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The solution-phase metal ion reactivity of ambs and mb-OB3b will be examined by electrospray ionization – IM-MS and density functional theory (DFT). Ultraviolet-visible and fluorescence spectral techniques will be used to check for correlations across gas- and solution-phase analyses to answer the following questions:

  1. Which solution-phase behaviors can be examined in the gas phase?
  2. How does pH affect the charge states and metal-ion binding?
  3. Which sequence residues affect the conformational structure and metal-ion binding?
  4. What are the dissociation mechanisms during CID?
  5. Can the metal-ion coordination sites be identified using energy-resolved CID and IM-MS?
  6. Can metal-ion displacement be monitored by collision cross-section (CCS) changes?
  7. Can IM-MS and theoretical CCS identify which type of coordination each metal ion prefers?
  8. How do IM-MS, DFT, UV-Vis, and fluorescence findings correlate?
Diagram describing location of lowest energy conformers
The location of the lowest energy conformers is determined by using two methods.

Figure 1. The lowest energy conformers are located using the B3LYP/LanL2DZ method a) [amb5A+H]+ and b) [amb5A+2H]2+. The dashed lines represent the salt-bridges and hydrogen bonds that stabilize the secondary structures and include a tight turn at Pro4 augmented with a hydrogen bond between the backbone carbonyl and amine groups of Gly3 and Tyr5, and a hydrogen bond between the substituent hydroxyl group of Tyr5 and the carbonyl of the N-terminus acetyl group. The 2+ conformer exhibits the most elongated structure, because of the charge repulsion between the two protonated substituent groups of His1 and His6 which are separated by a distance of almost 14 Å. This charge repulsion is mediated in the 1+ conformer, which exhibits a folded secondary structure, due to a salt-bridge between the imidazolium of His1 and the carboxylate of the C-terminus, and a hydrogen bond between the thiol of Cys2 and the C-terminus carboxylate. The theoretical collision-cross sections (Ω) of the 2+ and 1+ conformers are 234 ± 14 Å2 and 203 ± 6 Å2, respectively, which agree with the IM-MS measurements of 220 ± 8 Å2 and 201 ± 8 Å2 (uncertainties 2σ).

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