Fig. 2.

Cis-trans proline isomerization about the G193-P194 peptide bond in HSP27. a Native mass spectrum of 25 μM HSP27 in 200 mM ammonium acetate, pH 6.9. The y-axis depicts signal intensity and the x-axis displays the mass-to-charge ratio. b 2D ¹H-¹⁵N HSQC spectrum of 2 mM [U-¹³C,¹⁵N]-HSP27 at 298 K in 30 mM NaH₂PO₄, 100 mM NaCl, 2 mM EDTA, 2 mM NaN₃, pH 7. Each peak corresponds to an N–H bond and resonance assignments are listed next to each peak. Residues that are colored in red and labeled with an asterisk (*) indicate doubled peaks. The dashed circles indicate resonances that are weak and not observable at the plotted contour level. See Fig. S1 for a lower contour HSQC spectrum. c Residues that exhibit two peaks are shown with asterisks (*) above their one-letter amino acid code. A zoomed-in region from b, which shows the major (black) and minor (red) peaks that arise from E195. d 2D ¹H-¹³C strip plots taken from 3D HNCACB (i. and iv.) and C(CO)NH (ii. and iii.) spectra at the ¹⁵N frequency corresponding to E195 or E195*. In the HNCACB spectrum, the ¹³Cα and ¹³Cβ nuclei from E195 (red/black) and P194 (gray) are indicated with dashed lines to reveal their corresponding positions in the C(CO)NH spectrum taken at the E195 ¹⁵N frequency. The difference between ¹³Cβ and ¹³Cγ chemical shifts in proline residues is diagnostic of cis- or trans-proline bonds. In the minor state, the ¹³Cβ and ¹³Cγ chemical shift difference is ~10 ppm (red), indicative of a cis-conformation, whereas in the major state this difference is only ~5 ppm (black), indicative of a trans-conformation. e The population of cis-P194 at 298 K is shown for each residue with well-resolved cis and trans peaks in the ¹H-¹⁵N HSQC spectrum. Both the peak intensity (red) and integrated volume (gray) are shown, and the average value ±1 standard deviation is listed. f The natural logarithm of the equilibrium constant for cis-trans isomerization about the G193-P194 peptide bond is shown as a function of temperature. This analysis yields a change in enthalpy (ΔH) and change in entropy (ΔS) of 13.5 kJ mol⁻¹ and 3.5 J mol⁻¹ K⁻¹, respectively, upon formation of the cis-G193-P194 peptide bond