Methods for Solving Highly Symmetric De Novo Designed Metalloproteins

Ruckthong, Leela; Stuckey, Jeanne A.; Pecoraro, Vincent L.

doi:10.1016/bs.mie.2016.05.007

Cited by 6 publications

(4 citation statements)

References 39 publications

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“…Our laboratory has previously used the strategy of increasing α -helical length in three-stranded coiled coil constructs to, at least empirically, improve the success rate of crystallization, and this seems to have proven true of threehelical bundle designs as well. 79 …”

Section: Discussionmentioning

confidence: 99%

Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein

et al. 2018

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Cupredoxins are copper-dependent electron-transfer proteins that can be categorized as blue, purple, green, and red depending on the spectroscopic properties of the Cu(II) bound forms. Interestingly, despite significantly different first coordination spheres and nuclearity, all cupredoxins share a common Greek Key β-sheet fold. We have previously reported the design of a red copper protein within a completely distinct three-helical bundle protein, α3DChC2.1 While this design demonstrated that a β-barrel fold was not requisite to recapitulate the properties of a native cupredoxin center, the parent peptide α3D was not sufficiently stable to allow further study through additional mutations. Here we present the design of an elongated protein GRANDα3D (GRα3D) with ΔGu = −11.4 kcal/mol compared to the original design’s −5.1 kcal/mol. Diffraction quality crystals were grown of GRα3D (a first for an α3D peptide) and solved to a resolution of 1.34 Å. Examination of this structure suggested that Glu41 might interact with the Cu in our previously reported red copper protein. The previous bis(histidine)(cysteine) site (GRα3DChC2) was designed into this new scaffold and a series of variant constructs were made to explore this hypothesis. Mutation studies around Glu41 not only prove the proposed interaction, but also enabled tuning of the constructs’ hyperfine coupling constant from 160 to 127 × 10−4 cm−1. X-ray absorption spectroscopy analysis is consistent with these hyperfine coupling differences being the result of variant 4p mixing related to coordination geometry changes. These studies not only prove that an Glu41–Cu interaction leads to the α3DChC2 construct’s red copper protein like spectral properties, but also exemplify the exact control one can have in a de novo construct to tune the properties of an electron-transfer Cu site.

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Section: Discussionmentioning

confidence: 99%

Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein

et al. 2018

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“…The side chains of the model were included with the D-Leu residue at the twelfth position mutated to L-Leu. 73 Electron difference density maps ( F o – F c ) contoured at 3 σ show two possible metal sites at the 16th (16Cys) and the 30th (30His) positions corresponding to Hg(II) and Zn(II) ions, respectively. These positions were confirmed by solving the structure by single anomalous dispersion using AutoSol in Phenix.…”

Section: Methodsmentioning

confidence: 99%

“…The Hg(II) S Zn(II) N (GRAND-CSL16CL30H) 3 + structure has a Matthews’ coefficient (2.41) consistent with 49.05% solvent content per ASU and was solved using the five heptads of GRAND-CSL12 D L16C as a search model. The side chains of the model were included with the d -Leu residue at the twelfth position mutated to l -Leu . Electron difference density maps ( F o – F c ) contoured at 3σ show two possible metal sites at the 16th (16Cys) and the 30th (30His) positions corresponding to Hg(II) and Zn(II) ions, respectively.…”

Section: Materials and Methodsmentioning

confidence: 99%

A Crystallographic Examination of Predisposition versus Preorganization in de Novo Designed Metalloproteins

et al. 2016

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Preorganization and predisposition are important molecular recognition concepts exploited by nature to obtain site-specific and selective metal binding to proteins. While native structures containing an MS3 core are often unavailable in both apo- and holo-forms, one can use designed three-stranded coiled coils (3SCCs) containing tris-thiolate sites to evaluate these concepts. We show that the preferred metal geometry dictates the degree to which the cysteine rotamers change upon metal complexation. The Cys ligands in the apo-form are preorganized for binding trigonal pyramidal species (Pb(II)S3 and As(III)S3) in an endo conformation oriented toward the 3SCC C-termini, whereas the cysteines are predisposed for trigonal planar Hg(II)S3 and 4-coordinate Zn(II)S3O structures, requiring significant thiol rotation for metal binding. This study allows assessment of the importance of protein fold and side-chain reorientation for achieving metal selectivity in human retrotransposons and metalloregulatory proteins.

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“…The structure was solved using ap reviously published method. [48] The structure was refined to 1.42 (R working = 19.6 %, R free = 20.3 %).…”

Section: Data Collections and Refinementsmentioning

confidence: 99%

How Outer Coordination Sphere Modifications Can Impact Metal Structures in Proteins: A Crystallographic Evaluation

Ruckthong

Stuckey

Pecoraro

2019

Chemistry A European J

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A challenging objective of de Novo metalloprotein design is to control of the outer coordination spheres of an active site to fine tune metal properties. The well-defined three stranded coiled coils, TRI and CoilSer peptides, are used to address this question. Substitution of Cys for Leu yields a thiophilic site within the core. Metals such as Hg(II), Pb(II) and As(III) result in trigonal planar or trigonal pyramidal geometries; however, spectroscopic studies showed Cd(II) formed 3-, 4- or 5-coordinate Cd(II)S3(OH2)x (where x=0–2) when the outer coordination spheres were perturbed. Unfortunately, there has been little crystallographic examination of these proteins to explain the observations. Herein, we compare the high-resolution x-ray structures of apo- and mercurated proteins to explain the modifications that lead to metal coordination number and geometry variation.It reveals that Ala substitution for Leu opens a cavity above the Cys site allowing for water excess, facilitating Cd(II)S3(OH2).Replacement of Cys by Pen restricts thiol rotation, causing a shift in the metal binding plane that displaces water, forming Cd(II)S3.D-Leu, above the Cys site, reorients the side chain towards the Cys layer diminishing the space for water accommodation yielding Cd(II)S3, while D-Leu below opens more space, allowing for equal Cd(II)S3(OH2) and Cd(II)S3(OH2)2.These studies provide insights on how to control desired metal geometries in metalloproteins using coded and non-coded amino acids.

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Methods for Solving Highly Symmetric De Novo Designed Metalloproteins

Cited by 6 publications

References 39 publications

Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein

Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein

A Crystallographic Examination of Predisposition versus Preorganization in de Novo Designed Metalloproteins

How Outer Coordination Sphere Modifications Can Impact Metal Structures in Proteins: A Crystallographic Evaluation

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