Cyt Cb562 Research Articles

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.

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

AbstractTo 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.

Unfolding cytochromes c-b562 and Rd apo b562

We have analyzed the early stages of unfolding of cytochromes c-b562 (PDB ID: 2BC5) and Rd apo b562 (PDB ID: 1YYJ). Our geometrical approach proceeds from an analysis of the crystal structure reported for each protein. We quantify, residue-by-residue and region-by-region, the spatial and angular changes in the structure as the protein denatures, and quantify differences that result from the seven residues that differ in the two proteins. Using two independent analyses, one based on spatial metrics and the second on angular metrics, we establish the order of unfolding of the five helices in cyt c-b562 and the four helices in the apo protein. For the two helices nearest the N-terminal end of both proteins, the ones in the apo protein unfold first. For the two helices nearest the C-terminal end, the interior helix of the apo protein unfolds first, whereas the terminal helix of the holo protein unfolds first. Excluded-volume effects (repulsive interactions) are minimized in turning regions; the overall range in Δ values is Δ = 36.3 Å3 for cyt c-b562 and Δ = 36.6 Å3 for the apo protein, whereas the span for all 20 amino acids is Δ = 167.7 Å3. As our work indicates that the interior helix of cytochrome c-b562 is the first to fold, we suggest that this helix protects the heme from misligation, consistent with ultrafast folding over a minimally frustrated funneled landscape.

Design and Construction of Functional Supramolecular Metalloprotein Assemblies.

Nature puts to use only a small fraction of metal ions in the periodic table. Yet, when incorporated into protein scaffolds, this limited set of metal ions carry out innumerable cellular functions and execute essential biochemical transformations such as photochemical H2O oxidation, O2 or CO2 reduction, and N2 fixation, highlighting the outsized importance of metalloproteins in biology. Not surprisingly, elucidating the intricate interplay between metal ions and protein structures has been the focus of extensive structural and mechanistic scrutiny over the last several decades. As a result of such top-down efforts, we have gained a reasonably detailed understanding of how metal ions shape protein structures and how protein structures in turn influence metal reactivity. It is fair to say that we now have some idea-and in some cases, a good idea-about how most known metalloproteins function and we possess enough insight to quickly assess the modus operandi of newly discovered ones. However, translating this knowledge into an ability to construct functional metalloproteins from scratch represents a challenge at a whole different level: it is one thing to know how an automobile works; it is another to build one. In our quest to build new metalloproteins, we have taken an original approach in which folded, monomeric proteins are used as ligands or synthons for building supramolecular complexes through metal-mediated self-assembly (MDPSA, Metal-Directed Protein Self-Assembly). The interfaces in the resulting protein superstructures are subsequently tailored with covalent, noncovalent, or additional metal-coordination interactions for stabilization and incorporation of new functionalities (MeTIR, Metal Templated Interface Redesign). In an earlier Account, we had described the proof-of-principle studies for MDPSA and MeTIR, using a four-helix bundle, heme protein cytochrome cb562 (cyt cb562), as a model building block. By the end of those studies, we were able to demonstrate that a tetrameric, Zn-directed cyt cb562 complex (Zn4:M14) could be stabilized through computationally prescribed noncovalent interactions inserted into the nascent protein-protein interfaces. In this Account, we first describe the rationale and motivation for our particular metalloprotein engineering strategy and a brief summary of our earlier work. We then describe the next steps in the "evolution" of bioinorganic complexity on the Zn4:M14 scaffold, namely, (a) the generation of a self-standing protein assembly that can stably and selectively bind metal ions, (b) the creation of reactive metal centers within the protein assembly, and (c) the coupling of metal coordination and reactivity to external stimuli through allosteric effects.

Cytochrome unfolding pathways from computational analysis of crystal structures

We have developed a model to study the role of geometrical factors in influencing the early stages of unfolding in three cytochromes: cyt c′, cyt c-b562 and cyt c. Each stage in unfolding is quantified by the spatial extension λ̂i of n-residue segments, and by their angular extension 〈βn〉. Similarities and differences between and among the three cytochromes in the unfolding of helical and non-helical regions can be determined by analyzing the data for each signature separately. Definite conclusions can be drawn when spatial and angular changes are considered in tandem. To facilitate comparisons, we present graphical portraits of the three cytochromes at the same stage of unfolding, and in relation to their native state structures. We also display specific segments at different stages of unfolding to illustrate differences in stability of defined domains thereby allowing us to make specific predictions on the unfolding of corresponding internal and terminal helices in cyt c′ and cyt c-b562. Our work accords with an earlier experimental report on the presence and persistence of a hydrophobic core in cyt c.

Domain-swapped cytochrome cb562 dimer and its nanocage encapsulating a Zn-SO4 cluster in the internal cavity.

Protein nanostructures have been gaining in interest, along with developments in new methods for construction of novel nanostructures. We have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping. Herein, we show that a four-helix bundle protein cyt cb562, with the cyt b562 heme attached to the protein moiety by two Cys residues insertion, forms a domain-swapped dimer. Dimeric cyt cb562 did not dissociate to monomers at 4 °C, whereas dimeric cyt b562 dissociated under the same conditions, showing that heme attachment to the protein moiety stabilizes the domain-swapped structure. According to X-ray crystallographic analysis of dimeric cyt cb562, the two helices in the N-terminal region of one protomer interacted with the other two helices in the C-terminal region of the other protomer, where Lys51-Asp54 served as a hinge loop. The heme coordination structure of the dimer was similar to that of the monomer. In the crystal, three domain-swapped cyt cb562 dimers formed a unique cage structure with a Zn-SO4 cluster inside the cavity. The Zn-SO4 cluster consisted of fifteen Zn2+ and seven SO42- ions, whereas six additional Zn2+ ions were detected inside the cavity. The cage structure was stabilized by coordination of the amino acid side chains of the dimers to the Zn2+ ions and connection of two four-helix bundle units through the conformation-adjustable hinge loop. These results show that domain swapping can be applied in the construction of unique protein nanostructures.

Stability and Folding Kinetics of Structurally Characterized Cytochrome c-b562,

The four-helix-bundle protein fold can be constructed from a wide variety of primary amino acid sequences. Proteins with this structure are excellent candidates for investigations of the relationship between folding mechanism and topology. The folding of cytochrome b(562), a four-helix-bundle heme protein, is hampered by heme dissociation. To overcome this complication, we have engineered a variant of cytochrome b(562) (cyt c-b(562)) featuring a c-type linkage between the heme and the polypeptide chain. The replacement of the native cyt b(562) leader sequence in this protein with that of a c-type cytochrome (cyt c(556)) led to high yields of fully matured and correctly folded cyt c-b(562). We have determined the X-ray crystal structure of cyt c-b(562) at 2.25 A and characterized its physical, chemical, and folding properties. These measurements reveal that the c-type linkage does not perturb the protein fold or reduction potential of the heme group. The covalent attachment of the porphyrin to the polypeptide does, however, produce a substantial change in protein stability and folding kinetics.