Co-translational integration of the light harvesting complex II and its associated pigments into polymeric membranes

• Main Author: Zapf, Thomas
• Other Authors: Susana Geifman Shochat
• Format: Theses and Dissertations
• Language: English
• Published: 2016
• Subjects: DRNTU::Science::Biological sciences::Biochemistry; DRNTU::Science::Biological sciences::Molecular biology; DRNTU::Science::Chemistry::Organic chemistry::Polymers
• Online Access: https://hdl.handle.net/10356/65967
• Institution: Nanyang Technological University

One of nature’s most effective evolutionary concepts is to harvest and dissipate solar energy through the major light harvesting complex II (LHCII). This protein with its associated pigments is the main solar energy collector in higher plants. Our aim is to combine LHCII-pigment complexes with stable and highly controllable polymer-based membrane systems for future technological applications. We produced LHCII using wheat germ extract-based cell-free protein synthesis and show through Surface Plasmon Resonance (SPR,) Transmission Electron Microscopy (TEM) and Western blot the successful integration of LHCII and its pigments into polymersomic vesicles, called polymersomes. We further demonstrate by digestion assays an unidirectionality of LHCII insertion. Centrifugal microfiltration in means of polymersome purification as well as the development of a novel silica nanoparticles based purification method are further presented in this work. A silica nanoparticle based purification method was developed and optimized to meet our need of increased purification and harvesting efficiency of proteopolymersomes out of the crude cell-free lysate environment. Proper purification of synthesized proteopolymersomes was essential for further usage and subsequent characterization and analysis. Surface-modified silica nanoparticles with an antibody targeting the material of the polymer were able to bind and immunoprecipitate polymersomes and proteins by centrifugation. Analysis suggests that both purification methods did not compromise the polymersomic structure, nor their ability to retain integrated membrane protein. Comparison showed that immunoprecipitation was able to produce proteopolymersomes of greater purity and yield. Fluorescence measurements of purified proteopolymersomes indicate successful binding of pigments to the proteins within this new environment. Surface Plasmon Resonance after cell-free synthesis on tethered polymer membranes indicates that LHCII is able to integrate functionally into planar bilayers. Surface Plasmon enhanced Fluorescence Spectroscopy reveals energy transfer from Chl b to Chl a which strongly suggests a native folding of the protein. Regeneration experiments showed that pigments harmed in their function through surface plasmon induced bleaching can be exchanged through incubation with fresh pigment solution.