Site-directed mutagenesis of the highly perturbed copper site of auracyanin D

King, Jeremy D.; Harrington, Lucas B.; Lada, Bryan M.; He, Guannan; Cooley, Jason W.; Blankenship, Robert E.

doi:10.1016/j.abb.2014.10.003

Cited by 8 publications

(8 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although strategies based on the coupled distortion model allowed us to recapitulate green and blue optical absorption spectroscopic properties within a de novo construct, the correlation of Cu(II)–Cys bond strength to the optical absorption profile does not hold in our alpha helical scaffold. Recently, several green cupredoxins have been discovered that do not include the complication of a second type 2 copper site within the same protein. − EXAFS studies of WT and axial mutation variants of these new cupredoxins will be an important addition to our understanding of how structure dictates optical absorption spectroscopic properties in green copper proteins.…”

Section: Discussionmentioning

confidence: 99%

Traversing the Red–Green–Blue Color Spectrum in Rationally Designed Cupredoxins

et al. 2020

View full text Add to dashboard Cite

Blue copper proteins have a constrained Cu(II) geometry that has proven difficult to recapitulate outside native cupredoxin folds. Previous work has successfully designed green copper proteins which could be tuned blue using exogenous ligands, but the question of how one can create a self-contained blue copper site within a de novo scaffold, especially one removed from a cupredoxin fold, remained. We have recently reported a red copper protein site within a three helical bundle scaffold which we later revisited and determined to be a nitrosocyanin mimic, with a CuHis 2 CysGlu binding site. We now report efforts to rationally design this construct toward either green or blue copper chromophores using mutation strategies that have proven successful in native cupredoxins. By rotating the metal binding site, we created a de novo green copper protein. This in turn was converted to a blue copper protein by removing an axial methionine. Following this rational sequence, we have successfully created red, green, and blue copper proteins within an alpha helical fold, enabling comparisons for the first time of their structure and function disconnected from the overall cupredoxin fold.

show abstract

Section: Discussionmentioning

confidence: 99%

Traversing the Red–Green–Blue Color Spectrum in Rationally Designed Cupredoxins

et al. 2020

View full text Add to dashboard Cite

show abstract

“…3 indicates the presence of at least 3 absorption regions at 411 nm (24,331 cm 21 ), 581 (17,211 cm 21 ), and 721 nm (13,870 cm 21 ), which correspond to violet, yellow, and red light, respectively. Other spectroscopically characterized green copper proteins contain perturbed type I blue copper sites (32)(33)(34) where the relative intensity of transitions near 450 nm and 600 nm govern the apparent color. These are attributed to cysteine sulfur sor p-to-Cu charge transfer transitions (30).…”

Section: Discussionmentioning

confidence: 99%

The bacterial copper resistance protein CopG contains a cysteine-bridged tetranuclear copper cluster

Hausrath

Ramirez

et al. 2020

Journal of Biological Chemistry

View full text Add to dashboard Cite

CopG is an uncharacterized protein ubiquitous in gram-negative bacteria whose gene frequently occurs in clusters of copper-resistance genes and can be recognized by the presence of a conserved CxCC motif. To investigate its contribution to copper resistance, here we undertook a structural and biochemical characterization of the CopG protein from Pseudomonas aeruginosa.Results from biochemical analyses of CopG purified under aerobic conditions indicate that it is a green copper-binding protein that displays absorbance maxima near 411, 581, and 721 nm and is monomeric in solution. Determination of the three-dimensional structure by X-ray crystallography revealed that CopG consists of a thioredoxin domain with a C-terminal extension that contributes to metal binding. We noted that adjacent to the CxCC motif is a cluster of four copper ions bridged by cysteine sulfur atoms. Structures of CopG in two oxidation states support the assignment of this protein as an oxidoreductase. On the basis of these structural and spectroscopic findings and also genetic evidence, we propose that CopG has a role in interconverting Cu(I) and Cu(II) to minimize toxic effects and facilitate export by the Cus RND transporter efflux system.

show abstract

“…Cupredoxins are copper electron-transfer proteins found within bacteria, algae, animals, and plants − that can be characterized into blue, − green, − red, and binuclear purple − copper proteins depending on the spectroscopic properties of the Cu(II)-bound forms. These proteins quickly garnered the attention of the bioinorganic community due to their unique UV–vis absorption spectra with high-intensity bands at 400–450 and 550–600 nm (later assigned to σ and π ligand-to-metal charge-transfer (LMCT) bands, respectively) and unusual electron paramagnetic resonance (EPR) signals with compressed parallel hyperfine constant below 100 × 10 –4 cm 1 . ,, The ratio between σ and π LMCT intensities is often used as a qualifier between blue/perturbed blue or type 1 copper proteins, green/purple or type 1.5 copper proteins, and red or type 2 copper proteins. ,, …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

View full text Add to dashboard Cite

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.

show abstract

Site-directed mutagenesis of the highly perturbed copper site of auracyanin D

Cited by 8 publications

References 46 publications

Traversing the Red–Green–Blue Color Spectrum in Rationally Designed Cupredoxins

Traversing the Red–Green–Blue Color Spectrum in Rationally Designed Cupredoxins

The bacterial copper resistance protein CopG contains a cysteine-bridged tetranuclear copper cluster

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

Contact Info

Product

Resources

About