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Modelling of microbial polyhydroxyalkanoate surface binding protein PhaP for rational mutagenesis.

ABSTRACT: Phasins are unusual amphiphilic proteins that bind to microbial polyhydroxyalkanoate (PHA) granules in nature and show great potential for various applications in biotechnology and medicine. Despite their remarkable diversity, only the crystal structure of PhaPAh from Aeromonas hydrophila has been solved to date. Based on the structure of PhaPAh , homology models of PhaPAz from Azotobacter sp. FA-8 and PhaPTD from Halomonas bluephagenesis TD were successfully established, allowing rational mutagenesis to be conducted to enhance the stability and surfactant properties of these proteins. PhaPAz mutants, including PhaPAz Q38L and PhaPAz Q78L, as well as PhaPTD mutants, including PhaPTD Q38M and PhaPTD Q72M, showed better emulsification properties and improved thermostability (6-10°C higher melting temperatures) compared with their wild-type homologues under the same conditions. Importantly, the established PhaP homology-modelling approach, based on the high-resolution structure of PhaPAh , can be generalized to facilitate the study of other PhaP members.

SUBMITTER: Zhao H

PROVIDER: S-EPMC5658623 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Modelling of microbial polyhydroxyalkanoate surface binding protein PhaP for rational mutagenesis.

Zhao Hongyu H Yao Zhenyu Z Chen Xiangbin X Wang Xinquan X Chen Guo-Qiang GQ

Microbial biotechnology 20170825 6

Phasins are unusual amphiphilic proteins that bind to microbial polyhydroxyalkanoate (PHA) granules in nature and show great potential for various applications in biotechnology and medicine. Despite their remarkable diversity, only the crystal structure of PhaPAh from Aeromonas hydrophila has been solved to date. Based on the structure of PhaPAh , homology models of PhaPAz from Azotobacter sp. FA-8 and PhaPTD from Halom ...[more]

PMID: 28840964

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Project description:Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for binding to the polyhydroxyalkanoate (PHA) granule. BioF has been exploited for biotechnological purposes as an affinity tag in the functionalization of PHA beads with fusion proteins both in vivo and in vitro The structural model of this domain suggests an amphipathic α-helical conformation with the hydrophobic residues facing the PHA granule. In this work, we analyzed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on PHA, stability on the surface of the PHA granule against pH, temperature, and ionic strength, and their possible influence on PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP) that retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing the functionality and stability of the fusion proteins MinP-β-galactosidase and MinP-CueO from Escherichia coliIMPORTANCE Polyhydroxyalkanoates (PHAs) are biocompatible, nontoxic, and biodegradable biopolymers with exceptional applications in the industrial and medical fields. The complex structure of the PHA granule can be exploited as a toolbox to display molecules of interest on their surface. Phasins, the most abundant group of proteins on the granule, have been employed as anchoring tags to obtain functionalized PHA beads for high-affinity bioseparation, enzyme immobilization, diagnostics, or cell targeting. Here, a shorter module based on the previously designed BioF tag has been demonstrated to maintain the affinity for the PHA granule, with higher stability and similar functionalization efficiency. The use of a 67% shorter peptide, which maintains the binding properties of the entire protein, constitutes an advantage for the immobilization of recombinant proteins on the PHA surface both in vitro and in vivo.

Dataset Information

Modelling of microbial polyhydroxyalkanoate surface binding protein PhaP for rational mutagenesis.

Publications

Modelling of microbial polyhydroxyalkanoate surface binding protein PhaP for rational mutagenesis.

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