Thermostabilization of VPR, a kinetically stable cold adapted subtilase, via multiple proline substitutions into surface loops

Abstract

Protein stability is a widely studied topic, there are still aspects however that need addressing. In this paper we examined the effects of multiple proline substitutions into loop regions of the kinetically stable proteinase K-like serine protease VPR, using the thermostable structural homologue AQUI as a template. Four locations for proline substitutions were chosen to imitate the structure of AQUI. Variants were produced and characterized using differential scanning calorimetry (DSC), circular dichroism (CD), steady state fluorescence, acrylamide fluorescence quenching and thermal inactivation experiments. The final product VPR_ΔC_N3P/I5P/N238P/T265P was greatly stabilized which was achieved without any noticeable detrimental effects to the catalytic efficiency of the enzyme. This stabilization seems to be derived from the conformation restrictive properties of the proline residue in its ability to act as an anchor point and strengthen pre-existing interactions within the protein and allowing for these interactions to prevail when thermal energy is applied to the system. In addition, the results underline the importance of the synergy between distant local protein motions needed to result in stabilizing effects and thus giving an insight into the nature of the stability of VPR, its unfolding landscape and how proline residues can infer kinetic stability onto protein structures.

Figure 1

(A) The three-dimensional structure of VPR (PDB ID: 1SH7). Residues shown as sticks and balls and marked with red labels are the native residues mutated to prolines in this study. In addition, the catalytic triad Ser220, His70 and Asp37 are also shown along with all the Trp residues in the structure as well as calcium ion coordinators. Calcium ions are shown as green spheres. (B) Superimposed three-dimensional structures of VPR (light blue) (PDB ID: 1SH7) and AQUI (Orange) (PDB ID: 4DZT). The native prolines of AQUI are shown as sticks and balls along with the native VPR residues mutated to prolines. Calcium ions are shown as green spheres for VPR and golden spheres for AQUI. Atomic specifiers for side chains are as follows: carbon atoms are coloured same as the secondary structure; nitrogen atoms are coloured blue; oxygen atoms are coloured red and sulphur atoms coloured yellow.

Figure 2

Fluorescence emission of proline variants after excitation at 295 nm at pH 8.0. Intensities of emissions have been normalized against VPR_ΔC (dotted black line). (A) Emission of VPR_ΔC_N3P (Gold) and VPR_ΔC_I5P (purple). (B) Emission of VPR_ΔC_N238P (light blue) and VPR_ΔC_T265P (orange). (C) Emission of VPR_ΔC_N3P/I5P/N238P (blue) and VPR_ΔC_N3P/I5P/T265P (green). (D) Emission of VPR_ΔC_N3P/I5P (red) and VPR_ΔC_N3P/I5P/N238P/T265P (black).

Figure 3

Stern-Volmer graphs calculated form fluorescence quenching of proline variants between 310–410 nm at pH 8.0. VPR_ΔC (black boxes with a dotted black line). (A) Quenching of VPR_ΔC_N3P (Gold) and VPR_ΔC_I5P (purple). (B) Quenching of VPR_ΔC_N238P (light blue) and VPR_ΔC_T265P (orange). (C) Quenching of VPR_ΔC_N3P/I5P/N238P (blue) and VPR_ΔC_N3P/I5P/T265P (green). (D) Quenching of VPR_ΔC_N3P/I5P (red) and VPR_ΔC_N3P/I5P/N238P/T265P (black).

Figure 4

Normalized melting curves of proline variants in glycine buffer containing 15 mM CaCl₂ and 100 mM NaCl. VPR_ΔC (black dotted line). (A) Melting of VPR_ΔC_N3P (Gold) and VPR_ΔC_I5P (purple). (B) Melting of VPR_ΔC_N238P (light blue) and VPR_ΔC_T265P (orange). (C) Melting of VPR_ΔC_N3P/I5P/N238P (blue) and VPR_ΔC_N3P/I5P/T265P (green). (D) Melting of VPR_ΔC_N3P/I5P (red) and VPR_ΔC_N3P/I5P/N238P/T265P (black).

Figure 5

Arrhenius plots calculated form the thermal inactivation of proline variants in Tris buffer containing 15 mM CaCl₂. VPR_ΔC (black boxes with a dotted black line). (A) Thermal inactivation of VPR_ΔC_N3P (Gold) and VPR_ΔC_I5P (purple). (B) Thermal inactivation of VPR_ΔC_N238P (light blue) and VPR_ΔC_T265P (orange). (C) Thermal inactivation of VPR_ΔC_N3P/I5P/N238P (blue) and VPR_ΔC_N3P/I5P/T265P (green). (D) Thermal inactivation of VPR_ΔC_N3P/I5P (red) and VPR_ΔC_N3P/I5P/N238P/T265P (black).

Figure 6

Deconvoluted differential scanning thermograms showing the excess heat during the unfolding process of the proline variants in a glycine buffer containing 15 mM CaCl₂ and100 mM NaCl. VPR_ΔC (dotted black line). (A) Unfolding of VPR_ΔC_N3P (Gold) and VPR_ΔC_I5P (purple). (B) Unfolding of VPR_ΔC_N238P (light blue) and VPR_ΔC_T265P (orange). (C) Unfolding of VPR_ΔC_N3P/I5P/N238P (blue) and VPR_ΔC_N3P/I5P/T265P (green). (D) Unfolding of VPR_ΔC_N3P/I5P (red) and VPR_ΔC_N3P/I5P/N238P/T265P (black).

Figure 7

(A) Superimposed closeup of the N-terminals of VPR (light blue) (PDB ID: 1SH7) and AQUI (orange) (PDB ID: 4DZT) comparing the H-bond potential of both enzymes (red lines H-bonds in AQUI and blue lines H-bonds in VPR). (B) Closeup of the part of the protein in the closest vicinity of the N238P and T265P mutations in the structure of VPR. (C) Closeup highlighting the residues making up a part of the hydrophobic core and N-terminal interface to the main part of the protein in the structure of VPR. Calcium ions are shown as green spheres for VPR and golden spheres for AQUI. Atomic specifiers for side chains are as follows: carbon atoms are coloured same as the secondary structure; nitrogen atoms are coloured blue; oxygen atoms are coloured red and sulphur atoms coloured yellow.