In Vitro and Cellular Self-Assembly of a Zn-Binding Protein Cryptand via Templated Disulfide Bonds

Medina-Morales, A.M.; Pérez, Alfredo; Brodin, Jeffrey D.; Tezcan, F.A.

doi:10.1021/ja405318d

Cited by 35 publications

(57 citation statements)

References 77 publications

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“…24 The competitive binding isotherm showed the expected stoichiometry of four Zn 2+ ions per tetramer, and could be fit by a thermodynamic model with two pairs of equivalent Zn-binding sites and two corresponding equilibrium constants ( K d = 8.1±0.4 and 400±100 nM; overall ΔG Zn = – 164±1 kJ/mol) (Tables 1 and S1). These values indicate a significantly diminished Zn-binding affinity for C38/C81/C96 R1 4 compared to the previously characterized tetramers C96 R1 4 (ΔG Zn = –186±2 kJ/mol) and C81/C96 R1 4 (ΔG Zn = –183±1 kJ/mol), 24, 25 which feature one and two pairs of disulfide bonds, respectively. These findings indicate that the presence of all three pairs of interfacial disulfide bonds imparts a strain at the level of the quaternary architecture, manifested in the destabilization of Zn 2+ coordination by ∼20 kJ/mol.…”

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confidence: 51%

De Novo Design of an Allosteric Metalloprotein Assembly with Strained Disulfide Bonds

et al. 2016

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A major goal in metalloprotein design is to build protein scaffolds from scratch that allow precise control over metal coordination. A particular challenge in this regard is the construction of allosteric systems in which metal coordination equilibria are coupled to other chemical events that take place elsewhere in the protein scaffold. We previously developed a metal-templated self-assembly strategy (MeTIR) to build supramolecular protein complexes with tailorable interfaces from monomeric building blocks. Here, using this strategy, we have incorporated multiple disulfide bonds into the interfaces of a Zn-templated cytochrome cb562 assembly in order to create mechanical strain on the quaternary structural level. Structural and biophysical analyses indicate that this strain leads to an allosteric system in which Zn2+ binding and dissociation is remotely coupled to the formation and breakage of a disulfide bond over a distance of >14 Å. The breakage of this strained bond upon Zn2+ dissociation occurs in the absence of any reductants, apparently through a hydrolytic mechanism that generates a sulfenic acid/thiol pair.

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confidence: 51%

De Novo Design of an Allosteric Metalloprotein Assembly with Strained Disulfide Bonds

et al. 2016

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“…However, this task is complicated by the fact that proteins are complex macromolecules that do not possess many canonical interaction motifs with which to program their organization. A number of strategies have been devised toward this end, including computational design of associative surfaces (8), the construction of chimeric protein assemblies with preprogrammed symmetries (9,10), and those that use specific ligand-protein interactions (11)(12)(13)(14), metal coordination (15)(16)(17), disulfide bonds (18)(19)(20), or a combination of these strategies (21,22) to connect protein building blocks. These approaches have produced supramolecular architectures with increasing structural sophistication or improved functions.…”

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confidence: 99%

Exceptionally stable, redox-active supramolecular protein assemblies with emergent properties

Brodin

Carr

Sontz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The designed assembly of proteins into well-defined supramolecular architectures not only tests our understanding of proteinprotein interactions, but it also provides an opportunity to tailor materials with new physical and chemical properties. Previously, we described that RIDC3, a designed variant of the monomeric electron transfer protein cytochrome cb 562 , could self-assemble through Zn 2+ coordination into uniform 1D nanotubes or 2D arrays with crystalline order. Here we show that these 1D and 2D RIDC3 assemblies display very high chemical stabilities owing to their metal-mediated frameworks, maintaining their structural order in ≥90% (vol/vol) of several polar organic solvents including tetrahydrofuran (THF) and isopropanol (iPrOH). In contrast, the unassembled RIDC3 monomers denature in ∼30% THF and 50% iPrOH, indicating that metal-mediated self-assembly also leads to considerable stabilization of the individual building blocks. The 1D and 2D RIDC3 assemblies are highly thermostable as well, remaining intact at up to ∼70°C and ∼90°C, respectively. The 1D nanotubes cleanly convert into the 2D arrays on heating above 70°C, a rare example of a thermal crystalline-to-crystalline conversion in a biomolecular assembly. Finally, we demonstrate that the Zn-directed RIDC3 assemblies can be used to spatiotemporally control the templated growth of small Pt 0 nanocrystals. This emergent function is enabled by and absolutely dependent on both the supramolecular assembly of RIDC3 molecules (to form a periodically organized structural template) and their innate redox activities (to direct Pt 2+ reduction).protein self-assembly | supramolecular coordination chemistry | nanomaterials | biomaterials | inorganic nanoparticles T he enormous structural and chemical diversity of proteins makes them highly attractive building blocks for functional materials. Supramolecular protein assemblies constitute the major components of cellular machinery, and many of them [e.g., 0D ferritin (1), 1D silicatein filaments (2, 3), 2D S-layers (4)] have also found a number of applications in nano-and biotechnology (2, 4, 5). Such protein assemblies are typically highly ordered, yet they possess dynamic structures that respond to external stimuli (6). Distinctively, protein assemblies are innately functional: the individual building blocks of these assemblies often perform specific chemical functions. For instance, the monomeric components of silicatein filaments or ferritin cages each have catalytic sites that process the precursors (SiO 2 or Fe 2+ ) for the downstream biomineralization processes (1, 3). On self-assembly, these individual components can be significantly stabilized (allowing them to withstand the extracellular environment and perform their functions) (7), and their chemical function takes on an entirely different context within the supramolecular architecture (acting as the template for biomineralization in the cases of silicatein and ferritin). Thus, they display emergent properties, both structurally and functionally.Such a...

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“…In the case of Zn 4 :RIDC1 4 , the T96/T96’ residue pairs across the i2 interfaces and the E81/E81’ across the i3 interfaces were both suitable for this purpose. Accordingly, we generated a single-cysteine ( C96 RIDC1) (Brodin, Medina-Morales, Ni, Salgado, Ambroggio, & Tezcan, 2010) and a double-cysteine ( C81/C96 RIDC1) (Medina-Morales, Perez, Brodin, & Tezcan, 2013) variant which properly self-assembled into stable metal-free tetramers ( C96 RIDC1 4 and C81/C96 RIDC1 4 ). Moreover, these tetramers adopted the same structure as the parent tetramer Zn 4 :MBPC1 4 upon binding Zn 2+ .…”

Section: Metal-templated Interface Redesignmentioning

confidence: 99%

“…Importantly, the quadruply-disulfide-linked C81/C96 RIDC1 4 scaffold is sufficiently preorganized to permit altering the inner-coordination sphere of the templating Zn ions without perturbing the quaternary structure ( Fig. 5 A-C ) (Medina-Morales, et al, 2013). …”

Section: Metal-templated Interface Redesignmentioning

confidence: 99%

Metal-Directed Design of Supramolecular Protein Assemblies

Bailey

Subramanian

Churchfield

et al. 2016

Methods in Enzymology

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Owing to their central roles in cellular signaling, construction, and biochemistry, protein-protein interactions (PPIs) and protein self-assembly have become a major focus of molecular design and synthetic biology. In order to circumvent the complexity of constructing extensive non-covalent interfaces, which are typically involved in natural PPIs and protein self-assembly, we have developed two design strategies, Metal-Directed Protein Self-Assembly (MDPSA) and Metal-Templated Interface Redesign (MeTIR). These strategies, inspired by both the proposed evolutionary roles of metals and their prevalence in natural PPIs, take advantage of the favorable properties of metal coordination (bonding strength, directionality, and reversibility) to guide protein self-assembly with minimal design and engineering. Using a small, monomeric protein (cytochrome cb562) as a model building block, we employed MDPSA and MeTIR to create a diverse array of functional supramolecular architectures which range from structurally tunable oligomers to metalloprotein complexes that can properly self-assemble in living cells into novel metalloenzymes. The design principles and strategies outlined herein should be readily applicable to other protein systems with the goal of creating new PPIs and protein assemblies with structures and functions not yet produced by natural evolution.

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In Vitro and Cellular Self-Assembly of a Zn-Binding Protein Cryptand via Templated Disulfide Bonds

Cited by 35 publications

References 77 publications

De Novo Design of an Allosteric Metalloprotein Assembly with Strained Disulfide Bonds

De Novo Design of an Allosteric Metalloprotein Assembly with Strained Disulfide Bonds

Exceptionally stable, redox-active supramolecular protein assemblies with emergent properties

Metal-Directed Design of Supramolecular Protein Assemblies

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