Pfd 425s

Circular dichroism (CD) spectra were recorded on a Jasco J815 spectropolarimeter equipped with a PFD-425S Peltier cell holder in a 1 mm path length cuvette. The concentration for all protein samples was 25 µM in 50 mM Tris, 150 mM NaCl, pH 7.0. The samples were scanned in continuous mode from 180 to 260 nm in 0.5 nm increments, with a scan rate of 10 nm per minute and a digital integration time of 8 s. Ten spectral scans were averaged by the instrument software. For each protein sample, five independent replicate measurements were performed. CD-spectra were deconvoluted using spectral data ranging from 180 to 260 nm using the CONTIN algorithm in the DichroWeb software [34 (link),35 (link)]. Statistical analysis of secondary structure compositions was performed by pairwise Student’s t test.

Protein samples were adjusted to a final concentration of about 6 µM for far UV (200–260 nm) by diluting in storage buffer. For near UV (260–320 nm), proteins were concentrating using Amicon stirred cells to 230 µM and 170 µM for the experiment with ATP and ADP, respectively. CD spectra were generated on a Jasco J-815 Spectropolarimeter equipped with the Jasco PFD-425S temperature-control unit in a 1-mm path-length quartz cell at 25 °C. Each spectrum was from three accumulated scans with a 1 second response time at a scan speed of 100 nm per minute. Scans were performed with a bandwidth of 1 nm in far UV and at 0.5 nm increments in near UV. Data was recorded using Spectra Manager software (Jasco). For examining the effects of ligand, ATP and ADP (Sigma-Aldrich) was added at a final concentration of 0.1 mM. Data was plotted after background subtraction of identical scans of controls without the protein. ATP and ADP were used in this experiment because these T. thermophilus PilB variants has no ATPase activity at 25 °C (data not shown).

The secondary structure of recombinant HpEno was analysed by circular dichroism (CD) using a JASCO J-815 spectropolarimeter (Jasco Inc., Easton, MD) equipped with a PFD-425S Peltier-type cell holder for temperature control and magnetic stirring. CD spectra were recorded using 1 cm path length cells from 200 to 250 nm. Ellipticities are reported as the mean residue ellipticity, [θ]_MRW.

Far-UV Circular Dichroism (CD) spectra of hGCAP1 with and without Ca²⁺ were recorded in a Jasco J-810 spectropolarimeter (JASCO Europe, Cremella, Italy) equipped with a Jasco temperature controller module PFD-425S. All measurements were performed in a 0.1 cm path length quartz cuvette at 0.2 mg/mL protein concentration in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM DTT, added with 2 mM Ca²⁺ or 2 mM EGTA. Far-UV spectra were recorded from 190 to 260 nm, at 20 and 95 °C (temperature was increased in ramp mode at 1 °C/min).

The far-UV CD spectra of ThSwo were collected using a Jasco model J-815 CD spectropolarimeter (Japan Spectroscopic; Tokyo, Japan) coupled to a Peltier control system (PFD 425S-Jasco). The CD spectra were generated using the purified recombinant ThSwo protein at a concentration of approximately 4 µM in buffer A containing 1 mM of TCEP at 20 °C. A total of 12 accumulations within the range of 260–208 nm at a rate of 50 nm min⁻¹ were recorded and averaged using a quartz cuvette with a pathlength of 1 mm. The Dichroweb server (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) and CDNN deconvolution software were used to statistically analyze the CD spectra [50 (link)]. The ThSwo thermal-induced unfolding experiments followed by CD were measured at 218 nm from 20 to 90 °C, using approximately 10 µM ThSwo in buffer A containing 1 mM of TCEP with a 1 mm pathlength cell. The melting temperature (Tm) of ThSwo was obtained by fitting the CD data to a sigmoidal Boltzmann function as implemented using Origin 8.1 software (OriginLab, Northampton, MA, USA).

Fluorescence spectra were obtained using a LS-55 Spectrofluorometer (Perkin-Elmer), equipped with a water-jacketed cell holder for temperature control at 25°C. Fluorescence emission scans were recorded from 320 to 400 nm using an excitation wavelength of 280 nm (2.5 nm bandpass) with a 1 cm path-length cell. Samples were complemented with 8-Anilino-1-naphthalenesulfonic acid (ANS) at 1:50 molar ratio. ANS fluorescence was measured using an excitation and emission wavelengths of 360 and 460 nm respectively. Far-UV CD spectra were measured using a JASCO J-815 spectropolarimeter (Jasco Inc., Easton, MD) equipped with a PFD-425S Peltier-type cell holder for temperature control and magnetic stirring. Scans were taken between 200 to 250 nm, at a scan rate of 10 nm min^-1 using a 1.0 cm path-length cuvette. Ellipticities are reported as mean residue ellipticity (θ]_MRW).