PMC

Dynamic isomerization of duplex DNA containing two C-Hg^II-T base pairs. a Dynamic changes in local metal ion coordination. Rate constants of interconversion were determined by plotting changes in cross peaks (NH₂ and NH) and exchange cross peaks (NH₂' and NH') observed in z-z exchange [¹⁵N,¹H]-HSQC spectrum as a function of different delay times. Rate constants of interconversion were determined by global fitting of integrated peak volumes versus exchange delay times using Supplementary Equations -, where R₁, k₁, and k_-1 were constrained as being equal for all four curves with the assumption that all proton- and nitrogen atoms have the same relaxation rate (R₁) and that the NH proton is converted equally to both NH₂ protons during the exchange. b Representative example (G9H1') of duplex interconversion rate constants as determined by monitoring changes in exchange cross peaks and exchange-mediated NOE cross peaks in [¹H,¹H]-NOESY spectra with various mixing times (t_m). At longer mixing times, a decrease in signal intensity was observed due to auto relaxation (R₁). The sum of interconversion rate constants (k₁ + k_-1) was determined by fitting changes in cross peak intensity vs mixing time (t_m) (Supplementary Equation ). The individual rate constants of interconversion (k₁ and k_-1) were determined by fitting (Supplementary Equation ) cross peak intensities normalized by the intensities of diagonal peaks versus mixing times. See Supplementary Figs. , for [¹H,¹H]-NOESY spectra, and Supplementary Figs. – for the same types of analyses at other positions. c Summary of major- and minor duplex structures, structures of the metallo base pairs they contain, and the rate constants of their exchange. Rate constants values are given as mean ± standard deviation from 5 exchange cross peaks and exchange-mediated NOE cross peaks. The Hg^II-ligand complexes present in the major species are net positive (+1), whereas those of the minor species are neutral. The combined contributions of more favourable electrostatic interactions and near-canonical B-form helical structure present in the major species likely outweigh the stronger ligand–metal–ligand binding interactions present in the minor duplex

Example speed selection protocol for participants with chronic stroke. a Metabolic rate used to find the time delay in the body’s response time. The baseline metabolic rate was the average rate between minutes two and three. Data were fit with linear regression between minutes four to six. The time the regression crossed the 5% increase in metabolic rate was found. The time delay was the difference between the threshold crossing and the onset of the treadmill speed increase. b Linear regressions on heart rate data to find the time when heart rate reaches the 78% maximum heart rate threshold. The time delay was subtracted from the time the heart rate regression crossed the threshold. The heart rate did not cross the threshold in this participant. c Exponential model fit between minutes two to nine on respiratory exchange ratio. The threshold for respiratory exchange ratio was the larger of 0.8 respiratory exchange ratio or a 5% increase from baseline respiratory exchange ratio. The time delay was subtracted from the time the exponential fit crossed the threshold. d Normalized treadmill speed throughout the protocol to map threshold times to speed. 75% of the slower speed between respiratory exchange ratio and heart rate was the speed selected for participants with chronic stroke

RhoA nucleotide exchange in the presence of DH-PH^PRG monitored by NMR. A, schematic of RhoA nucleotide exchange mediated by DH-PH^PRG. ¹⁵N-labeled RhoA (blue, 20.4 kDa) with GDP and Mg²⁺ (purple) bound is in an inactive state. DH-PH^PRG (green, 43 kDa) interacts with residues in the RhoA switch regions, promoting the release of GDP. Subsequently, GTP binds RhoA, and the GEF is released to produce the activated form of RhoA. B, kinetics of RhoA-GDP nucleotide exchange to GTP (green dashed line) and GTPγS (blue) measured by the real-time NMR assay. A theoretical GTP exchange curve corrected for hydrolysis (see supplemental material) is also presented (green solid line) Error bars indicate S.D. for values reported by multiple peaks. C, snapshots of ¹H-¹⁵N HSQC spectra during the 75-min time course of RhoA-GDP to GTPγS nucleotide exchange, in the presence of DH-PH^PRG (6 nm). Black and blue boxes indicate the positions of the RhoA-GDP and RhoA-GTPγS cross peaks, respectively, for Gly-14 and Ser-73. D, RhoA-GDP to GTPγS nucleotide exchange, with increasing concentration of DH-PH^PRG. The intrinsic nucleotide exchange rate is shown in black. Nucleotide exchange rates k (10⁻⁴ min⁻¹) are 5.5, 19, 24, 39, 130, 267, 371, and 1,179 for GEF concentrations of 0, 0.36, 0.51, 0.9, 1.8, 4, 6, 25 nm, respectively. Lower panel, the hyperbolic (green curve) dependence, on GEF concentration, of the nucleotide exchange rate is indicative of a two-step binding model.

Individual intensity trajectories for 300 ε and τ foci were simulated in MATLAB 2014b. The simulation allows us to set k_on and k_off (in units of frames^–1) for ε exchanging into Pol III*, and k_on and k_off for Pol III* exchanging into the replisome. By changing these rate constants, we can simulate different exchange mechanisms. The black line represents the average cross-correlation function for Pol III* exchange (both ε and τ). Here, k_on and k_off for Pol III* were set to 0.01 and the rate constants for ε were set to k_on = 1 and k_off <<1 to simulate stable binding of core to Pol III*. A clear positive peak can be seen. The green line represents the average cross-correlation function for simulated trajectories without any exchange. Here, k_on = 1 and k_off<<1 for all rate constants, to simulate stable binding to the replisome. In this case, there is no positive cross correlation. The gray line represents the average cross-correlation function for core exchange. In this case k_on = 1 and k_off<<1 for Pol III* and k_on and k_off for ε were set to 0.01. Again, we do not observe a positive cross correlation. Cross-correlation functions have been vertically offset for clarity.
DOI: http://dx.doi.org/10.7554/eLife.23932.024