Design and assembly of a chemically switchable and fluorescently traceable light-driven proton pump system for bionanotechnological applications

Abstract

Energy-supplying modules are essential building blocks for the assembly of functional multicomponent nanoreactors in synthetic biology. Proteorhodopsin, a light-driven proton pump, is an ideal candidate to provide the required energy in form of an electrochemical proton gradient. Here we present an advanced proteoliposome system equipped with a chemically on-off switchable proteorhodopsin variant. The proton pump was engineered to optimize the specificity and efficiency of chemical deactivation and reactivation. To optically track and characterize the proteoliposome system using fluorescence microscopy and nanoparticle tracking analysis, fluorescenlty labelled lipids were implemented. Fluorescence is a highly valuable feature that enables detection and tracking of nanoreactors in complex media. Cryo-transmission electron microscopy, and correlative atomic force and confocal microscopy revealed that our procedure yields polylamellar proteoliposomes, which exhibit enhanced mechanical stability. The combination of these features makes the presented energizing system a promising foundation for the engineering of complex nanoreactors.

Synthetic Biology: Bottom-Up Assembly of Molecular Systems

“…Vesicles can also have more than one membrane, arranged in a concentric manner, and are then referred to as multilamellar vesicles (MLVs). Additional membranes affect the physicochemical, mechanical, and functional properties of the vesicles, including their permeability and stability, their interaction with surfaces, and their encapsulation efficiency. − MLVs are frequently used as vectors for the targeted delivery of drugs, ,− transport and presentation of antigens, , or as nanoparticles for applications in biotechnology . The membrane composition determines the ability of vesicles to encapsulate and release cargo as well as the potential to incorporate membrane transport proteins for selective permeability.…”

“…Reconstitutions from solubilized protein–detergent and lipid–detergent mixtures commonly yield proteoliposomes with randomly oriented membrane proteins ( i.e. , inside-in and inside-out) but may exhibit slight preferences. ,, The orientation of membrane proteins in vesicle membranes may be assessed by functional assays ,, or by exploiting the one-sided access to the protein. The latter can be done by analyzing proteolytic fragments or by evaluating the efficiency of a chemical labeling reaction before and after solubilization of reconstituted proteoliposomes. ,, Symmetric distribution of vectorial transport modules in the vesicle membrane results in a functional short-circuit, which prevents the establishment of a substrate gradient .…”

“…Simple modifications such as the addition of fluorescent molecules to the head groups of lipids already increase the possible applications of proteoliposomes by facilitating tracking in different environments. 57 Furthermore, fluorescently labeled lipids can be used to create proteoliposomes, which enhance the spectral absorption range of light-absorbing proteins. The lipid-linked fluorophore Texas Red was used in proteoliposomes containing the light-harvesting complex II to broaden the absorption range by efficient energy transfer from the chromophore to the protein (Figure 9a).…”

“…Currently, cryo-TEM is the most powerful microscopic tool to study vesicular systems at the nanoscale, yielding detailed information about their size, shape, and internal structure including lamellarity. 57,337,359,419 Statistical methods such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and tunable resistive pulse sensing (TRPS) offer a more quantitative analysis without providing any morphological information. 423,424 DLS is an ensemble technique, which describes the average vesicle size, whereas NTA and TRPS measure on a vesicle-by-vesicle basis and yield accurate size distributions even for polydisperse samples.…”

Rhodopsins: An Excitingly Versatile Protein Species for Research, Development and Creative Engineering

“…Several concepts to utilize rhodopsins in bioelectronic and biomimic nanotechnology have already been attempted, but did not yet really come to maturation ( Khodonov et al, 2000 ; Kuang et al, 2014 ; Hirschi et al, 2019 ; Aprahamian, 2020 ; Shim et al, 2021 ). With the rapidly growing insight in the structural and mechanistic potential of the rhodopsin pigments, this is expected to change at short notice.…”