Aromatic Side Chain−Porphyrin Interactions in Designed Hemoproteins
journal contributionposted on 07.12.1999, 00:00 by Dahui Liu, David A. Williamson, Michelle L. Kennedy, Todd D. Williams, Martha M. Morton, David R. Benson
Aromatic amino acid side chains are commonly observed to interact with the heme cofactors of natural hemoproteins. These interactions are of the types previously identified for pairs or groups of aromatic amino acid side chains in proteins: offset π-stacking and T-stacking (an edge-to-face arrangement). To evaluate how such interactions may influence structural stability of hemoproteins, we synthesized peptide-sandwiched mesohemes (PSMs) 2 and 3 in which the alanine-4 (Ala-4) residues in 1 have been replaced by phenylalanine (Phe) and tryptophan (Trp), respectively. The Co(III) analogues of 1, 2, and 3 (1-Co, 2-Co, and 3-Co, respectively) were also prepared. Histidine (His)-to-iron coordination in 1 had previously been shown to induce helical conformations in the peptides (helix content ∼50% at 8 °C). Molecular modeling studies suggested that Trp, but not Phe, could engage in edge-to-face interactions with the porphyrin if the peptides are fully helical. Replacing Ala-4 with Trp, but not with Phe, was thus predicted to favor enhanced peptide helix content. Circular dichroism spectra are consistent with significantly increased helix content in 3 relative to 1, but not in 2. Hydrogen−deuterium (H/D) exchange rates determined by electrospray ionization mass spectrometry, however, decrease in the order 1 ≫ 2 > 3, while pH titrations reveal that the stability of the model protein folds decreases in the order 3 > 2 ≫ 1. Furthermore, 1H NMR spectra of 2-Co and 3-Co indicate that the aromatic side chains in each compound are oriented within the shielding region of the porphyrin ring. Two-dimensional NOE and chemical shift data show that the helices in 3-Co are more highly organized than in 1-Co and span nearly the entire peptide sequence, while in 2-Co shorter helices of intermediate stability run between Phe-4 and Ala-13. The combined results indicate that aromatic side chain-porphyrin interactions in 2 and 3 stabilize their respective model protein folds, and suggest a similar role for the corresponding interactions in natural hemoproteins. Finally, the chemical shift patterns of the Trp side chains in 3-Co, the different effects of Phe and Trp on peptide architecture, and the pattern of chemical shifts exhibited by the α-NH and α-CH hydrogens in all three Co(III) PSMs demonstrate that the solution structures of these designed hemoproteins are similar to those predicted in molecular modeling studies.
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pH titrationshelix contentside chainspeptide sequencesolution structureschemical shift data showpeptide architecturechemical shift patternsacid side chainsTrp side chainsinteractionPSMNOENHheme cofactorsshielding regionHemoproteins AromaticCHmodel protein folds decreasesCoorder 3chemical shiftselectrospray ionization mass spectrometryMolecular modeling studies1 H NMR spectramodeling studiesmodel protein foldsPhepeptide helix contentCircular dichroism spectraporphyrin ringhelical conformationshemoprotein