pH-Dependent Compaction of the Intrinsically Disordered Poly-E Motif in Titin

Figures

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  Figure 1. Sequences of the designed poly-E peptides. The reference sequence (P1) and the six variants are shown, with deviations from the reference highlighted in bold text.

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  Figure 2. A peptide derived from the native poly-E sequence of titin shows a pH dependence as measured by CD. (a) CD spectrum of the poly-E peptide with an arrow indicating the direction of the change in the spectra with decreasing pH. (b) Molar ellipticity at 200 nm plotted against pH shows a transition to a more ordered state below pH 5.

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  Figure 3. Peptides showing disordered characteristics at pH 7. CD spectra were collected for each peptide at pH 7 as a baseline. The majority of the peptides exhibited the characteristic 200 nm minima associated with a disordered structure. The ∆proline peptide (a) has a reduced minimum, suggesting that this peptide might have a small degree of ordered structure. The aromatic peptide (b) did not show any minima at 200 nm. The concentration of all peptides is 80 µM.

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  Figure 4. Conformational changes in peptides with hydrophobic linkers observed using (a) size-exclusion chromatography and (b) circular dichroism. Circular dichroism values at 200 nm were taken and normalized to the highest pH point for the peptide.

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  Figure 5. Effects of proline on pH sensitivity as observed with (a) size-exclusion chromatography and (b) circular dichroism. Circular dichroism values at 200 nm were taken and normalized to the highest pH point for the peptide.

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  Figure 6. pH-dependent conformational changes as observed with (a) size-exclusion chromatography and (b) circular dichroism. Circular dichroism values at 200 nm were taken and normalized to the highest pH point for the peptide.

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  Figure 7. Hydrocarbon peptide, which precipitates at low pH, as seen by CD. The trough at 220 nm corresponds to pH 2.

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  Figure 8. pH-dependence of the average radius of gyration for each peptide studied. All peptides are found to elongate with pH. Some show an essentially linear trend (for example, P2), while others have a pH where there is a distinct “switch” to longer configurations (such as P3).

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  Figure 9. pH-dependence of the fraction of secondary structures for each peptide studied. The amount of secondary structures decreased with the increasing pH, which is consistent with the formation of an elongated conformation.

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  Figure 10. Representative structures of the wild-type peptide (P1) and the aromatic peptide (P3) show an increased secondary structure in P3. Representative structures of the P1 peptide at pH 1 (a) and pH 12 (b) demonstrate that the P1 peptide has a more extended conformation at pH 12. Representative structures of the P3 peptide at pH 1 (c) and pH 12 (d) demonstrate that the P3 has a significant secondary structure at pH 1. The distance between the initial and final α-carbons are reported in ångströms.

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  Figure 11. Average end-to-end distances for each peptide. End-to-end distance is defined as the center-of-mass distance between the first and last amino acid in the peptide. Errors in the mean are reported for each data point but are smaller than the symbols. This data show that the peptides all extend when the pH increases.

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  Figure 12. 1H-NMR spectra of the poly-E peptide at varying pHs, showing increased compaction at lower pHs. Spectra of our 28-amino acid peptide were collected at intervals of 0.5 pH units from pH 5.0 to pH 6.5, which spans the pH range where conformational transitions are observed in both CD and HPLC [25]. As it can be seen, the peaks in the methyl and aliphatic regions of the spectra become sharper, which is consistent with a more compact structure.