Cd spectrometer

The His124Phe and His192Phe mutants of DesB were prepared using the PCR method. In order to confirm the proper folding of these mutants, circular dichroism (CD) spectra were measured using a CD spectrometer (JASCO, Japan).

All natives sequences from PS2Aa1 and HCoV-229E were synthesized via F-moc solid-phase peptide synthesis (SPPS) [34 (link)] using the tea-bag procedure reported by Houghten [35 (link)]. Peptides were purified by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) (Jasco Corporation, Tokyo, Japan) using a Vydac C-18 preparative column using a mixture of (A) H₂O with 0.1% (v/v) TFA, and (B) acetonitrile (ACN) containing 0.1% (v/v) TFA as mobile phase. For the elution of peptides, the gradient program was: 30 min with 5–70% of B at 1 ml/min and detection at 220 nm. The molar mass of purified peptides was determined by ESI-MS mass spectrometry (MS) [36 (link),37 (link)] (Supplementary Figures S1–S5).
Circular dichroism (CD) of peptides was carried out at 25°C in a 1 mm path length cuvette and spectra were obtained over 190–260 nm in a CD Spectrometer (J-815 Jasco Corporation, Japan) using a 0.2 mM peptide solution dissolved in a mixture of 50 mM sodium phosphate buffer, pH 7.4, and 30% (v/v) 2,2,2-trifluoroethanol (TFE). Each spectrum was obtained as an average of three scans taken at a rate of 20 (nm/min) with a spectral band of 1 nm. Each experiment was repeated four times and averages were taken of the resulting data [38 (link)].

All proteins analyzed were produced in BL21 (DE3) pLysS Escherichia coli cells (Novagen) with expression induced with 0.5 mM IPTG for 18 h at 18°C. Murine PKI (α-isoform) and PKAcα1 (nonmyristoylatable) were cloned into the pET-30 Ek/LIC vector (Novagen) and purified as N-terminal His6-tag fusion proteins by immobilized metal affinity chromatography and size-exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated in 50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 10% (v/v) glycerol and 1 mM DTT. PKAc K72H and R133A point mutants were generated by PCR site-directed mutagenesis [21 (link)], expressed, and purified as above. N-terminal 6His-tagged RIIα subunit was expressed as described previously [53 (link)]. Glutathione-S-transferase (GST) tagged λ protein phosphatase (λPP) was cloned into pGEX-6P-1 and purified with Glutathione-Sepharose 4B (GE Healthcare). Secondary structure compositions of wild-type (WT) and mutant PKAc proteins (0.6 mg/ml) were analyzed by circular dichroism (CD) in the far UV range (180–260 nm) using a Jasco 1100 CD spectrometer with a path length of 0.1 cm, following exchange into 10 mM sodium phosphate (pH 7.4) and 25 mM NaF.

The secondary structure of each of the three peptides, CBP, HABP1, and CBP-HABP1 were measured through circular dichroism (CD) spectra. Measurements were made with CD spectrometer (JASCO, J-815) at room temperature, using a 1.0 mm cuvette. Each peptide sample was dissolved at 0.2 mg/mL in 10 mM potassium phosphate (pH 7.4) at 4 °C for 16 h. Spectra shown are averaged from three experimental repeats. The scans were acquired from 190 to 300 nm at a scanning speed of 60 nm/min. CD spectra were processed for secondary structure composition with the tools of CD Pro. For each peptide, the reference set selected was SMP50. The mean residue ellipticity (MRE) was analyzed with CD Pro software to compare likely conserved secondary structure features from the single domains (CBP and HABP1) in the chimeric peptide (CBP-HABP1), see Figure S1 [51 (link)]. Absorbance measurements were taken every 1 nm as the average of 5 technical replicates and smoothed by a 7-point Savitzky–Golay filter. The fractions of secondary structure (Regular Helix, Distorted Helix, Regular Sheet, Distorted Sheet, Turns, and Unordered) were averaged for all three CD Pro tools (SELCON3, CDSSTR, and CONTILL).

Circular dichroism (CD) spectra were recorded on a CD spectrometer (Jasco, Tokyo, Japan). The CA variants at a concentration of 20 μM in 20 mM phosphate buffer (pH 7.5) were heated from 30 °C to 100 °C inside a quartz cuvette at a rate of 2 °C/min. The temperature dependent changes of ellipticity were monitored at 220 nm. The obtained curves were smoothed by non-linear regression using Sigmaplot 10.0 (Systat Software, San Jose, CA, USA). The mid-point temperature (T_M), enthalpy (ΔH), and entropy (ΔS) of unfolding were analyzed according to a protocol by Greenfield31 (link).