Dynamic Factors Affecting Gaseous Ligand Binding in an Artificial Oxygen Transport Protein

Abstract

We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7 this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime which may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when when exposed to oxygen. Compared to HP7, distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off-rate. EPR comparison of these ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation greatly increases water penetration into the protein core. The inability of the mutant protein to bind oxygen may be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together these data underline the importance of the control of protein dynamics in the design of functional artificial proteins.

Elementary tetrahelical protein design for diverse oxidoreductase functions

“…Another way is to permit water access to the oxyferrous heme state in the protein. H-D exchange, done on an earlier maquette, indicated rapid water access into the interior of the maquette 13,21,32 . Reanalysis of this maquette reveals a brief but detectable oxyferrous heme state decay with a t 1/2 of less than 0.1 s (Supplementary Fig.…”

Rational design of a zinc phthalocyanine binding protein

“…We termed the resultant proteins FHHH and HHHF, the H and F designating whether a histidine or a phenylalanine resides at the seventh position of each helix (see Figure 1B and 1C). We chose to remove these histidines as opposed to the ones on the second and third helices to reduce the probability of ZnPcS-mediated protein dimerization brought about by the rotation of ligand helix to a position facing solution, a motion more likely to occur on the less constrained terminal helices (Zhang et al, 2013). As shown below, both proteins are capable of forming the hemin:ZnPcS heterocomplex with the ZnPcS bound as a monomer.…”

“…We have chosen to start with the homodimeric four α-helix bundle protein HP7, which binds two heme cofactors at two bis-histidine heme ligation sites with one histidine ligand originating from each helix (see Figure 1) (Koder et al, 2009; Zhang et al, 2013; Zhang et al, 2011a). This protein is derived from a series of progenitor proteins which started with the prototype maquette protein H10H24 (Robertson et al, 1994), a tetrameric four α-helix bundle protein and progressed through the design intermediates BB (Gibney et al, 1997) and HP-1 (Huang et al, 2004).…”

Fast, cheap and out of control — Insights into thermodynamic and informatic constraints on natural protein sequences from de novo protein design

“…Some of the earliest experiments in biocatalyst design involved the creation of catalytic antibodies which catalyzed reactions originally catalyzed by enzymes with substantially different folds than that of an antibody [22]. We have implanted the oxygen transport function into a four alpha helix bundle fold [23–25], a function previously associated only with the globin fold (Figure 1). Both light-activated electron transfer [26, 27] and ligand-activated conformational switching [28] have similarly been implanted into four alpha helix bundles, functions only seen before in much more complex structures.…”