Specific anion effect on properties of HRV 3C protease

Dušeková, Eva; Berta, Martin; Sedláková, Dagmar; Rooney, David; Veronika, Dzurillová; Shaposhnikova, Anastasiia; Fadaei, Fatemeh; Tomková, Mária; Minofar, Babak; Sedlák, Erik

doi:10.1016/j.bpc.2022.106825

Cited by 4 publications

(5 citation statements)

References 82 publications

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“…The observed slopes of thermodynamic parameters obtained for different salts (e.g., ΔΔH/Δ[salt]) are specific for a given chloride salt and may be correlated with some of the physicochemical properties of the cation. Similar correlations were found in different studies of proteins in the presence of salts of the Hofmeister series [ 42 , 43 , 44 , 45 ]. In our case, we could not find any evidence for a robust correlation between the salt-specific values of the slopes and ion properties such as hydration entropy, hydration number, charge density, polarizability, and viscosity B coefficient (see Figures S6–S17 , Supplementary Materials , which includes also pairwise correlations between thermodynamic parameters of domains).…”

Section: Discussionsupporting

confidence: 88%

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Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2

Džupponová

Tomášková

Antošová

et al. 2023

IJMS

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Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (Ttrs, hot 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from Thermus thermophilus. IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNAfMet during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA.

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Section: Discussionsupporting

confidence: 88%

“…( d ) Distribution of surface electrostatic potential in the presence of 0.1 M salt (left) and 2 M salt (right). ( e ) The frustration index analysis performed by [ 43 , 44 , 45 ]. The color of the lines indicates whether a given contact is minimally (green) or highly (red) frustrated.…”

Section: Discussionmentioning

confidence: 99%

Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2

Džupponová

Tomášková

Antošová

et al. 2023

IJMS

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“…CD spectra arising from β-sheets can be highly variable and dependent on subtle changes in sheet structure and twist ( 23 ), and in this context, the PV 3C pro spectrum is best described as being of the β II type that has a weak maximum around 190 nm and a strong minimum in the 200 nm region ( 24 ). Interestingly, such β II type CD spectra have been observed for trypsin and chymotrypsin ( 24 ) that have the same overall fold as 3C pro , but more typical β-sheet spectra have been reported for human rhinovirus 14 and Coxsackievirus B3 3C proteins ( 25 , 26 ). This prompted us to express and purify 3C from three other enteroviruses (EV71 and Coxsackieviruses A21 and B3) and compare their CD spectra obtained under identical conditions.…”

Section: Resultsmentioning

confidence: 89%

“…3 D ). Canonical spectra have previously been observed for human rhinovirus 14 and Coxsackievirus B3 3C pro proteins ( 25 , 26 ), but interestingly β II type CD spectra have been observed for trypsin and chymotrypsin ( 24 ). The 3C pro proteases have a chymotrypsin-like fold and catalytic triad mechanism that uses a cysteine instead of serine in the active site, and they similarly select for the amino acid at the C-terminal side of the cleaved peptide bond via a substrate binding pocket.…”

Section: Discussionmentioning

confidence: 93%

Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors

Campagnola,

Peersen

2023

Journal of Biological Chemistry

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“…The options for cleaving the purification tag at high salt concentrations are limited due to the salt sensitivity of commonly tested proteases, including enterokinase and Tobacco Etch Virus (TEV) protease. Other frequently used proteases, such as human rhinovirus 3C protease (HRV 3C) and thrombin, also exhibit salt sensitivity ( Jenny et al, 2003 ; Dušeková et al, 2022 ). However, salt-insensitive alternatives like SUMO protease and Factor Xa exist ( Jenny et al, 2003 ).…”

Section: Discussionmentioning

confidence: 99%

A systematic analysis of affinity tags in the haloarchaeal expression system, Haloferax volcanii for protein purification

Karan,

Renn,

Allers

et al. 2024

Front. Microbiol.

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Extremophilic proteins are valuable in various fields, but their expression can be challenging in traditional hosts like Escherichia coli due to misfolding and aggregation. Haloferax volcanii (H. volcanii), a halophilic expression system, offers a solution. This study examined cleavable and non-cleavable purification tags at both the N- and C-termini when fused with the superfolder green fluorescent protein (sfGFP) in H. volcanii. Our findings reveal that an N-terminal 8xHis-tag or Strep-tag®II significantly enhances protein production, purity, and yield in H. volcanii. Further experiments with mCherry and halophilic alcohol dehydrogenase (ADH) showed improved expression and purification yields when the 8xHis-tag or Strep-tag®II was positioned at the C-terminus for mCherry and at the N-terminus for ADH. Co-positioning 8xHis-tag and Twin-Strep-tag® at the N-terminus of sfGFP, mCherry, and ADH yielded significantly enhanced results. These findings highlight the importance of thoughtful purification tag design and selection in H. volcanii, providing valuable insights for improving protein production and purification with the potential to advance biotechnological applications.

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Specific anion effect on properties of HRV 3C protease

Cited by 4 publications

References 82 publications

Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2

Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2

Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors

A systematic analysis of affinity tags in the haloarchaeal expression system, Haloferax volcanii for protein purification

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