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, but is otherwise very similar in sequence to hIAPP, does not assemble into fibrils. In collaboration with Rudolpho Ghirlando of the LMB, we have carried out sedimentation studies which show that oligomers containing less than about 100 hIAPP molecules are unstable relative to the monomer of either 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 the 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. ? ? Both hIAPP and rIAPP contain an N-terminal disulfide loop, so as a control for these studies we synthesized a peptide C-C(AGQ)_9W which contains this loop as the quencher. In 6 M GdmCl, a good solvent, the decrease in the loop formation rate for hIAPP and rIAPP relative to our control peptide CC(AGQ)9W can be fully accounted for by chain expansion. If the chain stiffness is ordered as rIAPP > hIAPP > C-C(AGQ)_9W, where the increases in persistence length for the IAPP sequences result from the presence of bulkier sidechains in both sequences and the three prolines of rIAPP. The fibril- forming hIAPP is more compact than rIAPP in 6 M GdmCl. In buffer all three molecules collapse more than C(AGQ)9W which we studied previously. An unexpected result was that the collapse of C-C(AGQ)_9W is signicantly greater than the other sequences, which rules out the naive interpretation that attractive intermolecular interactions among hydrophobic residues are responsible for the decrease in the end-to-end distances.? ? In an effort to understand these results, we have carried out molecular dynamics simulations of these three peptides in water. The results suggest that the collapse of C-C(AGQ)_9W results from interactions between the disulfide loop and residues of the chain which are immediately C-terminal of the loop, which act to effectively decrease the chain length and increase the rate of loop formation. Preliminary results on hIAPP and rIAPP, however, suggest that these interactions nmay not be the origin of the collapse of these peptides. ? ? We anticipate 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 |
