Computational protocol: Structural Characterization of Hsp12, the Heat Shock Protein from Saccharomyces cerevisiae, in Aqueous Solution Where It Is Intrinsically Disordered and in Detergent Micelles Where It Is Locally α-Helical*

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Protocol publication

[…] We recorded all NMR spectra at the National Magnetic Resonance Facility at Madison on Varian VNMRS (600 MHz and 900 MHz) spectrometers equipped with triple-resonance cryogenic probes. The temperature of the sample was regulated at 25 °C for Hsp12 in aqueous solution and 40 °C for Hsp12 in the presence of 200 mm SDS (Hsp12_SDS) or 200 mm DPC (Hsp12_DPC). We collected a series of two- and three-dimensional NMR spectra () at 600 MHz for Hsp12, Hsp12_SDS, and Hsp12_DPC for use in assignments. All three samples contained 0.7 mm [U-13C,15N]Hsp12 in NMR buffer with 10 mm MOPS, 100 mm NaCl, 3 mm NaN3, 5 mm DTT, pH 7.0, 95% H2O, 5% D2O; the Hsp12_SDS and Hsp12_DPC samples contained in addition, respectively, 200 mm SDS or 200 mm DPC. We collected 13C-resolved three-dimensional 1H-1H NOESY and 15N-resolved three-dimensional 1H-1H NOESY data on 900 MHz for Hsp12_SDS and 600 MHz for Hsp12_DPC for deriving distance constraints. Raw NMR data were processed with NMRPipe () and analyzed using the program XEASY (). Two-dimensional 1H-15N HSQC and three-dimensional HNCO datasets were used to identify the number of spin systems, and these identifications plus three-dimensional HNCACB and three-dimensional CBCA(CO)NH datasets were used as input to the PINE server () to determine sequence specific backbone resonance assignments. In addition, backbone resonance assignments were confirmed on the basis of 15N-resolved 1H-1H NOESY data for Hsp12_SDS and Hsp12_DPC. Two-dimensional 1H-13C HSQC, three-dimensional HBHA(CO)NH, three-dimensional HC(CO)NH, and three-dimensional C(CO)NH experiments were used to assign the side chain and HB and HA resonances for Hsp12_SDS and Hsp_DPC. Three-dimensional 15N-edited 1H-1H NOESY (100-ms mixing time), and three-dimensional 13C-edited 1H-1H NOESY (120 ms) experiments were used to derive the distance constraints to determine the three-dimensional structure of the protein (). We used standard pulse sequences () to record steady-state [1H]-15N NOE and 15N relaxation (T1, T2) data at 600 MHz. We acquired multiple interleaved NMR spectra with relaxation delays of 0, 50, 100, 150, 210, 280, 340, 440, 600, 800, and 1100 ms for T1 and 10, 30, 50, 70, 90, 110, 130, 150, 170, 190, and 210 ms for T2 determinations. The relaxation rates were calculated by least squares fitting of peak heights versus relaxation delay to a single exponential decay. The reported error estimates are standard deviations derived from fitting the data. Steady-state [1H]-15N NOE values were calculated from the ratio of peak heights in a pair of NMR spectra acquired with and without 3-s proton saturation. The signal-to-noise ratio in each spectrum was used to estimate the experimental uncertainty. [...] We derived 1H-1H distance restraints from 15N-resolved three-dimensional 1H-1H NOESY and 13C-resolved three-dimensional 1H-1H NOESY spectra. We used TALOS+ software () to obtain backbone dihedral angle restraints (ϕ and ψ) from assigned 1Hα, 15N, 13Cα, 13Cβ, and 13C′ chemical shifts and CYANA software version 3.0 () for automated NOESY peak assignments and structure calculations. We used MOLMOL () and PyMOL () software to calculate the root mean square deviation (r.m.s.d.) and for graphical analysis. We used the PSVS server () to check structure quality. […]

Pipeline specifications

Software tools TALOS+, CYANA, MOLMOL, PyMOL, PSVS
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Saccharomyces cerevisiae
Chemicals Carbon