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

Abstract

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).

SARS-CoV-2 NSP1 C-terminal (residues 131–180) is an intrinsically disordered region in isolation

“…Tryptophan residue usually has a maximal fluorescence emission wavelength at 340–350 ​nm when exposed to water. In contrast, in our results, a blue shift was observed, which indicates buried Trp residue in the hydrophobic core of SDS micelle ( Tulumello and Deber, 2009 ; Hou et al., 2020 ). This fluorescence-based study was further validated by the lifetime measurement of the Trp fluorescence.…”

Linear Epitopes Play an Important Role in the Immunoglobulin G (IgG)/Immunoglobulin E (IgE)-Binding Capacity of Scy p 4

“…According to this study, DTT reduced the IgE-binding capacity of Scy p 4, suggesting the presence of disulfide bonds in Scy p 4, and it could affect the IgE-binding capacity of Scy p 4. Meanwhile, urea and GdnHCl destroy the hydrogen-bond network of water and protein molecules, thus affecting the stability and activity of proteins in solution . In this study, these two factors had no significant effect on allergenicity of Scy p 4.…”

“…Meanwhile, urea and GdnHCl destroy the hydrogen-bond network of water and protein molecules, thus affecting the stability and activity of proteins in solution. 38 In this study, these two factors had no significant effect on allergenicity of Scy p 4. In addition, the immunobinding capacity of Scy p 4 cannot be completely disabled, even at the maximum concentrations of DTT used.…”

Biophysical characteristics of lipid‐induced Aβ oligomers correlate to distinctive phenotypes in transgenic mice

“…Although the debate on whether the relative intensity of ellipticity ([θ]) between λ 222 and λ 208 indicates the extent of α-helix or 3 10 helix seem to be unsettled, 65 it is clear that in the presence of SDS denaturant, helix displays a value of [θ] 222 -[θ] 208 < 1.0. [47][48][49] This criterion was used in normalizing the data (see Methods). Based on this analysis, GM1Os displayed complete melting followed by 14LPOs, while LFAOs and 16LPOs, which showed partial melting but to the same extent of the helix ( Figure 4F).…”

“…The normalized values of fully melted samples were calculated by correlating the 90°C data with the post-melt scans wherein the maximum difference in ellipticity between 222 and 208 nm to be <0.1 was considered to be 100% helix based on established observations on SDS-protein systems. [47][48][49] The data obtained was fit using the Boltzman function in OriginLab 8.0 program to derive T m values.…”