Engineering a switch-based biosensor for arginine using a Thermotoga maritima periplasmic binding protein

Donaldson, Teraya; Iozzino, Luisa; Deacon, Lindsay J.; Billones, Hilbert; Ausili, Alessio; D’Auria, Sabato; Dattelbaum, Jonathan D.

doi:10.1016/j.ab.2017.02.021

Cited by 16 publications

(9 citation statements)

References 40 publications

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“…2c), although no exogenous arginine was added to the purification and crystallization media. This finding indicates that the TmArgBP P235GK monomer tightly binds arginine and that this protein variant could be used for arginine sensing (Donaldson et al, 2017).…”

Section: Resultsmentioning

confidence: 81%

The non-swapped monomeric structure of the arginine-binding protein from Thermotoga maritima

et al. 2019

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Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate-binding proteins, non-monomeric members have rarely been reported. The arginine-binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain-swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C-terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variety of oligomeric states (monomers, dimers, trimers and larger aggregates). With the aim of defining the structural determinants of domain swapping in TmArgBP, the monomeric form of the P235GK mutant has been structurally characterized. Analysis of this arginine-bound structure indicates that it consists of a closed monomer with its C-terminal helix folded against the rest of the protein, as typically observed for substrate-binding proteins. Notably, the two terminal helices are joined by a single nonhelical residue (Gly235). Collectively, the present findings indicate that extending the hinge region and conferring it with more conformational freedom makes the formation of a closed TmArgBP monomer possible. On the other hand, the short connection between the helices may explain the tendency of the protein to also adopt alternative oligomeric states (dimers, trimers and larger aggregates). The data reported here highlight the importance of evolutionary control to avoid the uncontrolled formation of heterogeneous and potentially harmful oligomeric species through domain swapping.

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Section: Resultsmentioning

confidence: 81%

The non-swapped monomeric structure of the arginine-binding protein from Thermotoga maritima

et al. 2019

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“…Substrate-binding proteins represent promising candidates to be used as scaffold for biosensors, although substantial engineering may be required. This approach has been applied with some success also for the arginine detection [ 16 , 20 , 32 , 33 ]. Here, we exploited the extraordinary stability of TmArgBP to generate novel and simplified scaffolds whose tryptophan fluorescence depends on their arginine binding state.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, any attempt to devise a TmArgBP-based sensor must proceed through modifications of the wild-type protein. Interesting variants of the protein have been hitherto developed by using as a scaffold the TmArgBP dimer [ 16 , 20 ]. Taking into account the relative inhomogeneity of the wild-type TmArgBP that presents trimeric and higher oligomeric forms along with the dimeric state and the virtually irreversible arginine binding to the protein, we here attempted to generate novel TmArgBP-derived scaffolds using simplified variants.…”

Section: Introductionmentioning

confidence: 99%

Development of a Protein Scaffold for Arginine Sensing Generated through the Dissection of the Arginine-Binding Protein from Thermotoga maritima

Smaldone

Ruggiero

Balasco

et al. 2020

IJMS

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Arginine is one of the most important nutrients of living organisms as it plays a major role in important biological pathways. However, the accumulation of arginine as consequence of metabolic defects causes hyperargininemia, an autosomal recessive disorder. Therefore, the efficient detection of the arginine is a field of relevant biomedical/biotechnological interest. Here, we developed protein variants suitable for arginine sensing by mutating and dissecting the multimeric and multidomain structure of Thermotoga maritima arginine-binding protein (TmArgBP). Indeed, previous studies have shown that TmArgBP domain-swapped structure can be manipulated to generate simplified monomeric and single domain scaffolds. On both these stable scaffolds, to measure tryptophan fluorescence variations associated with the arginine binding, a Phe residue of the ligand binding pocket was mutated to Trp. Upon arginine binding, both mutants displayed a clear variation of the Trp fluorescence. Notably, the single domain scaffold variant exhibited a good affinity (~3 µM) for the ligand. Moreover, the arginine binding to this variant could be easily reverted under very mild conditions. Atomic-level data on the recognition process between the scaffold and the arginine were obtained through the determination of the crystal structure of the adduct. Collectively, present data indicate that TmArgBP scaffolds represent promising candidates for developing arginine biosensors.

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“…Thermotoga neapolitana is a hyperthermophilic Gram-negative bacterium of the order of Thermotogales [94][95][96], which are excellent models for genetic engineering and biotechnological applications [97][98][99][100]. The CTN1690 ORF shows a clear homology of the O 6 -alkylguanine-DNAalkyl-transferase.…”

Section: Pyrococcus Furiosus and Thermotoga Neapolitana Ogtmentioning

confidence: 99%

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

Mattossovich

Merlo

Miggiano

et al. 2020

IJMS

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The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.

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Engineering a switch-based biosensor for arginine using a Thermotoga maritima periplasmic binding protein

Cited by 16 publications

References 40 publications

The non-swapped monomeric structure of the arginine-binding protein from Thermotoga maritima

The non-swapped monomeric structure of the arginine-binding protein from Thermotoga maritima

Development of a Protein Scaffold for Arginine Sensing Generated through the Dissection of the Arginine-Binding Protein from Thermotoga maritima

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

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