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 how PTPase 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.

Figure 1

The relation of the relative residue activity of PTPase and SDS concentration.

Figure 2

(A) Intrinsic fluorescence spectra of PTPase in the presence of 0–100 μM SDS. SDS concentrations for curves 1–6 were 0, 6.25, 12.5, 25, 50 and 100 μM, respectively. Relative changes of Imax (B) and λmax (C) vs SDS concentration.

Figure 3

(A) ANS fluorescence spectra of PTPase in the presence of 0–50 μM SDS. SDS concentrations for curves 1–7 were 0, 1.56, 3.125, 6.25, 12.5, 25 and 50 μM, respectively. (B) Relative changes of Imax vs SDS concentration.

Figure 4

(A) Far-UV CD spectra of PTPase in the presence of 0–6 mM SDS. SDS concentrations for curves from top to bottom were 0, 0.35, 0.70, 1.40, 1.80, 2.00, 2.40, 2.80, 3.00, 3.50, 4.00, 5.00, 5.50 and 6.00 mM, respectively. (B) Relative changes of the elipticity at 222 nm vs SDS concentration.

Figure 5

(A) Cartoon structure of Tt1001 protein. α-helix, β-sheet and loops were colored in red, yellow and green, respectively. P-loop was highlighted in marine. (B) Essential residues Cys11, Cys16, Arg17 and Asp123 were showed as sticks. (C) Tryptophan and tyrosine residues were showed as sticks and lines, respectively. (D) Polar and charged residues located on loop 3 and loop 5 are showed as sticks. All the structures are prepared by PyMOL (DeLano, Warren L., The PyMOL Molecular Graphics System (2008). DeLano Scientific, California, USA).