A8G. An A8G mutation exchanges the methyl sidechain for a hydrogen atom. Due to the size reduction, it is expected that helices would shift to fill in the hole caused by the mutation, and in turn alter the Co(II) coordination sphere. The SC-XRD of the purple crystals of Co-MAHF-9 A8G (Figure ) reveals a similar framework to Co- MAHF-9, but with a lower crystallographic symmetry. In the asymmetric unit (ASU) there are two identical peptide sequences that have different sidechain conformations. While in Co-MAHF-9, both His residues coordinate via their Nε atom and the Cɣ-Cδ-O-Co dihedral of both Glu residues are in a cis-conformation, in Co- MAHF-9 A8G, one of the His coordinates with Nδ and one Glu coordinates with a trans Cɣ-Cδ-O-Co dihedral. This difference is caused by a ~1.7 Å shortened helix-helix distance along the channel in the A8G mutant. As a result, the metal coordination geometry is slightly closer to tetrahedral, as indicated by τδ of 0.85. A8F. An A8F mutation introduces a bulkier aromatic sidechain. Analysis of SC-XRD data shows a coordination site and pore shape that are nearly identical to that of Co-MAHF-9 (Figure ), despite the drastic size increase of the sidechain. It appears that the void space between helices above A8 in Co-MAHF-9 accommodates the phenyl ring of Phe by filling the gap with very minimal steric clashes. A8V. Introduction of a β-branched sidechain from an A8V mutation induces a significant 8.28 Å translation of the helices along the channel axis (Table ) relative to those in Co-MAHF-9 (Figure ). This reorganization prevents a potential steric clash between the βmethyl of Val8 and Leu6. Furthermore, the pores are much larger in Co-MAHF-9 A8V, being egg-shaped and approximately 7.8 Å wide. Despite, the large shifting of the helices, each peptide maintains the two Co(II) bridging centers (μ2). Akin to Co-MAHF-9, the Co sites are 4-coordinate with two Glu4 and two His7 ligation, however the geometry is now nearly an ideal tetrahedral (τδ = 0.96). Overall, the shifting of the helices reveals a new degree of freedom for the peptide assembly to adjust in response to mutation and alter the pore shape without compromising the metal coordination. A8I. Like Val, Ile is also a β-branched hydrocarbon sidechain. Only microcrystalline purple powder was obtainable for Co-MAHF-9 A8I. The unit cell was determined via powder X-ray diffraction (PXRD) by means of Pawley refinement and differs from other frameworks in this report (Figure , Table ). Virtual mutagenesis in PyMOL suggests that the A8I mutation would not be compatible in the Co-MAHF-9 A8V framework due to a steric clash with the Co(II) center. A8M. Methionine has a long and flexible sidechain that could induce major structural changes. Analysis of SC-XRD data reveals that the resulting framework (Figure ) has a significantly different structure than Co-MAHF-9. The shape of the channel is wider than Co- MAHF-9 A8V (8.3 Å vs 7.8 Å). Co-MAHF-9 A8M features a 6-coordinate trigonal prismatic geometry, with coordination of two κ 2 -Glu and two His (2His-2Glu), where the peptides are no longer bridging metal centers (μ1). Though the helix arrangement is similar to that of Co-MAHF-9 A8V, the structural differences are due to Met•••His S-aromatic interactions 32 that straddle the His sidechains and prevent bridging metal centers between chains. This mutation highlights how secondary sphere interactions with the metal-binding residues can drastically alter the primary sphere. A8L. The Leu sidechain is comparable in size to that of Met but is branched. Analysis of SC-XRD data of Co-MAHF-9 A8L (Figure ) presents both a new pore shape and metal coordination site. Yet another new, intriguing feature is the switch from parallel to antiparallel packing of the chains along the channel axis. The metal coordination is tetrahedral with Co•••Co centers in close proximity, with the shortest being 5.3 Å apart. It appears the significant restructuring upon mutation is due to the need to bury the highly hydrophobic, pore-facing Leu8 residues away from solvent, as evidenced by Leu8-Leu8 van der Waals contacts. A8P. Only microcrystalline powder was obtainable for Co-MAHF- 9 A8P. The powder is crystalline as evidenced by the peaks in its powder X-ray diffraction (PXRD) pattern, but there are too few peaks to allow for accurate unit cell indexing (Figure ). The presence of proline and its inability to donate a backbone H-bond is expected to substantially interfere with the peptide folding into a helix, as it is known that proline introduces kinks in alpha helices. Mutations with polar neutral sidechains. Introducing mutants with uncharged sidechains capable of H-bonding could influence metal coordination, for example by fixing the rotational states of the Glu or His sidechains through secondary interactions. A8S. The A8S mutation provides a small polar hydroxyl group in the vicinity of side chains Glu4 and His7. The structure from SC-XRD analysis of Co-MAHF-9 A8S contains distorted tetrahedral Co(II) centers coordinated by 2His-2Glu (Figure ). However, unlike in Co-MAHF-9, the carboxylates are coordinated to the metal with a trans Cɣ-Cδ-O-Co orientation in Co-MAHF-9 A8S. There is Hbonding between the hydroxyl group of Ser8 and Nε of His7, forcing His to use the Nδ for Co coordination. Though the alignment of the helices along and across the channel is the same as in Co-MAHF-9, in Co-MAHF-9 A8S the Ser8-His7 H-bond prevents the peptide from bridging metal centers.