Dataset Information

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?C_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.

SUBMITTER: Oskarsson KR

PROVIDER: S-EPMC6978356 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Óskarsson K R KR Sævarsson A F AF Kristjánsson M M MM

Scientific reports 20200123 1

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), circula ...[more]

PMID: 31974391

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Project description:Termite mounds have recently been confirmed to mitigate approximately half of termite methane (CH4) emissions, but the aerobic CH4 oxidising bacteria (methanotrophs) responsible for this consumption have not been resolved. Here, we describe the abundance, composition and CH4 oxidation kinetics of the methanotroph communities in the mounds of three distinct termite species sampled from Northern Australia. Results from three independent methods employed show that methanotrophs are rare members of microbial communities in termite mounds, with a comparable abundance but distinct composition to those of adjoining soil samples. Across all mounds, the most abundant and prevalent methane monooxygenase sequences were affiliated with upland soil cluster α (USCα), with sequences homologous to Methylocystis and tropical upland soil cluster (TUSC) also detected. The reconstruction of a metagenome-assembled genome of a mound USCα representative highlighted the metabolic capabilities of this group of methanotrophs. The apparent Michaelis-Menten kinetics of CH4 oxidation in mounds were estimated from in situ reaction rates. Methane affinities of the communities were in the low micromolar range, which is one to two orders of magnitude higher than those of upland soils, but significantly lower than those measured in soils with a large CH4 source such as landfill cover soils. The rate constant of CH4 oxidation, as well as the porosity of the mound material, were significantly positively correlated with the abundance of methanotroph communities of termite mounds. We conclude that termite-derived CH4 emissions have selected for distinct methanotroph communities that are kinetically adapted to elevated CH4 concentrations. However, factors other than substrate concentration appear to limit methanotroph abundance and hence these bacteria only partially mitigate termite-derived CH4 emissions. Our results also highlight the predominant role of USCα in an environment with elevated CH4 concentrations and suggest a higher functional diversity within this group than previously recognised.

Dataset Information

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

Publications

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

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