Towards unidirectional reconstitution of membrane proteins into liposomes

Amati, Andrea Marco (2020). Towards unidirectional reconstitution of membrane proteins into liposomes (Unpublished). (Dissertation, Universität Bern, Philosophisch-naturwissenschaftliche Fakultät)

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Investigating functions of membrane proteins (MPs) in their natural environment, i. e.
in the membranes of various cellular compartments, is not straight forward, as these
are packed with many other components which disturb direct measurement of the MP
of interest, complicating the quantitative analysis. A common strategy to circumvent
this dilemma is a bottom-up approach, in which the protein of interest is isolated,
purified, and reconstituted into synthetic vesicles. Reconstitution is necessary as
after successful expression, extraction from their native environment, and purification,
isolated MPs display increased flexibility and reduced stability. Further, a wide
variety of MPs, especially such with a vectorial function, can only execute their
correct natural function in a membrane. Reconstitution into membrane mimetic
systems (symmetric or asymmetric) resolves these problems.
Functional investigations on MPs with vectorial function require compartmentbased
model membrane systems, the most common of which are liposomes. Detergentmediated
reconstitution of MPs into liposomes however constitutes a difficult task,
requiring optimization for every individual MP.
Orientation of the reconstituted MP in the liposomal membrane is influenced by
various factors. Consequently, reconstitution scarcely ever results in homogeneous
orientation of the MPs in liposomes, raising difficulties in evaluation of functional
assays, which are even further tightened if investigating on consecutive processes of
several different MPs coreconstituted in the same vesicle.
The aim of my PhD thesis was therefore to develop a tool kit to unidirectionally
reconstitute MPs in a predefined orientation into liposomes, notwithstanding all other
factors influencing orientation.
As a basis for our approach we decided to focus on reconstitution of MPs into
preformed, detergent-destabilized liposomes, since other reconstitution methods
appeared to be less promising. The paradigm of our strategy is the F1F0 ATP
synthase, which was reported to unidirectionally reconstitute into liposomes with
its F1 part facing outwards, the size and the hydrophilic nature of F1 disabling it
from passing the hydrophobic core of membranes. Hence, MPs were modified by
attachment of large soluble units (LSUs) purposed to guide unidirectional orientation
into liposomes, similarly as the F1 part for the ATP synthase.
To link the LSUs to the MP, we generally followed two different approaches. On
one hand, two model MPs (cytochrome bo3 ubiquinol oxidase, bo3 oxidase; green
light-absorbing proteorhodopsin, pR) were genetically modified to introduce different
IX
linker tags, and on the other hand, a more generally applicable approach was pursued
exploiting the His-tag used for purification.
In the former approach, as linker systems between the MP of interest and the LSU,
either the interaction between biotin and streptavidin via in vitro biotinylation of an
AviTag, or covalent bonding of the recently reported SpyTag-SpyCatcher system were
employed. In the more generally applicable approach, for the specific interaction with
His-tagged MPs the high affinity interaction between His-tags and trisNTA moieties
coupled to LSUs were exploited.
In this thesis, unidirectional reconstitution of pR in a predefined manner is reported
by using the SpyTag-SpyCatcher system. Application of the same system and
additionally the AviTag system to bo3 oxidase resulted in promising trends, however
orientation of bo3 oxidase seems to be much more difficult to be influenced than orientation
of proteorhodopsin. Further, a dendritic version of trisNTA was synthesized,
characterized, and coupled to different LSUs. Since direct coupling of trisNTA to
LSUs was not particularly successful, trisNTA was coupled to a synthesized SpyTag
peptide (trisNTA-Spy), offering the possibility to couple SpyCatcher-containing LSUs
indirectly to trisNTA.
These approaches to unidirectionally orientate MPs are not easy to be implemented,
however they are very promising. The most promising of the developed approaches are
the SpyTag-SpyCatcher-based and the trisNTA-Spy-based systems. However, further
investigation to optimize reconstitution of MPs into preformed, detergent-destabilized
liposomes is required. It is further very important to have at hand a method to
analyze the orientation of reconstituted MPs in the liposomal membrane, which is not
depending on the function of the MP. Such a method is currently being developed in
our lab.

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