Overcoming universal restrictions on metal selectivity by protein design

Metalation calculators forE. colistrain JM109 (DE3): Aerobic, anaerobic and hydrogen peroxide exposed cells cultured in LB media

“…An inference might be that Mn 2+ ribonucleotide reductase is metalated under conditions where Mn 2+ is not more available than Fe 2+ : notably this state is approached in Figure 3 and perhaps might be detected in anaerobically grown cells after calibration of a weaker Fur-regulated promoter. Synthetic proteins have been designed which do not select metals according to the order of metal-selectivity given by the Irving-Williams series and as commonly observed in nature 43 . With some analogy to ribonucleotide reductase, this is achieved through the introduction of steric selection through the cooperative binding of multiple metals.…”

“…This raises questions as to whether evolution could have achieved greater selectivity bypassing the necessity to maintain metal availabilities as the inverse of the Irving-Williams series. It has been suggested that such enhanced selectivity comes at the price of increased rigidity and an impaired catalytic landscape 43 . Importantly, for artificial metalloproteins to acquire the correct metal in a cell, engineered selectivity need only be sufficient to confer correct metalation as predicted by metalation calculators based on the prevailing intracellular thermodynamic landscape.…”

Metalation calculators for E. coli strain JM109 (DE3): aerobic, anaerobic, and hydrogen peroxide exposed cells cultured in LB media

“… 43 Synthetic proteins have been designed which do not select metals according to the order of metal selectivity given by the Irving–Williams series and as commonly observed in nature. 44 With some analogy to ribonucleotide reductase, this is achieved through the introduction of steric selection through the cooperative binding of multiple metals. This raises questions as to whether evolution could have achieved greater selectivity bypassing the necessity to maintain metal availabilities as the inverse of the Irving–Williams series.…”

Designed Rubredoxin miniature in a fully artificial electron chain triggered by visible light

“…The combination of powerful computational tools 46,47 , and more recently machine learning 48,49 , together with the genome palette (e.g., directed evolution and phage/yeast display) 50,51 is significantly helping protein designers in increasing success rate. However, direct correlation between single point mutations and metal-dependent function still remains elusive when large scaffolds are adopted 50,52 . Design of synthetic metalloproteins by miniaturization helps circumventing this problem by limiting the metal surroundings to only a few crucial residues 53 .…”

“…They bind a single iron ion through four Cys Sg with an almost tetrahedral geometry and they can cycle between the oxidation states (II) and (III). Rds (45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55) amino acids) adopt a C2-pseudo-symmetric fold constituted by two symmetry-related CXXCX αturns 19 . Despite well-conserved backbones and sequences (50-60% sequence identity), their reduction potential varies in the range -100/+50 mV in prokaryots and could reach 125 mV (vs SHE) in eukaryots, as well as in the closely related rubrerythrin 16 .…”