A comparison between experimental cross-sections and theoretical cross-sections () shows excellent agreement between experiment and theory. Deviations fall below 3% in all cases and below 2% in almost all cases. Substitutions at E22 do little to alter the turn-bend structure of the decapeptide, with subtle changes in the factors that stabilize this structure, as discussed here. Both E22Q and E22G show the same D23 Oδ backbone NH hydrogen bonds, which act to preserve the bend in residues 23-28. Still, these bonds are not as highly populated as in the wild type. E22Q has the lowest RMSD from WT, consistent with the very similar resistance to proteolysis displayed by the two peptides[12
]. Hydrogen bonding between D23 and N27 is present in E22Q and WT but not present in E22G. Conversely E22G maintains a hydrogen bond between D23 and G25, which is not present in E22Q. Since both mutations have similar RMSD to the WT we reiterate that these bonding differences have little impact on the core structure. The structure of E22K contains a dissimilar network of backbone hydrogen bonds to the WT but, perhaps surprisingly, has a similar structure.
The Iowa substitution, D23N, on the other hand, does result in a change of backbone structure. Here, a negatively charged aspartic acid residue is replaced with a neutral asparagine residue. With this substitution, the hydrogen bonding between residue 23 and the surrounding residues is disrupted, suggesting that the D23 network of hydrogen bonds is primarily responsible for maintaining the backbone structure in the WT peptides, as well as in the E22X mutants. This is further emphasized by the decreased population of the C1 cluster of the D23N mutant, as compared to the E22X mutants (). With a mutation at residue 23, the peptide able to access slightly more conformational space than the WT and other E22X mutants.
These results confirm the presence of a turn in the 24-28 region of the peptide, as previously reported in NMR experiments11
. Proteolysis experiments on the mutant forms of Aβ(21-30) by Grant et al.[12
] showed that substitutions at E22 and D23 increase the peptide’s susceptibility to digestion, suggesting that these changes act to destabilize the core structure, perhaps encouraging higher order Aβ assembly. Our results for the Iowa form support this hypothesis, showing a change from a bend at G25-K28 with a turn at D23-S26 for the wild-type Aβ(21-30) to two turns between residues N23-N27 and E22-S26 in the mutant. On the other hand, our calculations on substitutions at E22 show that none of these FAD associated mutations significantly alter the backbone structure of WT Aβ(21-30). Regardless, subtle changes in structure for the various E22X mutants may either alter the intramolecular interactions between the Aβ(21-30) segment and the rest of the full-length peptide or the intermolecular interactions between the Aβ monomers. Consequently, the kinetics of monomer folding and the ensuing peptide assembly processes may be altered by these mutations.
This hypothesis however, does not explain the differences between the results presented here and a previous proteolysis study on Aβ(21-30)[12
]. In those studies, the E22G, E22Q and E22K mutants all showed decreased resistance to digestion relative to the WT. There can be several reasons for the differences. First, the proteolysis occurs at K28, which is at the C-terminal end of the conserved core structure of the peptide. Hence, other factors may dominate the trypsin-induced proteolysis. Second, the proteolysis is a relatively slow process and may occur from minor structural components that are not considered in our structural analysis of . In fact, a central feature of the kinetic analysis of the proteolysis experiments was the equilibrium among protease-sensitive and protease-insensitive conformers. Finally, proteolysis occurs in solution and solution structures23
are more variable than our solvent free structures, where the vast majority of the structures fall in a single cluster family. For example, the population of cluster C1 for the gas phase structure of WT Aβ(21-30) is 99%, while the population in solution is 44%.
Overall, however, the above conclusions are consistent with those drawn from our solution phase molecular dynamics simulations[24
, 30] even though some important differences result from the loss of solvent. The small size of the peptide ensures that it is completely desolvated during the IM-MS experiments. Without the presence of solvent, electrostatic interactions and salt bridges play a significant structural role. The presence of stable salt bridges in the gas phase is primarily responsible for the simultaneous existence of the bend and turn that is found in the gas phase for the WT and the E22X mutants. In solvent, the D23-K28 salt bridge is not present. As a consequence, the fraction of structures containing a backbone bend is substantially reduced, although the bend remains the dominant structural element for the WT and E22X mutants.
Conversely, the D23N structure consists of a single turn in both the gas and solution phase. This provides further evidence that the D23-K28 salt bridge is the cause of the structural difference upon transitioning from solution to gas phase for the WT and E22X mutants.
Simulations in explicit solvent[24
] show that a network of D23 hydrogen bonds serves as the primary source of structural stabilization for the bend in the peptide backbone of the WT and E22X mutants. This same network of D23 hydrogen bonds still exists in the gas phase even in the presence of the D23-K28 salt bridge. Even though the gas phase structure is completely desolvated, the important interactions from the biologically-relevant solution phase structure remain intact. These results speak to the persistence of the fundamental intramolecular interactions in these peptides and why they are so resistant to proteolysis.