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.

Multi-modal engineering ofBstDNA polymerase for thermostability in ultra-fast LAMP reactions

“…By further using machine-learning methods and supercharging we sought to further improve the stability and functionality of the enzyme in additive ways (46). In fact, mutations introduced at multiple sites around the enzyme and its fusion partner generally proved additive, as has previously been observed for structurally distant mutations in other proteins, including transcription factors (47), kinesin (48), and serine proteases (49). Ultimately, we were eventually able to generate enzymes (Br512g3.1 and Br512g3.2) that could perform high temperature (74ºC), ultra-fast (6 minute) LAMP reactions, and still provide reliable and consistent outputs.…”

In vitro application of bacteriocin produced by Lactiplantibacillus plantarum for the biopreservation of meat at refrigeration temperature

“…According to literature review, proline is present mostly in thermostable proteins where it provides resistance to proteins against high temperatures because of its distinct cyclic nature (Perálvarez‐Marín et al., 2008). Another study demonstrated some restrictive properties of the proline residue due to which proline strengthen the pre‐existing interactions within the protein when thermal energy is applied to the system (Óskarsson et al., 2020). Among polar group, aspartic acid and glutamic acid are negatively charged amino acids, lysine and arginine are positively charged while threonine and serine are uncharged.…”

Multistrategy Engineering of an Inulosucrase to Enhance the Activity and Thermostability for Efficient Production of Microbial Inulin

“…The enhanced thermostability observed in mutants G531P and H434P, located in Region 13 and Region 9, respectively, may be attributed to the introduction of proline, known for its rigidifying effect on flexible regions in proteins. 35,36 Thus, the improved rigidity of Region 9 and Region 13 could contribute to the enhanced thermostability of these mutants. Moreover, compared to S344E, S344D exhibited slightly higher thermostability.…”