Oligomerization of mono-D-amino acid substituted Aβ
We next synthesized a series of mono-D-substituted peptides (13 for Aβ40, 24 for Aβ42) (Fig. 4). These peptides were chosen based on the results of the PICUP and ThT experiments, which revealed the dipeptide regions of Aβ that most affected oligomerization and β-sheet formation. For each of these dipeptide regions, we synthesized the two corresponding mono-substituted peptides. We then studied oligomerization behaviors using PICUP, SDS-PAGE, and silver staining. We included non-cross-linked peptides in each case because prior studies had shown that Aβ42, but not Aβ40, formed SDS-induced trimers (predominately) and tetramers 10. We wanted to determine if and how D-amino acid substitution might affect this process. Notwithstanding the prior reported lack of SDS effects on Aβ40 oligomer formation during SDS-PAGE, we included the non-cross-linked Aβ40 peptides as a control and to determine if the prior results were reproducible.
Non-cross-linked Aβ40 displayed an intense monomer band, along with a faint doublet band migrating at an Mr consistent with that of dimer (Fig. 5a). This faint doublet also was observed in the lanes of the D-H14, D-N27, D-I31, and D-I32 peptides, but was not readily visible in other lanes. We also observed a band migrating below the monomer band that may represent an intramolecularly cross-linked monomer. This band was most apparent with the D-H14 and D-Q15 peptides
Cross-linked Aβ40 produced an oligomer distribution comprising predominately dimers→tetramers, with less intense pentamer and hexamer bands (Fig. 5, Aβ40, XL). D-N27 showed the greatest difference in oligomer distribution compared to Aβ40, with smeared tetramer, pentamer, and hexamer bands. D-H14 and D-Q15 displayed less intense tetramer and pentamer bands, as well as a band at an Mr lower than monomer, consistent with what we observed in Aβ40 D-[H14,Q15] (Fig. 5, XL). This band was present in D-26, D-N27, D-I31, and D-I32 albeit less intense. Relatively small amounts of monomer were observed in D-A21, D-S26, D-I31, and D-I32 peptides. Although the nominal amounts of peptide loaded into each well were identical, variations in total band intensity can occur. For this reason, we performed densitometry on each lane and calculated the normalized intensities of bands (Fig. S?). These intensities were consistent with the observations presented above.
Non-cross-linked Aβ42 produced prominent monomer and trimer bands, and a sharp tetramer band of lower intensity. The trimer band had a characteristic trapezoidal shape 10. The substituted peptides displayed a variety of patterns, ranging from indistinguishable from Aβ42 D-V24, D-S26, D-N27, and D-K28: black circles) to the presence of only an intense monomer band (D-F20, D-I31, D-I32, D-M35, D-V39, D-V40, and D-I41: red diamonds). Some peptides had relatively little trimer and tetramer (D-D01, D-H14, D-E22, D-D23, and D-L34: blue squares), only small amounts of trimer (D-A30 and D-A42: grey stars), or monomer and a faint band migrating just above (D-Q15, D-A21, and D-V36: green triangles) (Fig. 5c non-XL).
Cross-linking of Aβ42 produced a characteristic oligomer distribution comprising predominately dimers→heptamers with a node at pentamer/hexamer 10. The oligomer distribution of D-F20 differed most from wild type Aβ42 (as it did in its un-cross-linked state), exhibiting only faint monomer, relatively darker dimer, and faint trimer bands. Interestingly, this variant was the most difficult to solubilize and its oligomer distribution was unique amongst all the peptides studied. The extent of the effects of D-amino acid substitution in the cross-linked states was very similar to that in the non-cross-linked states (cf. Figs. 5c non-XL vs. XL). If a particular peptide demonstrated essentially complete elimination of oligomers in the non-cross-linked states, this same peptide, when cross-linked, produced distributions in which the predominant oligomer states were dimer and trimer (e.g., see the substitutions from positions 30→42). Tetramer, pentamer, and hexamer bands were seen in these samples, but they were faint relative to their counterparts in WT Aβ42. Virtually no heptamer bands were visible in these samples. Relative to the oligomer distribution produced by WT Aβ42, the D-01, D-H14, and D-Q15 variants displayed more intense dimer and trimer bands with less intense tetramer, pentamer, and hexamer. Several oligomer distributions were devoid of a tetramer band (D-Q15 and D-A21). Others had very faint monomer bands compared to Aβ42 (D-F20, D-D23, D-M35, D-V36, and D-V40). The D-S26, D-N27, and D-K28 peptides produced distributions that were quite similar to that of WT Aβ42. Densitometric analyses of the normalized band intensities were consistent with these observations (Fig. ?????????). Fig. ?????? presents histograms of representative examples for each of the five classes is shown in Fig. 5d.