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:
- Which solution-phase behaviors can be examined in the gas phase?
- How does pH affect the charge states and metal-ion binding?
- Which sequence residues affect the conformational structure and metal-ion binding?
- What are the dissociation mechanisms during CID?
- Can the metal-ion coordination sites be identified using energy-resolved CID and IM-MS?
- Can metal-ion displacement be monitored by collision cross-section (CCS) changes?
- Can IM-MS and theoretical CCS identify which type of coordination each metal ion prefers?
- How do IM-MS, DFT, UV-Vis, and fluorescence findings correlate?
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).