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Thermal stabilization of an endoglucanase by cyclization.

ABSTRACT: An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-?-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins.

SUBMITTER: van Lieshout JF

PROVIDER: S-EPMC3416981 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Thermal stabilization of an endoglucanase by cyclization.

van Lieshout Johan F T JF Pérez Gutiérrez Odette N ON Vroom Wietse W Planas Antoni A de Vos Willem M WM van der Oost John J Koutsopoulos Sotirios S

Applied biochemistry and biotechnology 20120601 7

An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of ...[more]

PMID: 22653681

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Project description:Proteins with high catalytic efficiency and selectivity under mild conditions have long been appreciated by industrial and medicinal fields. These proteins, which are commonly multimeric, often possess low stability, impeding wider application. Currently, strategies to improve the stability of multimeric proteins concentrate on enhancing the interaction at internal interface of the subunits. In this report, we confirmed that the largely underestimated subunit terminal ends are as significant as the internal interface for protein stability. By connecting both the terminal ends and internal interface of subunits, the tetrameric Leifsonia alcohol dehydrogenase (LnADH) protein can been cyclized into a rigid form with significantly improved thermostability and resilience. The improvement in the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment (T5015) and melting temperature (Tm ) of the modified protein was 18°C and 23.3°C, respectively, which is superior to the results achieved by normal protein engineering. Our study provided a novel strategy to effectively improve the stability of multimeric proteins, which is suitable not only for the short-chain dehydrogenase/reductase (SDR) family but also other classes of proteins with close terminal ends.IMPORTANCE Industrially interesting proteins are generally multimeric proteins; however, their applications are often restricted due to low stability caused by the natural tendency of subunit disassociation. Current approaches targeting this problem mainly focus on enhancing the internal interfaces of the subunits to avoid their disassociation. In this study, we identified and confirmed the external interface to be significant for improving the stability of multimeric proteins. By connecting the terminal ends and internal interface with disulfide bonds, we found that the multimeric protein LnADH cyclized into a robust monomeric-like form, resulting in superior thermostability compared to traditional protein engineering. This intersubunit cyclization approach is efficient and easy to perform, providing a novel method for engineering many important classes of multimeric proteins.

Project description:Enzymatic degradation is a major concern in peptide analysis. Postmortem metabolism in biological samples entails considerable risk for measurements misrepresentative of true in vivo concentrations. It is therefore vital to find reliable, reproducible, and easy-to-use procedures to inhibit enzymatic activity in fresh tissues before subjecting them to qualitative and quantitative analyses. The aim of this study was to test a benchtop thermal stabilization method to optimize measurement of endogenous opioids in brain tissue. Endogenous opioid peptides are generated from precursor proteins through multiple enzymatic steps that include conversion of one bioactive peptide to another, often with a different function. Ex vivo metabolism may, therefore, lead to erroneous functional interpretations. The efficacy of heat stabilization was systematically evaluated in a number of postmortem handling procedures. Dynorphin B (DYNB), Leu-enkephalin-Arg(6) (LARG), and Met-enkephalin-Arg(6)-Phe(7) (MEAP) were measured by radioimmunoassay in rat hypothalamus, striatum (STR), and cingulate cortex (CCX). Also, simplified extraction protocols for stabilized tissue were tested. Stabilization affected all peptide levels to varying degrees compared to those prepared by standard dissection and tissue handling procedures. Stabilization increased DYNB in hypothalamus, but not STR or CCX, whereas LARG generally decreased. MEAP increased in hypothalamus after all stabilization procedures, whereas for STR and CCX, the effect was dependent on the time point for stabilization. The efficacy of stabilization allowed samples to be left for 2 hours in room temperature (20°C) without changes in peptide levels. This study shows that conductive heat transfer is an easy-to-use and efficient procedure for the preservation of the molecular composition in biological samples. Region- and peptide-specific critical steps were identified and stabilization enabled the optimization of tissue handling and opioid peptide analysis. The result is improved diagnostic and research value of the samples with great benefits for basic research and clinical work.

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Thermal stabilization of an endoglucanase by cyclization.

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Thermal stabilization of an endoglucanase by cyclization.

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