Porphyrin distortion during affinity maturation of a ferrochelatase antibody, monitored by Resonance Raman spectroscopy.
Resonance Raman (RR) spectra are reported for mesoporphyrin IX bound to the Fab fragment of the ferrochelatase antibody 7G12. Binding induces activation of a Raman band at 680 cm(-1), which is assigned to an out-of-plane porphyrin vibration, gamma15. This is exactly the predicted effect of distorting mesoporphyrin to the geometry of N-methylmesoporphyrin IX, the 7G12 hapten, based on DFT/CIS modeling of the RR spectrum. The modeling also shows that the pyrrole ring that is tilted out of the porphyrin plane bears a nitrogen lone pair, which is therefore available for coordination by an incoming metal ion. The 680 cm(-1) band intensity is approximately 3 times higher for the affinity-matured antibody than for the germline precursor antibody, while intermediate values are found for variants in which germline residues are mutated to mature residues or vice versa. Thus, RR spectroscopy reveals an evolution from weak substrate distortion in the germline antibody to strong substrate distortion in the affinity-matured antibody, and supports the view that catalysis involves a substrate strain mechanism.
Venkateshrao
Swarnalatha
S
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Yin
Jun
J
Jarzecki
Andrzej A
AA
Schultz
Peter G
PG
Spiro
Thomas G
TG
D003198:Computer Simulation; D005294:Ferrochelatase / Q000737:chemistry / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D008652:Mesoporphyrins / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D011758:Pyrroles / Q000737:chemistry; D013059:Spectrum Analysis, Raman / Q000379:methods
D007128:Immunoglobulin Fragments; D008652:Mesoporphyrins; D011758:Pyrroles; C065420:N-methylmesoporphyrin IX; D005294:Ferrochelatase