Regulation of Poly-E Motif Flexibility by pH, Ca2+ and the PPAK Motif

Figures

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  Figure 1. Schematic of the three constructs developed for this study.

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  Figure 2. Constructs containing poly-E motif sequences exhibit pH-dependent conformational shifts. (a) Constructs were separated on a TOSOH TSKgel G3000s sizing column at various pHs. No retention time shifts were observed with the PPAK-containing protein (PEVK1) while both proteins containing poly-E motif sequences (PEVK2 and PEVK3) increase in retention time as pH decreases, consistent with the protein assuming a more flexible conformation. (b) The Circular Dichroism (CD) signal at 200 nm was measured at a range of pHs. The normalized signal shifts as a function of pH for the PEVK2 and PEVK3 constructs, consistent with the hypothesis that the glutamate content in these peptides modulates conformational flexibility. Each measurement was conducted in triplicate and error bars are included to show the standard deviation of each measurement.

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  Figure 3. Increasing calcium concentrations induce conformational flexibility in PEVK2 construct but not PEVK3 construct. (a) CD signal at 200 nm was measured for all three constructs over a range of calcium concentrations. The PEVK2 construct exhibits a conformational shift that is consistent with the shift observed with shifts in pH. A similar shift is not observed in the PEVK3 construct, suggesting that the PPAK motifs alter calcium binding; (b) CD signal at 200 nm was measured at pH 3.5, where the glutamate motifs are protonated, and no conformational change is observed.

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  Figure 4. PEVK2 and PEVK3 constructs do not exhibit conformational fluctuations in the presence of Mg2+. CD signal at 200 nm was measured in the presence of increasing magnesium. No significant conformational fluctuations were observed over the concentration range tested.

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  Figure 5. Na+ and K+ induce increased conformational flexibility at higher ionic strengths. CD signal was measured at 200 nm in the presence of increasing concentrations of K+ (a) or Na+ (b). Both constructs exhibit a decrease in the CD signal as ionic strength increases, consistent with the constructs assuming a more flexible conformation at higher ionic strengths. This supports our hypothesis that increased ionic strength will induce electrostatic shielding of the glutamates, reducing charge repulsion.