We have recently focussed on charcterizing the thermodynamics and the dynamics of the unfolded states of human (hIAPP) and rat (rIAPP) amylin. It is well known that rIAPP, which contains three prolines at positions 25, 28 and 29, does not assemble into fibrils. In collaboration with Rudolpho Ghirlando of the LMB, we have carried out sedimentation studies which show that there are no detectable small aggregates which coexist with monomers in aqueous solutions of both rat and human amylin. This result is surprising given that species with intermediate degrees of assembly have been observed is a number of other amyloid-forming systems? ? To carry out these experiments, solutions are prepared at 4C and cleared of traces of pre-formed seeds by high-speed (g=268,000 ) sedimentation. These conditions slow spontaneous formation of new fibrils sufficiently that samples can be equilibrated and analyzed in the analytical centrifuge. We find that both molecules sediment as monomers. For rIAPP these experiments can be carried out at physiological pH and temperatures up to 37 C, but for hIAPP, sedimentation can only be carried out at low temperatures where the polymerization reaction is slow.? ? To study the solution conformation of the monomers, we make use of the triplet quenching method developed in our laboratory (Lapidus et al 2000). The C-terminal tyrosine (Y37)is replaced by tryptophan, the triplet excited state of which is quenched by an internal disulfide formed between residues C2 and C7 in both sequences. By measuring loop formation dynamics as a function of the concentration of guanidinium chloride, which solubilizes the these hydrophobic peptides, and temperature we are able to observe the effects of both backbone stiffness and intramolecular interactions on their chain dynamics. ? ? In 6 M GdmCl, a good solvent, the decrease in the loop formation rate for rIAPP relative to our control peptide C(AGQ)9W can be fully accounted for by chain expansion, that is the difference in sequence increases the persistence length and expands the distribution of end-to-end distances, P(r). In buffer rIAPP collapses much more than C(AGQ)9W. Attractive intermolecular interactions among hydrophobic residues decrease the end-to-end distances and also decrease the rate at which the chain diffuses. The fibril-forming hIAPP is more compact than rIAPP in 6 M GdmCl. The sequence of hIAPP is similar to rIAPP in hydrophobic residue content, so a similar compaction is expected as denaturant concentration is decreased. We anticipate, however, that these attractive intra-chain interactions also contribute to aggregation, so the chain dynamics may reveal interesting ? differences between hIAPP and rIAPP.
| Vaiana, Sara M; Ghirlando, Rodolfo; Yau, Wai-Ming et al. (2008) Sedimentation studies on human amylin fail to detect low-molecular-weight oligomers. Biophys J 94:L45-7 |
