Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues

Site-selective protein-protein coupling has long been a goal of chemical biology research. In recent years, that goal has been realized to varying degrees through a number of techniques, including the use of tyrosinase-based coupling strategies. Early publications utilizing tyrosinase from Agaricus bisporus showed the potential to convert tyrosine residues into ortho-quinone functional groups, but this enzyme is challenging to produce recombinantly and suffers from some limitations in substrate scope. Initial screens of several tyrosinase candidates revealed that the tyrosinase from Bacillus megaterium (megaTYR) as an enzyme that possesses a broad substrate tolerance. We use the expanded substrate preference as a starting point for protein design experiments and show that single point mutants of megaTYR are capable of activating tyrosine residues in various sequence contexts. We leverage this new tool to enable the construction of protein trimers via a charge-directed sequential activation of tyrosine residues (CDSAT).

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Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues

Journal of the American Chemical Society ◽

10.1021/jacs.1c03079 ◽

2021 ◽

Author(s):

Casey S. Mogilevsky ◽

Marco J. Lobba ◽

Daniel D. Brauer ◽

Alan M. Marmelstein ◽

Johnathan C. Maza ◽

...

Keyword(s):

Protein Complexes ◽

Tyrosine Residues ◽

Sequential Activation

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Rational thermostabilisation of four-helix bundle dimeric de novo proteins

Scientific Reports ◽

10.1038/s41598-021-86952-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shin Irumagawa ◽

Kaito Kobayashi ◽

Yutaka Saito ◽

Takeshi Miyata ◽

Mitsuo Umetsu ◽

...

Keyword(s):

Protein Design ◽

De Novo ◽

Protein Complexes ◽

Salt Bridge ◽

Dynamics Simulation ◽

Saturation Mutagenesis ◽

Helix Bundle ◽

Stable Mutant ◽

Four Helix Bundle ◽

The Stability

AbstractThe stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

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Metal-free site-selective C–H cyanoalkylation of 8-aminoquinoline and aniline-derived amides with azobisisobutyronitrile

RSC Advances ◽

10.1039/d1ra06013a ◽

2021 ◽

Vol 11 (49) ◽

pp. 30719-30724

Author(s):

Mengfei Zhao ◽

Zengxin Qin ◽

Kaixin Zhang ◽

Jizhen Li

Keyword(s):

Metal Free ◽

Good Substrate ◽

Substrate Tolerance ◽

Site Selective

An efficient metal-free cyanoalkylation of 8-aminoquinoline and aniline-derived amides was achieved in the presence of K2S2O8. The method showed good substrate tolerance and also suitable for bromination and dimerization reactions.

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Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation

10.26434/chemrxiv.7250201 ◽

2018 ◽

Author(s):

Shang Jia ◽

Christopher Chang

Keyword(s):

Protein Function ◽

Living Cells ◽

Covalent Modification ◽

Native Protein ◽

Selective Labeling ◽

Protein Residues ◽

Mild Conditions ◽

Starting Point ◽

Histidine Phosphorylation ◽

Site Selective

Site-selective bioconjugation to native protein residues is a powerful tool for protein functionalization, with cysteine and lysine side chains being the most common points for attachment owing to their high nucleophilicity. We now report a strategy for histidine modification using thiophosphorodichloridate reagents that mimic post-translational histidine phosphorylation, enabling fast and selective labeling of protein histidines under mild conditions where various payloads can be introduced via copper-assisted alkyne-azide cycloaddition (CuAAC) chemistry. We establish that these reagents are particularly effective at covalent modification of His-tags, which are common motifs to facilitate protein purification, as illustrated by selective attachment of polyarginine cargoes to enhance the uptake of proteins into living cells. This work provides a starting point for probing and enhancing protein function using histidine-directed chemistry.

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Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

10.26434/chemrxiv.13634021.v1 ◽

2021 ◽

Author(s):

Klara Markova ◽

Antonin Kunka ◽

Klaudia Chmelova ◽

Martin Havlasek ◽

Petra Babkova ◽

...

Keyword(s):

Protein Folding ◽

Protein Design ◽

Energy Landscape ◽

Single Point ◽

Three Dimensional ◽

Protein Stabilization ◽

Dimensional Structure ◽

Evolutionary Analysis ◽

Folding Energy

The functionality of a protein depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase enzyme (Tm = 73.5°C; ΔTm > 23°C) affected the protein folding energy landscape. Introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded catalytically active domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations target cryptic hinge regions and newly introduced secondary interfaces, where they make extensive non-covalent interactions between the intertwined misfolded protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively in vivo. Our findings uncovered hidden protein-folding consequences of computational protein design, which need to be taken into account when applying a rational stabilization to proteins of biological and pharmaceutical interest.

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Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

10.26434/chemrxiv.13634021 ◽

2021 ◽

Author(s):

Klara Markova ◽

Antonin Kunka ◽

Klaudia Chmelova ◽

Martin Havlasek ◽

Petra Babkova ◽

...

Keyword(s):

Protein Folding ◽

Protein Design ◽

Energy Landscape ◽

Single Point ◽

Three Dimensional ◽

Protein Stabilization ◽

Dimensional Structure ◽

Evolutionary Analysis ◽

Folding Energy

The functionality of a protein depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase enzyme (Tm = 73.5°C; ΔTm > 23°C) affected the protein folding energy landscape. Introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded catalytically active domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations target cryptic hinge regions and newly introduced secondary interfaces, where they make extensive non-covalent interactions between the intertwined misfolded protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively in vivo. Our findings uncovered hidden protein-folding consequences of computational protein design, which need to be taken into account when applying a rational stabilization to proteins of biological and pharmaceutical interest.

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A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.699203 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jeffrey A. Purslow ◽

Jolene N. Thimmesch ◽

Valeria Sivo ◽

Trang T. Nguyen ◽

Balabhadra Khatiwada ◽

...

Keyword(s):

Protein Design ◽

Active Site ◽

Single Point ◽

Conformational Dynamics ◽

Phosphorylation Site ◽

Phosphoryl Group ◽

Single Point Mutation ◽

Phosphotransferase System ◽

Function Relationship ◽

Enzyme I

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

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