Rational Heme Protein Design: All Roads Lead to Rome

Heme proteins are among the most abundant and important metalloproteins, exerting diverse biological functions including oxygen transport, small molecule sensing, selective C? H bond activation, nitrite reduction, and electron transfer. Rational heme protein designs focus on the modification of the heme‐binding active site and the heme group, protein hybridization and domain swapping, and de novo design. These strategies not only provide us with unique advantages for illustrating the structure–property–reactivity–function (SPRF) relationship of heme proteins in nature but also endow us with the ability to create novel biocatalysts and biosensors.     

Functional tuning and expanding of myoglobin by rational protein design

Rational protein design is a powerful strategy, not only for revealing the structure and function relationship of natural metallo-proteins, but also for creating artificial metalloproteins with improved properties and functions. Myoglobin (Mb), a small heme protein created by nature with diverse functions, has been shown to be an ideal scaffold for rational protein design. The progress reviewed herein includes fine-tuning its native functions of O₂ binding and transport, peroxidase activity and nitrite reductase (NIR) activity, and rational expanding its functionalities to peroxygenase, heme-copper oxidase (HCO), nitric oxide reductase (NOR), as well as hydroxylamine reductase. These studies have enhanced our understanding of how metalloproteins work in nature, and provided insights for rational design of functional metalloproteins for practical applications in the future.     

Catalysis and Electron Transfer in De Novo Designed Helical Scaffolds

The relationship between protein structure and function is one of the greatest puzzles within biochemistry. De novo metalloprotein design is a way to wipe the board clean and determine what is required to build in function from the ground up in an unrelated structure. This Review focuses on protein design efforts to create de novo metalloproteins within alpha‐helical scaffolds. Examples of successful designs include those with carbonic anhydrase or nitrite reductase activity by incorporating a ZnHis₃ or CuHis₃ site, or that recapitulate the spectroscopic properties of unique electron‐transfer sites in cupredoxins (CuHis₂Cys) or rubredoxins (FeCys₄). This work showcases the versatility of alpha helices as scaffolds for metalloprotein design and the progress that is possible through careful rational design. Our studies cover the invariance of carbonic anhydrase activity with different site positions and scaffolds, refinement of our cupredoxin models, and enhancement of nitrite reductase activity up to 1000‐fold.     

Metal-Templated Design of Chemically Switchable Protein Assemblies with High-Affinity Coordination Sites

To mimic a hypothetical pathway for protein evolution, we previously tailored a monomeric protein (cyt cb₅₆₂) for metal-mediated self-assembly, followed by re-design of the resulting oligomers for enhanced stability and metal-based functions. We show that a single hydrophobic mutation on the cyt cb₅₆₂ surface drastically alters the outcome of metal-directed oligomerization to yield a new trimeric architecture, (TriCyt1)_3. This nascent trimer was redesigned into second and third-generation variants (TriCyt2)₃ and (TriCyt3)₃ with increased structural stability and preorganization for metal coordination. The three TriCyt variants combined furnish a unique platform to 1) provide tunable coupling between protein quaternary structure and metal coordination, 2) enable the construction of metal/pH-switchable protein oligomerization motifs, and 3) generate a robust metal coordination site that can coordinate all mid-to-late first-row transition-metal ions with high affinity.     

De novo design, synthesis, and function of semiartificial myoglobin conjugated with coiled-coil two-alpha-helix peptides

The introduction of a flavin chromophore on the myoglobin (Mb) surface and an effective electron-transfer (ET) reaction through the flavin were successfully achieved by utilizing the self-assembly of heterostranded coiled-coil peptides. We have prepared a semiartificial Mb, named Mb-1alphaK, in which an amphiphilic and cationic alpha-helix peptide is conjugated at the heme propionate (Heme-1alphaK). Heme-1alphaK has a covalently bound iron-protoporphyrin IX (heme) at the N terminus of a 1alphaK peptide sequence. This sequence was designed to form a heterostranded coiled-coil in the presence of a counterpart amphiphilic and anionic 1alphaE peptide sequence in a parallel orientation. Two peptides, Fla(1)-1alphaE and Fla(31)-1alphaE, both incorporating a 10-methylisoalloxazine moiety as an artificial flavin molecule, were also prepared (Fla=2-[7-(10-methyl)isoalloxazinyl]-2-oxoethyl). Heme-1alphaK was successfully inserted into apomyoglobin to give Mb-1alphaK. Mb-1alphaK recognized the flavin-modified peptides and a two-alpha-helix structure was formed. In addition, an efficient ET from reduced nicotinamide adenine dinucleotide to the heme center through the flavin unit was observed. The ET rate was faster in the presence of Fla(1)-1alphaE than in the presence of Fla(31)-1alphaE or the equivalent molecule that has no peptide chain. These results demonstrate that the introduction of a functional chromophore on the Mb surface can be achieved by using specific peptide-peptide interactions. Moreover, the dependence of the ET rate on the position of the flavin indicated that the distance between the heme active site and the flavin chromophore was regulated by the three-dimensional structure of the designed polypeptide.     

Regulating the Coordination State of a Heme Protein by a Designed Distal Hydrogen-Bonding Network

Heme coordination state determines the functional diversity of heme proteins. Using myoglobin as a model protein, we designed a distal hydrogen-bonding network by introducing both distal glutamic acid (Glu29) and histidine (His43) residues and regulated the heme into a bis-His coordination state with native ligands His64 and His93. This resembles the heme site in natural bis-His coordinated heme proteins such as cytoglobin and neuroglobin. A single mutation of L29E or F43H was found to form a distinct hydrogen-bonding network involving distal water molecules, instead of the bis-His heme coordination, which highlights the importance of the combination of multiple hydrogen-bonding interactions to regulate the heme coordination state. Kinetic studies further revealed that direct coordination of distal His64 to the heme iron negatively regulates fluoride binding and hydrogen peroxide activation by competing with the exogenous ligands. The new approach developed in this study can be generally applicable for fine-tuning the structure and function of heme proteins.     

Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique

Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme‐protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high‐yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non‐canonical amino acid p‐carboxymethyl‐L ‐phenylalanine (pCMF) and its efficient site‐specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe?Met bond in the ferric form of cytochrome c, thereby lowering the pK_a value for the alkaline transition of the heme‐protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.     

Design of Peptidic Foldamer Helices: A Stereochemical Patterning Approach

Assembly language : The programmed sequences of stereochemical building blocks lead to novel biomimetic helices. The rational design approach offers new possibilities for creating periodic secondary structures.

     

Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy

Magic‐angle spinning solid‐state NMR spectroscopy has been applied to study the dynamics of CBM3b–Cbh9A from Clostridium thermocellum (ctCBM3b), a cellulose binding module protein. This 146‐residue protein has a nine‐stranded β‐sandwich fold, in which 35 % of the residues are in the β‐sheet and the remainder are composed of loops and turns. Dynamically averaged ¹H‐¹³C dipolar coupling order parameters were extracted in a site‐specific manner by using a pseudo‐three‐dimensional constant‐time recoupled separated‐local‐field experiment (dipolar‐chemical shift correlation experiment; DIPSHIFT). The backbone‐Cα and Cβ order parameters indicate that the majority of the protein, including turns, is rigid despite having a high content of loops; this suggests that restricted motions of the turns stabilize the loops and create a rigid structure. Water molecules, located in the crystalline interface between protein units, induce an increased dynamics of the interface residues thereby lubricating crystal water‐mediated contacts, whereas other crystal contacts remain rigid.