![The E91K substitution causes a remarkable structural alteration in α[0-1]. (A) CD wavelength scanning spectra for α[0-1] (left panel) having E91 (α[0-1]E91) or K91 (α[0-1]K91) and mini-Sp having E91 (mini-SpE91, right panel) or K91 (mini-SpK91). α[0-1] had an α-helix content of 56.3%, whereas α[0-1]K91 had a less α-helix content (46.7%). (B) CD thermal stability analyses for the recombinant proteins found in panel A at 222 nm. (C) NMR-1H-15N HSQC spectra of 15N-α[0-1]E91 (α[0-1]E91) and 15N-α[0-1]K91 (α[0-1]K91) at 25°C (left panel) or 40°C (right panel). The resonances from 2 tryptophan residues (W59 and W131) were detected only for α[0-1]E91 at 25°C (inserted figures). (D) Representative emission spectra for α[0-1]E91 (E91) and α[0-1]K91 (K91) (left panels), α[0-1]E91/W59F (E91/W59F) and α[0-1]K91/W59F (K91/W59F) (middle panels), and α[0-1]E91/W131F (E91/W131F) and α[0-1]K91/W131F (K91/W131F) (right panels) in the presence of various concentrations of acrylamide (0-250 mM). (E) The data from each recombinant were fit to a linear Stern-Volmer plot, and the KSV value was calculated from the slope of the plot. (F) The KSV values for each recombinant were obtained from 3 independent measurements and are expressed as the means ± SD. Fluorescence intensity is found in arbitrary unit. Statistical significance between E91 and K91 was calculated by unpaired Student t test. ∗P < .05; ∗∗P < .01. Statistical significance among the wild-type and variants of E91 and the wild-type and variants of K91 was determined by 1-way ANOVA with the Tukey multiple comparison test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodrci/1/1/10.1016_j.brci.2025.100003/2/brci_rci-2025-000113-gr4.jpeg?Expires=1753905552&Signature=oOjMeF~bsaDPq1Xt~~NjmJC-trDijzf-RBGDpOsP4yUkeQkkCyR-T33QlUr1CL-L9FNMGP10WLFgaGvXK23xRjtPzmm6eLEdd3Bs3nwk35SOugfffBop4Sh6QWfLqs4wcvJlQXXgdziax84m9X~eSbRSowpkf-8J~jlnk~cuH82YuZziWzyzQYG1vOM4bZ3LUDx6uj6JEVyjcjQtCOpP7b1JNBNoi74xNh~6KlK-GMPU5WEv1byNECb6~JebzU~iJ~oC3yh6a6RnP-yMSdOw5qkeW8QBnMd4FqDwWInDOBbNM-un1OtzSkZIXlEBhwtyivTot4SnudT-ovpzQ9~vLQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The E91K substitution causes a remarkable structural alteration in α[0-1]. (A) CD wavelength scanning spectra for α[0-1] (left panel) having E91 (α[0-1]E91) or K91 (α[0-1]K91) and mini-Sp having E91 (mini-SpE91, right panel) or K91 (mini-SpK91). α[0-1] had an α-helix content of 56.3%, whereas α[0-1]K91 had a less α-helix content (46.7%). (B) CD thermal stability analyses for the recombinant proteins found in panel A at 222 nm. (C) NMR-1H-15N HSQC spectra of 15N-α[0-1]E91 (α[0-1]E91) and 15N-α[0-1]K91 (α[0-1]K91) at 25°C (left panel) or 40°C (right panel). The resonances from 2 tryptophan residues (W59 and W131) were detected only for α[0-1]E91 at 25°C (inserted figures). (D) Representative emission spectra for α[0-1]E91 (E91) and α[0-1]K91 (K91) (left panels), α[0-1]E91/W59F (E91/W59F) and α[0-1]K91/W59F (K91/W59F) (middle panels), and α[0-1]E91/W131F (E91/W131F) and α[0-1]K91/W131F (K91/W131F) (right panels) in the presence of various concentrations of acrylamide (0-250 mM). (E) The data from each recombinant were fit to a linear Stern-Volmer plot, and the KSV value was calculated from the slope of the plot. (F) The KSV values for each recombinant were obtained from 3 independent measurements and are expressed as the means ± SD. Fluorescence intensity is found in arbitrary unit. Statistical significance between E91 and K91 was calculated by unpaired Student t test. ∗P < .05; ∗∗P < .01. Statistical significance among the wild-type and variants of E91 and the wild-type and variants of K91 was determined by 1-way ANOVA with the Tukey multiple comparison test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.
