Circular Dichroism Analysis of MBP-CHIP Proteins

Circular dichroism far-UV and thermal denaturation measurements were recorded using a Jasco J-810 spectropolarimeter equipped with a CDF-426S Peltier temperature control unit (Jasco Products, OK, USA). The wild-type and mutant MBP–CHIP proteins were prepared at 2.5–5.8 µM concentration in a buffer containing 10 mM potassium phosphate and 100 mM sodium fluoride at pH 7.4. The protein concentrations were determined using a NanoDropTM One Microvolume UV–Vis spectrophotometer (Thermo Scientific, MA, USA), with theoretical extinction coefficients ε₂₈₀ = 96,720 (wild-type MBP–CHIP, G249V, and R51_I53delinsPA) and 91,220 (K143_W147del) M⁻¹cm⁻¹. Far-UV spectra were acquired in the range of 185–260 nm at a scan rate of 50 nm/min at 20 °C, using a quartz cell with a path length of 1 mm. Three scans were accumulated for each spectrum and three spectra were buffer subtracted and then averaged. Thermal denaturation profiles were obtained by recording the decrease in ellipticity at 222 nm as a function of temperature in the range of 20–90 °C with a scan rate of 40 °C/h. The results are expressed in mean residue ellipticity [θ]_mrw = θ(deg × cm² × dmol⁻¹), and BeSTSel (Beta Structure Selection) was used to estimate the secondary structure content [30 (link)]. Final graphs were prepared by using GraphPad Prism software (San Diego, CA, USA).