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Binding of chimeric metal-binding green fluorescent protein to lipid monolayer

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Abstract

Membrane-based bioanalytical devices for metal determination using green fluorescent protein as the sensor molecule may be a useful future biomimetic material. However, in order to develop such a device, it is necessary first to understand the interaction of the protein with lipid membranes. Thus we have investigated the interaction between chimeric cadmium-binding green fluorescent proteins (CdBPGFPs) and lipid monolayers, using a film-balance technique complemented with epifluorescence microscopy. The binding avidity was monitored from the surface pressure vs. area isotherms or from the measured increase in the lateral pressure upon injection of the chimeric CdBPGFPs beneath the lipid monolayer. Increased fluidization as well as expansion of the surface area were shown to depend on the concentration of the CdBPGFPs. The kinetics of the protein-induced increase in lateral pressure was found to be biphasic. The chimeric CdBPGFPs possessed high affinity to the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer with a dissociation constant of K d=10−8M. Epifluorescence measurements showed that this affinity is due to the presence of the Cd-binding peptide, which caused the GFP to incorporate preferentially to the liquid phase and defect part of the rigid domain at low interfacial pressure. At high compression, the Cd-binding peptide could neither incorporate nor remain in the lipid core. However, specific orientation of the chimeric CdBPGFPs underneath the air–water interface was achieved, even under high surface pressure, when the proteins were applied to the metal-chelating lipid-containing surfaces. This specific binding could be controlled reversibly by the addition of metal ions or metal chelator. The reversible binding of the chimeric CdBPGFPs to metal-chelating lipids provided a potential approach for immobilization, orientation and lateral organization of a protein at the membrane interface. Furthermore, the feasibility of applying the chelator lipids for the codetermination of metal ions with specific ligands was also revealed. Our finding clearly demonstrates that a strong interaction, particularly with fluid lipid domains, could potentially be used for sensor development in the future.

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Abbreviations

GFP :

green fluorescent protein

CdBPGFPs :

cadmium-binding green fluorescent protein

DPPC :

1,2-dipalmitoyl-sn-glycero-3-phosphocholine

AAS :

atomic absorption spectrometry

Cd 2+ :

cadmium (II)

Zn 2+ :

zinc (II)

Cu 2+ :

copper (II)

Ni 2+ :

nickel (II)

E. coli :

Escherichia coli

NTA-DOGS :

1,2-dioleoyl-sn-glycero-3-(N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl)

His6GFP :

hexahistidine green fluorescent protein

CdBP4GFP :

four-repeat cadmium-binding peptide green fluorescent protein

His6CdBP4GFP :

hexahistidine four-repeat cadmium-binding peptide green fluorescent protein

IMAC :

immobilized-metal-affinity chromatography

PBS :

phosphate-buffered saline

mN/m :

millinewton per metre

le :

liquid expanded

lc :

liquid condensed

PE :

phosphatidyl ethanolamine

PI :

phosphatidyl inositol

NTA :

nitrilotriacetic acid

EDTA :

ethylenediamine tetraacetic acid

RESA :

ring-infected erythrocyte surface antigen

CdBP :

cadmium-binding peptide

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) and the Bundesministerium für wirtschaftliche Zusammenarbeit und Entwicklung (Federal Ministry for Economic Cooperation and Development; BMZ) (grant no.GA233/19–1,2). This project was also supported by a grant from the annual budget of Mahidol University for collaborative research between Germany and Thailand.

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Authors and Affiliations

  1. Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, 2 Prannok Road, Bangkok-Noi, 10700, Bangkok, Thailand

    Chartchalerm Isarankura Na Ayudhya & Virapong Prachayasittikul

  2. Institute of Biochemistry, Westfälische Wilhelms Universität, Wilhelm-Klemm-Str. 2, 48149, Muenster, Germany

    Chartchalerm Isarankura Na Ayudhya & Hans-Joachim Galla

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  1. Chartchalerm Isarankura Na Ayudhya
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  2. Virapong Prachayasittikul
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  3. Hans-Joachim Galla
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Correspondence to Virapong Prachayasittikul or Hans-Joachim Galla.

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Isarankura Na Ayudhya, C., Prachayasittikul, V. & Galla, HJ. Binding of chimeric metal-binding green fluorescent protein to lipid monolayer. Eur Biophys J 33, 522–534 (2004). https://doi.org/10.1007/s00249-004-0393-4

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