Cobalt(II) and zinc(II) binding to a ferredoxin maquette

Kennedy, Michelle; Petros, Amy K.; Gibney, Brian R.

doi:10.1016/j.jinorgbio.2004.01.002

Cited by 13 publications

(11 citation statements)

References 34 publications

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“…In our experience, Fe 4 S 4 cubane clusters readily insert into Cys rich maquette loops containing a ferredoxin consensus motif CIGCGAC. Indeed, a short and flexible 16 amino acid sequence KLCEGGCIACGACGGW will spontaneously incorporate Fe 4 S 4 clusters (Gibney et al, 1996) as well as other metals such as Co (Kennedy, Petros, & Gibney, 2004). This sequence was inspired by short hexadecapeptide (Frausto da Silva & Williams, 2001) akin to sequences found in natural proteins.…”

Section: Iron–sulfur Cluster Cofactorsmentioning

confidence: 99%

De Novo Construction of Redox Active Proteins

Moser

Sheehan

Ennist

et al. 2016

Methods in Enzymology

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Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways.

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Section: Iron–sulfur Cluster Cofactorsmentioning

confidence: 99%

De Novo Construction of Redox Active Proteins

Moser

Sheehan

Ennist

et al. 2016

Methods in Enzymology

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“…Elucidation of the metal-binding thermodynamics using coordination chemistry methods provides the fundamental energetics relevant to the field of not only metalloprotein design 4 but also metal-induced protein folding and metal-ion storage, uptake, and delivery . Having employed the ferredoxin maquettes to reveal the essential engineering requirements of [4Fe−4S] proteins, herein we use these synthetic analogues to investigate the thermodynamics of thiolate-rich metalloprotein active-site metal-ion specificity and selectivity . The random-coil structure of the ferredoxin maquette presents a model system in which to study the inherent thermodynamic preferences of a Cys 4 metal-ion binding site in the absence of protein-folding contributions.…”

Section: Metal-ion Selectivity and Specificity In Thiolate-rich Desig...mentioning

confidence: 99%

Femtomolar Zn(II) Affinity in a Peptide-Based Ligand Designed To Model Thiolate-Rich Metalloprotein Active Sites

et al. 2006

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Metal-ligand interactions are critical components of metalloprotein assembly, folding, stability, electrochemistry, and catalytic function. Research over the past 3 decades on the interaction of metals with peptide and protein ligands has progressed from the characterization of amino acid-metal and polypeptide-metal complexes to the design of folded protein scaffolds containing multiple metal cofactors. De novo metalloprotein design has emerged as a valuable tool both for the modular synthesis of these complex metalloproteins and for revealing the fundamental tenets of metalloprotein structure-function relationships. Our research has focused on using the coordination chemistry of de novo designed metalloproteins to probe the interactions of metal cofactors with protein ligands relevant to biological phenomena. Herein, we present a detailed thermodynamic analysis of Fe(II), Co(II), Zn(II), and[4Fe-4S]2(+/+) binding to IGA, a 16 amino acid peptide ligand containing four cysteine residues, H2N-KLCEGG-CIGCGAC-GGW-CONH2. These studies were conducted to delineate the inherent metal-ion preferences of this unfolded tetrathiolate peptide ligand as well as to evaluate the role of the solution pH on metal-peptide complex speciation. The [4Fe-4S]2(+/+)-IGA complex is both an excellent peptide-based synthetic analogue for natural ferredoxins and is flexible enough to accommodate mononuclear metal-ion binding. Incorporation of a single ferrous ion provides the FeII-IGA complex, a spectroscopic model of a reduced rubredoxin active site that possesses limited stability in aqueous buffers. As expected based on the Irving-Williams series and hard-soft acid-base theory, the Co(II) and Zn(II) complexes of IGA are significantly more stable than the Fe(II) complex. Direct proton competition experiments, coupled with determinations of the conditional dissociation constants over a range of pH values, fully define the thermodynamic stabilities and speciation of each MII-IGA complex. The data demonstrate that FeII-IGA and CoII-IGA have formation constant values of 5.0 x 10(8) and 4.2 x 10(11) M-1, which are highly attenuated at physiological pH values. The data also evince that the formation constant for ZnII-IGA is 8.0 x 10(15) M-1, a value that exceeds the tightest natural protein Zn(II)-binding affinities. The formation constant demonstrates that the metal-ligand binding energy of a ZnII(S-Cys)4 site can stabilize a metalloprotein by -21.6 kcal/mol. Rigorous thermodynamic analyses such as those demonstrated here are critical to current research efforts in metalloprotein design, metal-induced protein folding, and metal-ion trafficking.

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“…The Dutton and Gibney groups have used four-helix bundles as maquettes to study heme containing proteins 6–9. Gibney and coworkers also used the maquette motifs to develop a prototype ferredoxin maquette, a peptide-based synthetic analogue of natural [4Fe-4S] 2+/+ proteins 10. DeGrado,11,12 Ghirlanda,13 Lombardi and Pavone,14 Mihara,15,16 and their co-workers also used this approach to prepare different coiled coils and helical bundles which can bind between one and four heme units depending on the design.…”

Section: Introductionmentioning

confidence: 99%

Controlling and Fine Tuning the Physical Properties of Two Identical Metal Coordination Sites in De Novo Designed Three Stranded Coiled Coil Peptides

et al. 2010

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Herein we report how de novo designed peptides can be used to investigate whether the position of a metal site along a linear sequence that folds into a three stranded α-helical coiled coil defines the physical properties of Cd(II) ions in either CdS 3 or CdS 3 O (O-being an exogenous water molecule) coordination environments. Peptides are presented that bind Cd(II) into two identical coordination sites that are located at different topological positions at the interior of these constructs. The peptide GRANDL16PenL19IL23PenL26I binds two Cd(II) as trigonal planar 3-coordinate CdS 3 structures whereas GRANDL12AL16CL26AL30C sequesters two Cd(II) as pseudotetrahedral 4-coordinate CdS 3 O structures. We demonstrate how for the first peptide, having a more rigid structure, the location of the identical binding sites along the linear sequence does not affect the physical properties of the two bound Cd(II). However, because these are rigid aggregates, the sites are not completely independent as Cd(II) bound to one of the sites ( GRANDL12AL16CL26AL30C shows a completely different behavior. The physical properties of the two bound Cd(II) ions indeed depend on the position of the metal center, having pK a2 values for the equilibrium [Cd(pep)(Hpep) 2 ] + → [Cd(pep) 3 ] − + 2H + (corresponding to deprotonation and coordination of cysteine thiols) that range from 9.9 to 13.9. In addition, the L26AL30C site shows dynamic behavior, which is not observed for the L12AL16C site. These results indicate that for Correspondence to: Vincent L. Pecoraro. # These authors contributed equally. Supporting Information Available:Model and derivation of the pK a2 fitting equations for the dual site peptides, Table of the thermodynamic parameters determined from GuHCl-induced unfolding curves of peptides GRANDL26AL30C, GRANDL12AL16C, and GRANDL12AL16CL26AL30C, GuHCl denaturation titration curves of GRANDL12AL16C, GRANDL26AL30C, GRANDL12AL16CL26AL30C and GRANDL12AL16CL26AL30CL33I at pH 6.5, 113 Cd NMR of [Cd(II)(H 2 O)] 16 [apo] 30 (GRANDL12AL16CL26AL30C) 3 − at pH 6.0, 113 Cd NMR of (GRANDL12AL16CL26AL30C) with 1.0 eq 113 Cd(II) at pH 8.5, NOESY spectrum of GRANDL12AL16CL26AL30C showing sequential assignments of backbone amide protons, sections of NOESY spectra for GRANDL12AL16CL26AL30C in the presence of 2.0 equivalents of Cd(II) at different pH values, overlay of simulated and experimental UV-Vis pH titration curves of GRANDL12AL16CL26AL30C and part of the assignment. This material is available free of charge via the Internet at http://pubs.acs.org.It should be noted that there is not a 100% correspondence between the 113 Cd NMR shifts and the 111m Cd PAC assessments of the CdS 3 /CdS 3 O ratio. Quantum chemical calculations show that a change in Cd-S bond length of 0.01 Å can cause a change in 113 Cd NMR chemical shift of ~20 ppm (Hemmingsen et.al. unpublished results). Thus, the PAC data often indicate a higher percentage of CdS 3 in these samples than one would predict using the strict NMR correlation. NIH Public Access

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Cobalt(II) and zinc(II) binding to a ferredoxin maquette

Cited by 13 publications

References 34 publications

De Novo Construction of Redox Active Proteins

De Novo Construction of Redox Active Proteins

Femtomolar Zn(II) Affinity in a Peptide-Based Ligand Designed To Model Thiolate-Rich Metalloprotein Active Sites

Controlling and Fine Tuning the Physical Properties of Two Identical Metal Coordination Sites in De Novo Designed Three Stranded Coiled Coil Peptides

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