The Poly-E motif in Titin's PEVK region undergoes pH dependent conformational changes

Figures

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  Fig. 1. Poly-E peptides exhibit pH-dependent changes in retention times. Peptides were separated on a TSK-GEL G2500PWXL column and the retention times were determined as a function of varying pH. The PPAK peptide (P1+W) shows no significant change in retention time. All poly-E peptides (P2+W – P6+W) have increased retention time at lower pHs. The P2+W and P3+W peptides show similar shifts, suggesting overall charge density is more important than clustering.

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  Fig. 2. P2+W peptide decreases fluorescence intensity with decreased pH. Tryptophan fluorescence was measured for the P1+W and P2+W peptides at varying pH and the intensity at 360 nm was plotted as a function of pH. The intensity of the P2+W peptide exhibits a sigmoidal shape while the P1+W intensity is best fit as a line.

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  Fig. 3. P2+W peptide shows a red shift with decreased pH. P2+W exhibits CoM shifts between pH 3 and pH 6. In contrast, P1+W has a significant pH shift between pH 3 and 4, followed by a small shift between pH 4 and 5 and then no significant CoM after pH 5. Insert. Normalized Center of Mass curves showing magnitude of CoM shifts as a function of pH.

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  Fig. 4. P2+W peptide undergoes more quenching at higher pHs. The Ksv values of P2+W shows a sigmoidal shift with pH where a major transition occurs between pH 4 and pH 7. The Ksv of free tryptophan amino acid remain constant between that region of pH.

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  Fig. 5. P2 peptide undergoes a pH dependent conformational change. A. CD spectra of P2 peptide from 250 nm to 190 nm. Spectra were collected between pH 2 and 9 at increments of 1 pH unit. The arrow represents the change in the minima at 200 nm with decreasing pH. B. Molar ellipticity at 200 nm for P1 and P2 peptides. P2 peptide decreases molar ellipticity between pH 4 and 6 while P1 peptide does not exhibit a similar conformational change.

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  Fig. 6. P2 peptide exhibits coil-like structure that becomes more pre-molten globule like at lower pHs. The ME200 verse ME222 values for the P2 peptide are in the region associated with a coil-like structure while the P1 values are in a region of undefined structure on the plot. The P2 conformation exhibits a shift below pH 6 toward the region associated with a pre-molten globule conformation, potentially due to reduced PII helical content.