Complementary analysis of peptide aggregation by NMR and time-resolved laser spectroscopy

Aggregation is an important area of scientific investigation because of the consequences of this process for many aspects of protein and peptide chemi stry. Previous studies of the aggregation of the beta A4 peptide fragment, beta(12-28), and synthetic analogues in low pH aqueous solution show that r eplacing either or both phenylalanines with glycine reduces the tendency of this peptide to form aggregates. In this investigation, several beta(12-28 ) analogues have been synthesized in which the phenylalanine residues 19 an d/or 20 have been substituted with the nonnative amino acid, naphthylalanin e, to produce the peptides [napAla(19,20)], [napAla(19),Gly(20)], and [Gly( 19),napAla(20)] and allowing the aggregation behavior of these peptides to be examined in aqueous solution at low pH with both NMR and fluorescence sp ectroscopy. The NMR chemical shift, diffusion coefficients and relaxation t imes as well as rotational correlation times measured with both NMR and flu orescence spectroscopy are concentration dependent providing evidence that [napAla(19,20)]beta(12-28) forms soluble aggregates. Similar results obtain ed for [napAla(19),Gly(20)]beta(12-28) and [Gly(19),napAla(20)]beta(12-28) suggest that these peptides have a greatly reduced tendency to aggregate. I n addition, [napAla(19,20)]beta(12-28) produces excimer fluorescence emissi on in a concentration-dependent manner with essentially no excimer detected in the fluorescence spectra of the singly substituted naphthylalanine anal ogues. Fluorescence lifetimes were measured, and unlike naphthylalanine, th e free amino acid, the excimer fluorescence decay of [napAla(19,20)]beta(12 -28) does not exhibit a rise time component, suggesting a ground-state prea ssociation of the peptides through naphthyl pi-pi interactions that stabili ze the aggregates. Fluorescence spectroscopy, due to its concentration sens itivity, permits measurements of peptide solutions at much lower concentrat ion 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 t o the analysis of these naphthyl-substituted peptides produces a more compl ete picture of their aggregation behavior than could be obtained using eith er 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.