| Abstrakt: |
Aggregation is an important area of scientific investigation because of the consequences of this process for many aspects of protein and peptide chemistry. Previous studies of the aggregation of the βA4 peptide fragment, β(12−28), and synthetic analogues in low pH aqueous solution show that replacing either or both phenylalanines with glycine reduces the tendency of this peptide to form aggregates. In this investigation, several β(12−28) analogues have been synthesized in which the phenylalanine residues 19 and/or 20 have been substituted with the nonnative amino acid, naphthylalanine, to produce the peptides [napAla19,20], [napAla19,Gly20], and [Gly19,napAla20] and allowing the aggregation behavior of these peptides to be examined in aqueous solution at low pH with both NMR and fluorescence spectroscopy. The NMR chemical shift, diffusion coefficients and relaxation times as well as rotational correlation times measured with both NMR and fluorescence spectroscopy are concentration dependent providing evidence that [napAla19,20]β(12−28) forms soluble aggregates. Similar results obtained for [napAla19,Gly20]β(12−28) and [Gly19,napAla20]β(12−28) suggest that these peptides have a greatly reduced tendency to aggregate. In addition, [napAla19,20]β(12−28) produces excimer fluorescence emission in a concentration-dependent manner with essentially no excimer detected in the fluorescence spectra of the singly substituted naphthylalanine analogues. Fluorescence lifetimes were measured, and unlike naphthylalanine, the free amino acid, the excimer fluorescence decay of [napAla19,20]β(12−28) does not exhibit a rise time component, suggesting a ground-state preassociation of the peptides through naphthyl π−π interactions that stabilize the aggregates. Fluorescence spectroscopy, due to its concentration sensitivity, permits measurements of peptide solutions at much lower concentration than NMR, allowing direct measurement of the peptide monomer. However, NMR spectroscopy, through the measurement of nuclear relaxation times, can provide complementary information about the differential regional mobility of the peptide. The application of both NMR and fluorescence spectroscopy to the analysis of these naphthyl-substituted peptides produces a more complete picture of their aggregation behavior than could be obtained using either method alone. An advantage of using the combination of these methods is that their different time scales make them sensitive to different ranges of molecular motion. |
