Sample Preparation. Human calmodulin (CaM) was expressed and purified in Escherichia coli BL21(DE3) cells, as described F ▇▇▇▇▇://▇▇▇.▇▇▇/10.1021/jacs.2c02201 previously.9 Two site-directed mutations at A17C and A128C in the NTD and CTD domains of CaM, respectively, were introduced as sites of attachment for the nitroxide spin-labels.9 Full deuteration was achieved by growing the bacteria in deuterated minimal medium, with U-[12C/2H]-glucose as the sole carbon source. R1 nitroxide labeling was carried out with S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H- pyrrol-3-yl)methyl methanesulfonothioate (MTSL; Toronto Research Chemicals), as described previously.9 The purity of A17C-R1/ A128C-R1 nitroxide-labeled calmodulin was verified by mass spectrometry. Solutions for DEER EPR comprised 50 μM CaM (A17C-R1/ A128-R1), 8 mM CaCl2, 100 mM NaCl, 25 mM d-HEPES pH 6.4, and 20% (v/v) d8-glycerol, placed in 1.0 mm inner diameter (1.2 mm outer diameter) quarts EPR tubes (VitoCom). Freezing of the DEER samples was carried out using three different approaches: slow rate freezing (∼40 s) by placing the EPR tube in a −80 °C freezer;43 intermediate rate freezing (∼1.5 s) by directly placing the EPR tube in liquid N2;43 and rapid freeze-quenching (∼0.5 ms) by spraying a high- speed jet onto a spinning copper plate cooled to 77 K, as described previously.18,45 Q-Band DEER. Pulsed EPR data were collected at Q-band (33.8 GHz) and 50 K on a Bruker E-580 spectrometer equipped with a 150 W traveling-wave tube amplifier, a model ER5107D2 resonator, and a cryofree cooling unit, as described previously.52 DEER experiments were acquired using a conventional four-pulse sequence (Figure S1).53 The observer and ▇▇▇▇▇ pump pulses were separated by ca. 90 MHz, with the observer π/2 and π pulses set to 12 and 24 ns, respectively, and the ▇▇▇▇▇ π pulse to 10 ns. The pump frequency was centered at the Q-band nitroxide spectrum located at +40 MHz from the center of the resonator frequency. The τ1 value of 350 ns for the first echo period time was incremented eight times in 16 ns steps to average 2H modulation; the position of the ▇▇▇▇▇ pump pulse was incremented in steps of Δt = 10 ns. The bandwidth of the overcoupled resonator was 120 MHz. All DEER echo curves were acquired for tmax = 7.5 μs, with the exception of the DEER echo curve for τ2 < 7.5 μs, where tmax was set to the value of τ2. DEER data were recorded with values of the dipolar evolution time T (=2τ2) set to 10, 15, 20, 25, 30, and 35 μs. Measurement times were approximately as follows: for T = 10 μs, 3 h; 15−25 μs, 12 h; and 30−35 μs, 36 h. Calculation of P(r) Distance Distributions from Molecular Coordinates. P(r) distance distributions between pairs of R1 nitroxide spin-labels were calculated in the program Xplor-NIH38,54 from molecular coordinates, as recently described,18 using a rotamer library of R1 side-chain conformations whose populations consist of an intrinsic component modulated by overlap with nearby backbone atoms such that the population of a particular rotamer goes to zero when it closely approaches a backbone atom. Because the R1 nitroxide labels are represented as an ensemble, the shape of the P(r) distribution can be non-Gaussian and can include shoulders and/or separate peaks that correspond to distinct regions of conformational space occupied by one or both nitroxide labels.
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Sample Preparation. Human calmodulin (CaM) was expressed and purified in Escherichia coli BL21(DE3) cells, as described F ▇▇▇▇▇://▇▇▇.▇▇▇/10.1021/jacs.2c02201 previously.9 Two site-directed mutations at A17C and A128C in the NTD and CTD domains of CaM, respectively, were introduced as sites of attachment for the nitroxide spin-labels.9 Full deuteration was achieved by growing the bacteria in deuterated minimal medium, with U-[12C/2H]-glucose as the sole carbon source. R1 nitroxide labeling was carried out with S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H- pyrrol-3-yl)methyl methanesulfonothioate (MTSL; Toronto Research Chemicals), as described previously.9 The purity of A17C-R1/ A128C-R1 nitroxide-labeled calmodulin was verified by mass spectrometry. Solutions for DEER EPR comprised 50 μM CaM (A17C-R1/ A128-R1), 8 mM CaCl2, 100 mM NaCl, 25 mM d-HEPES pH 6.4, and 20% (v/v) d8-glycerol, placed in 1.0 mm inner diameter (1.2 mm outer diameter) quarts EPR tubes (VitoCom). Freezing of the DEER samples was carried out using three different approaches: slow rate freezing (∼40 s) by placing the EPR tube in a −80 °C freezer;43 intermediate rate freezing (∼1.5 s) by directly placing the EPR tube in liquid N2;43 and rapid freeze-quenching (∼0.5 ms) by spraying a high- speed jet onto a spinning copper plate cooled to 77 K, as described previously.18,45 Q-Band DEER. Pulsed EPR data were collected at Q-band (33.8 GHz) and 50 K on a Bruker E-580 spectrometer equipped with a 150 W traveling-wave tube amplifier, a model ER5107D2 resonator, and a cryofree cooling unit, as described previously.52 DEER experiments were acquired using a conventional four-pulse sequence (Figure S1).53 The observer and ▇▇▇▇▇ pump pulses were separated by ca. 90 MHz, with the observer π/2 and π pulses set to 12 and 24 ns, respectively, and the ▇▇▇▇▇ π pulse to 10 ns. The pump frequency was centered at the Q-band nitroxide spectrum located at +40 MHz from the center of the resonator frequency. The τ1 value of 350 ns for the first echo period time was incremented eight times in 16 ns steps to average 2H modulation; the position of the ▇▇▇▇▇ pump pulse was incremented in steps of Δt = 10 ns. The bandwidth of the overcoupled resonator was 120 MHz. All DEER echo curves were acquired for tmax = 7.5 μs, with the exception of the DEER echo curve for τ2 < 7.5 μs, where tmax was set to the value of τ2. DEER data were recorded with values of the dipolar evolution time T (=2τ2) set to 10, 15, 20, 25, 30, and 35 μs. Measurement times were approximately as follows: for T = 10 μs, 3 h; 15−25 μs, 12 h; and 30−35 μs, 36 h. Calculation of P(r) Distance Distributions from Molecular Coordinates. P(r) distance distributions between pairs of R1 nitroxide spin-labels were calculated in the program Xplor-NIH38,54 from molecular coordinates, as recently described,18 using a rotamer library of R1 side-chain conformations whose populations consist of an intrinsic component modulated by overlap with nearby backbone atoms such that the population of a particular rotamer goes to zero when it closely approaches a backbone atom. Because the R1 nitroxide labels are represented as an ensemble, the shape of the P(r) distribution can be non-Gaussian and can include shoulders and/or separate peaks that correspond to distinct regions of conformational space occupied by one or both nitroxide labels.
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