Synthesis and characterization of inner-sphere substitution products in azide-containing сobalt(III) dioximates

Coropceanu, Eduard B.; Bologa, O. A.; Arsene, Ion; Vitiu, A. A.; Bulhac, I.; Gorinchioy, N.; Боурош, П. Н.

doi:10.1134/s1070328416070046

Cited by 7 publications

(5 citation statements)

References 32 publications

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“…21 As previously described, 16 a parameter file for [Co(N 3 )] was derived from an existing small molecule structure. 22 For integration of [Co-(N 3 )] into our protein structure, the electronic Ligand Builder and Optimization Workbench, eLBOW, 23 in Phenix was subsequently employed to generate a coordinate file for the ligand. The geometry restraints for [Co(N 3 )] were strictly maintained due to the higher than average B-factors and presumably more disordered nature of the ligand atoms relative to those of the protein.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Using protein chain A of the available glutamine-free GlnBP structure (PDB: 1GGG) as a molecular replacement model, Phaser-MR calculated a single solution containing all six monomeric units with a log-likelihood gain (LLG) score of 1048 and a translation function Z score (TFZ) of 23.2 . As previously described, a parameter file for [Co(N 3 )] was derived from an existing small molecule structure . For integration of [Co(N 3 )] into our protein structure, the electronic Ligand Builder and Optimization Workbench, eLBOW, in Phenix was subsequently employed to generate a coordinate file for the ligand.…”

Section: Methodsmentioning

confidence: 99%

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Conformation-Dependent Hydrogen-Bonding Interactions in a Switchable Artificial Metalloprotein

Fatima,

Mehrafrooz,

Boggs

et al. 2024

Biochemistry

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Hydrogen-bonding (H-bonding) interactions in metalloprotein active sites can critically regulate enzyme function. Changes in the protein structure triggered by interplay with substrates, products, and partner proteins are often translated to the metallocofactor by way of specific changes in H-bond networks connected to the active site. However, the complexities of metalloprotein architecture and mechanism often preclude our ability to define the precise molecular interactions giving rise to these intricate regulatory pathways. To address this shortcoming, we have developed conformationally switchable artificial metalloproteins (swArMs) in which allosteric Gln-binding triggers protein conformational changes that impact the microenvironment surrounding an installed metallocofactor. Herein, we report a combined structural, spectroscopic, and computational approach to enhance the conformation-dependent changes in H-bond interactions surrounding the metallocofactor site of a swArM. Structure-informed molecular dynamics simulations were employed to predict point mutations that could enhance active site H-bond interactions preferentially in the Gln-bound holo-conformation of the swArM. Testing our predictions via the unique infrared spectral signals associated with the metallocofactor site, we have identified three key residues capable of imparting conformational control over the metallocofactor microenvironment. The resultant swArMs not only model biologically relevant structural regulation but also provide an enhanced Gln-responsive biological probe to be leveraged in future biosensing applications.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Conformation-Dependent Hydrogen-Bonding Interactions in a Switchable Artificial Metalloprotein

Fatima,

Mehrafrooz,

Boggs

et al. 2024

Biochemistry

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show abstract

“…Using Phenix-MR, a solution which contained all 12 monomeric subunits was determined that had an LLG score of 6276 and a Z score of 34. 34 For use in refinement, a small molecule parameter file for the [Co(N3)] complex was derived from a published structure (CCDC:1414770) 35 and the modeled ligand was generated using the electronic Ligand Builder and Optimization Workbench, eLBOW, 36 in Phenix. COOT 37 was used to manually refine the positions and geometry of the protein residues and add the [Co(N3)] ligand in the early stages of refinement.…”

Section: Methodsmentioning

confidence: 99%

Engineering a Conformationally Switchable Artificial Metalloprotein

Fatima

Boggs

Thompson

et al. 2022

Preprint

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Most naturally occurring metalloenzymes are gated by rate-limiting conformational changes and there exists a critical inter-play between macroscopic structural rearrangements of the protein, and subatomic changes affecting the electronic struc-ture of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structur-ally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conforma-tionally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime, X = N3–, H3C–, iPr–). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co‒S cysteine ligation within the E. coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction (2.99 Å resolu-tion) unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence and circular dichroism spectroscopies, along with isothermal titration calorimetry reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co‒S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.

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“…Using Phenix-MR, a solution which contained all 12 monomeric subunits was determined that had an LLG score of 6276 and a Z score of 34. 34 For use in refinement, a small-molecule parameter file for the [Co(N 3 )] complex was derived from a published structure (CCDC: 1414770), 35 and the modeled ligand was generated using the electronic Ligand Builder and Optimization Workbench, eLBOW, 36 in Phenix. COOT 37 was used to manually refine the positions and geometry of the protein residues and add the [Co(N 3 )] ligand in the early stages of refinement.…”

Section: ■ Introductionmentioning

confidence: 99%

Engineering a Conformationally Switchable Artificial Metalloprotein

et al. 2022

View full text Add to dashboard Cite

Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime and X = N3 –, H3C–, and i Pr–). We used UV–vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co–S cysteine ligation within the Escherichia coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co–S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.

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Synthesis and characterization of inner-sphere substitution products in azide-containing сobalt(III) dioximates

Cited by 7 publications

References 32 publications

Conformation-Dependent Hydrogen-Bonding Interactions in a Switchable Artificial Metalloprotein

Conformation-Dependent Hydrogen-Bonding Interactions in a Switchable Artificial Metalloprotein

Engineering a Conformationally Switchable Artificial Metalloprotein

Engineering a Conformationally Switchable Artificial Metalloprotein

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