Mutter, A. C. | bioRxiv Science

bioRxiv2026

Loop-Directed Cofactor Binding Enables Programmable Multico-factor Protein Design

The emergence of respiratory, photosynthetic, and assimilatory complexes in evolution required proteins capable of binding multiple catalytic and electron-transfer cofactors while exerting fine control over their spatial arrangement. Across natural systems these cofactors are preferentially positioned in loop regions. In contrast, most protein design strategies have focused on installing cofactor-binding sites within helical elements. Here we show that introducing only a pair of appropriately placed histidine ligands into the interhelical loop regions of a canonical single-chain four-helix bundle is sufficient to create new well-defined high affinity heterocofactor binding sites. This simple modification enables the self-assembly of complexes containing up to three distinct cofactors in a single designed domain with positional specificity. Using this strategy, we created constructs containing one or two hemes in combination with Zn(II)-phthalocyanine monosulfonate, Zn-heme, and the light-harvesting Zn(II)-tetraphenylporphyrin tetrasulfonate. Fluorescence measurements of constructs containing the latter show efficient energy transfer between photoactive donor cofactors. By demonstrating that loop-embedded ligands support robust, modular, and evolutionarily plausible cofactor recruitment, this work provides a mechanistic explanation for the widespread placement of redox and catalytic cofactors in loops in natural proteins: only limited packing complementarity is needed, meaning that just a few mutations can introduce a functional cofactor binding site, after which additional mutations can tune affinity, reactivity, and specificity. More importantly, it establishes a straightforward path toward constructing functional protein domains that mirror the complexity of biological energy-conversion architectures. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/734619v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@e48a58org.highwire.dtl.DTLVardef@36b289org.highwire.dtl.DTLVardef@1034495org.highwire.dtl.DTLVardef@18bb284_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Protein Dynamics Govern the Oxyferrous State Lifetime of an Artificial Oxygen Transport Protein

It has long been known that the alteration of protein side chains which occlude or expose the heme cofactor to water can greatly affect the stability of the oxyferrous heme state. Here we demonstrate that the rate of dynamically-driven water penetration into the core of an artificial oxygen transport protein also correlates with oxyferrous state lifetime by reducing global dynamics, without altering the structure of the active site, via the simple linking of the two monomers in a homodimeric artificial oxygen transport protein using a glycine-rich loop. The tethering of these two helices does not significantly affect the active site structure, pentacoordinate heme binding affinity, reduction potential, or gaseous ligand affinity. It does, however, significantly reduce the hydration of the protein core as demonstrated by resonance Raman spectroscopy, backbone amide hydrogen exchange, and pKa shifts in buried histidine side chains. This further destabilizes the charge-buried entatic state and nearly triples the oxyferrous state lifetime. These data are the first direct evidence that dynamically-driven water penetration is a rate-limiting step in the oxidation of these complexes. It furthermore demonstrates that structural rigidity which limits water penetration is a critical design feature in metalloenzyme construction and provides an explanation for both the failures and successes of earlier attempts to create oxygen-binding proteins. SignificanceThis communication sheds light on one of the more controversial areas in protein folding and design: the dynamic nature of the hydrophobic core and its relationship to metalloprotein function, in particular the relationship between dynamic solvent penetration into the protein core and the stability of metalloenzyme intermediates. We demonstrate that the basic tetrameric scaffold that is the classic helical bundle model for cofactor binding and activation can be easily upgraded to a more rigid, less dynamic, single chain helical bundle by merely taking the same helical sequences and converting it to a single chain protein connected by simple, nonoptimized glycine-rich loops. Importantly, our results explain the decades-long history of failure in the design of proteins capable of stably forming an oxyferrous state - the requirement for a protein large enough to protect the heme porphyrin surface with both structural specificity and sufficient structural rigidity to restrict water penetration into the protein core. Finally, we believe this is the first use of Deep UV Resonance Raman spectroscopy to monitor dynamic water penetration in a functional protein. This method may prove useful moving forward to many research groups.

biophysics↗