixture of ¹⁵N-Dsy0195-S52C/Dsy0195-S52C-MTSL (each 0.3 mM) at exchange times of 6 (c), 70 (d), and 180 minutes (e), respectively. Three cross peaks corresponding to residues L79, Y81 and E84 are labeled, for which peak broadening from PREs is clearly evident. The final averaged, normalized cross peak intensities, and associated errors, versus exchange time from amino acids L79, Y81 and E84 were fitted and shown in (f), (g), and (h).

Time-domain DEER signals and fits of modulation depth decay curve versus mixing time to determine k₋₁ values. (a) Overlay of the scaled refocused echo intensity for five repeated DEER experiments of 0.1 mM Dsy0195-S36C-MTSL. (b) Time-domain DEER signals of 1:1 mixtures of Dsy0195-S36C-MTSL/¹⁵N-Dsy0195-S36C at a series of exchange time points indicated in order, from bottom to top, at right. Exchange took place at room temperature (293 K). The repeated DEER experiments are shown in (c). Fits of the experimental data to a single exponential decay for data in (b) and (c) are shown in (d) and (e), and the best fit of the exponential decay to the average of the modulation depth versus exchange time is shown with error bars in (f).

General strategy for measurement of rate constants for homodimer subunit exchange. (a) represents the equilibrium between MTSL-labeled protein homodimer (MM) and MTSL-labeled monomer (M). (b) represents the equilibrium between ¹⁵N-labeled protein homodimer (NN) and ¹⁵N-labeled protein monomer (N). (c) represents the equilibrium between the MTSL-/¹⁵N-labeled protein heterodimer (MN) and the individual monomer species (M) + (N). The 1:1 ratio indicates that equal concentrations of (a) and (b) are mixed in equal volumes. After mixing, the MN heterodimer depicted in (c) will be produced as the subunits undergo exchange. Under these conditions, three distinct dimer species, MM, MN, and NN, will exist in a 1:2:1 ratio at equilibrium. The k₁ and k₋₁ refer to the association and dissociation rate constants for each dimer/monomer equilibrium, respectively. DEER+/NMR+ means detectable by DEER/NMR, and DEER-/NMR- means not detectable by either technique.