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Lu, Wei; Li, S.; Li, G.-F.; Gong, Yu; Zhao, Nanming; Zhang, R.-X.; Zhou, Hai‐Meng

doi:10.1023/a:1019978923575

Cited by 6 publications

(2 citation statements)

References 20 publications

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“…3A,B), indicating more and more ANS bound with the hydrophobic residues of PTPase 40 . The exposure of the hydrophobic residues probably arose from the conformational transition induced partially by the hydrophobic interactions of PTPase and SDS 41 . As shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27

Hou

Wang

2020

Sci Rep

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Deciphering the activity-conformation relationship of PTPase is of great interest to understand howPTPase activity is determined by its conformation. Here we studied the activity and conformational transitions of PTPase from thermus thermophilus HB27 in the presence of sodium dodecyl sulfate (SDS). Activity assays showed the inactivation of PTPase induced by SDS was in a concentration-dependent manner. Fluorescence and circular dichroism spectra suggested SDS induced significant conformational transitions of PTPase, which resulted in the inactivation of PTPase, and the changes of α-helical structure and tertiary structure of PTPase. Structural analysis revealed a number of hydrophobic and charged residues around the active sites of PTPase may be involved in the hydrophobic and ionic bonds interactions of PTPase and SDS, which are suggested to be the major driving force to result in PTPase inactivation and conformational transitions induced by SDS. Our results suggested the hydrophobic and charged residues around the active sites were essential for the activity and conformation of PTPase. Our study promotes a better understanding of the activity and conformation of PTPase.Although we have discussed the conformational transitions and the inactivation of PTPase induced by SDS in detail, more experimental evidences such as the complex structure of PTPase-SDS and the dynamics of PTPase-SDS interactions are still required to clarify the details of molecular interaction. Our study is valuable toward the long-term goal to better understand the activity and conformation of PTPase. Materials and MethodsReagents and materials. All chemicals used in this research such as para-nitrophenyl phosphate (pNPP), Isopropyl-β-D-1-thiogalactopyranoside (IPTG), Dithiothreitol (DTT), SDS and 1-anilinonaphtalene-8-sulfonate (ANS) were of the highest purity commercially available. PTPase from thermus thermophilus HB27 was cloned into pET-28a (+) vector (Novagen, Germany) and overexpressed in E. coli BL21 (DE3). PTPase was purified as described 12 . The concentration of recombinant PTPase was determined by BCA protein assay kit (Pierce, USA).PTPase activity assay. PTPase activity was assayed as described previously 25,44 . Briefly, pNPP (10 mM) and PTPase (2.4 μM) were added into 200 µL, 50 mM acetic acid-sodium acetate buffer (pH 3.8) plus 5 mM DTT. After incubation at 30 °C for 10 min, NaOH (1 M, 1 mL) was added into the mixture to terminate the reaction. The absorption change at 405 nm was recorded on a Helios-γ UV-VIS spectrophotometer (Thermo Scientific, USA).

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Section: Discussionmentioning

confidence: 99%

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27

Hou

Wang

2020

Sci Rep

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“…The anionic surfactant SDS is well-known to be a strong denaturant for many proteins [24,29]. The sulfate anion of SDS is the hydrophilic head group, while the long aliphatic dodecyl chain forms the tail group [30].…”

Section: Discussionmentioning

confidence: 99%

Effects of hydroxypropyl cyclodextrins on the reactivation of SDS-denatured aminoacylase

Kim

Zhou

Yan

2007

International Journal of Biological Macromolecules

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Solid-phase Assisted Refolding of Carbonic Anhydrase Using β-Cyclodextrin-Polyurethane Polymer

Esmaeili

Yazdanparast

2008

Protein J

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Artificial chaperone-assisted refolding has been shown to be an effective approach for improving the refolding yield of denatured proteins. Independent refolding of several structurally diverse proteins by this approach has provided promising results regarding significant suppression of aggregation along with enhanced refolding yields. However, from the industrial point of view, some modifications seem to be essential for making the technique more efficient. In that regard and with a cost-cutting goal we designed, for the first time, a beta-cyclodextrin-polyurethane polymer to replace the soluble beta-cyclodextrin as the stripping agent for refolding of carbonic anhydrase. Our results indicated that under the optimally developed refolding environment, the denatured carbonic anhydrase was refolded with a yield of 75% using 15 mg/mL of the beta-cyclodextrin-polyurethane polymer, a yield near to stripping by soluble beta-CD. This new stripping approach seems to constitute an ideal approach for refolding of proteins at much lower industrial costs compared to stripping with soluble beta-cyclodextrin. However, further-improvements in solid-phase artificial chaperone assisted technique are demanded either through synthesizing better stripping agents or by optimizing and defining better refolding environments.

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Untitled

Cited by 6 publications

References 20 publications

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27

Effects of hydroxypropyl cyclodextrins on the reactivation of SDS-denatured aminoacylase

Solid-phase Assisted Refolding of Carbonic Anhydrase Using β-Cyclodextrin-Polyurethane Polymer

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