Erythrocyte invasion-neutralising antibodies prevent Plasmodium falciparum RH5 from binding to basigin-containing membrane protein complexes.

Basigin is an essential host receptor for invasion of Plasmodium falciparum into human erythrocytes, interacting with parasite surface protein PfRH5. PfRH5 is a leading blood-stage malaria vaccine candidate and a target of growth-inhibitory antibodies. Here, we show that erythrocyte basigin is exclusively found in one of two macromolecular complexes, bound either to plasma membrane Ca2+-ATPase 1/4 (PMCA1/4) or to monocarboxylate transporter 1 (MCT1). PfRH5 binds to each of these complexes with a higher affinity than to isolated basigin ectodomain, making it likely that these are the physiological targets of PfRH5. PMCA-mediated Ca2+ export is not affected by PfRH5, making it unlikely that this is the mechanism underlying changes in calcium flux at the interface between an erythrocyte and the invading parasite. However, our studies rationalise the function of the most effective growth-inhibitory antibodies targeting PfRH5. While these antibodies do not reduce the binding of PfRH5 to monomeric basigin, they do reduce its binding to basigin-PMCA and basigin-MCT complexes. This indicates that the most effective PfRH5-targeting antibodies inhibit growth by sterically blocking the essential interaction of PfRH5 with basigin in its physiological context.

Structural and biochemical insights into His-tag-induced higher-order oligomerization of membrane proteins by cryo-EM and size exclusion chromatography.

Structural and functional characterization of proteins as well as the design of targeted drugs heavily rely on recombinant protein expression and purification. The polyhistidine-tag (His-tag) is among the most prominent examples of affinity tags used for the isolation of recombinant proteins from their expression hosts. Short peptide tags are commonly considered not to interfere with the structure of the tagged protein and tag removal is frequently neglected. This study demonstrates the formation of higher-order oligomers based on the example of two His-tagged membrane proteins, the dimeric arginine-agmatine antiporter AdiC and the pentameric light-driven proton pump proteorhodopsin. Size exclusion chromatography revealed the formation of tetrameric AdiC and decameric as well as pentadecameric proteorhodopsin through specific interactions between their His-tags. In addition, single particle cryo-electron microscopy (cryo-EM) allowed structural insights into the three-dimensional arrangement of the higher-order oligomers and the underlying His-tag-mediated interactions. These results reinforce the importance of considering the length and removal of affinity purification tags and illustrate how neglect can lead to potential interference with downstream biophysical or biochemical characterization of the target protein.

Synthetic Biology: Bottom-Up Assembly of Molecular Systems.

The bottom-up assembly of biological and chemical components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their composition and can thus provide specifically optimized environments for synthetic biological processes. This review aims to inspire future endeavors by providing a diverse toolbox of molecular modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important technical and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technological achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biology. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biological systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.

Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin.

The green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.

Cryo-electron microscopic and X-ray crystallographic analysis of the light-driven proton pump proteorhodopsin reveals a pentameric assembly.

The green-light absorbing proteorhodopsin (GPR) is the prototype of bacterial light-driven proton pumps. It has been the focus of continuous research since its discovery 20 years ago and has sparked the development and application of various biophysical techniques. However, a certain controversy and ambiguity about the oligomeric assembly of GPR still remains. We present here the first tag-free purification of pentameric GPR. The combination of ion exchange and size exclusion chromatography yields homogeneous and highly pure untagged pentamers from GPR overexpressing Escherichia coli. The presented purification procedure provides native-like protein and excludes the need for affinity purification tags. Importantly, three-dimensional protein crystals of GPR were successfully grown and analyzed by X-ray crystallography. These results together with data from single particle cryo-electron microscopy provide direct evidence for the pentameric stoichiometry of purified GPR.

Engineering and Production of the Light-Driven Proton Pump Bacteriorhodopsin in 2D Crystals for Basic Research and Applied Technologies.

The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.

Purification of Membrane Proteins by Affinity Chromatography with On-Column Protease Cleavage.

A protocol is described for the isolation of recombinant polyhistidine-tagged membrane proteins from overexpressing Escherichia coli cells. The gene encoding a target membrane protein is cloned into an expression plasmid and then introduced into E. coli cells for overexpression. Membranes from bacterial cells are isolated and the tagged target membrane protein is solubilized in detergent and subsequently bound to an affinity matrix. Tagged proteins are commonly eluted by an excess of a solute that competes for the binding to the matrix. Alternatively, amino acid sequence-specific proteases can be used to cleave off the affinity purification tag directly on the purification column (i.e., on-column cleavage). This selectively releases the target protein and allows subsequent elution. Importantly, this step represents an additional purification step and can significantly increase the purity of the isolated protein.

Expression, purification and crystallization of an SLC16 monocarboxylate transporter family homologue specific for l-lactate.

l-lactate plays an important role as metabolite and signaling molecule in eukaryotes and bacteria. Monocarboxylate transporters (MCTs) of the SLC16 solute carrier family are responsible for the transport of l-lactate across eukaryotic and bacterial cell membranes. Here we report an efficient protocol for the expression and purification of an SLC16 family homologue in milligram amounts. The purified protein is stable and can thus be used for biochemical and structural studies as shown by successful crystallization.

Directed Insertion of Light-Activated Proteorhodopsin into Asymmetric Polymersomes from an ABC Block Copolymer.

Nanoscopic artificial vesicles containing functional protein transporters are fundamental for synthetic biology. Energy-providing modules, such as proton pumps, are a basis for simple nanoreactors. We report on the first insertion of a functional transmembrane protein into asymmetric polymersomes from an ABC triblock copolymer. The polymer with the composition poly(ethylene glycol)-poly(diisopropylaminoethyl methacrylate)-poly(styrenesulfonate) (PEG-PDPA-PSS) was synthesized by sequential controlled radical polymerization. PEG and PSS are two distinctively different hydrophilic blocks, allowing for a specific orientation of our protein, the light-activated proton pump proteorhodopsin (PR), into the final proteopolymersome. A very interesting aspect of the PEG-PDPA-PSS triblock copolymers is that it allowed for simultaneous vesicle formation and oriented insertion of PR simply by adjusting the pH. The intrinsic positive charge of PR's intracellular surface was enhanced by a His-tag, which aligns readily with the negative charges of the PSS on the outside of the polymersomes. The directed insertion of PR was confirmed by a light-dependent pH change of the proteopolymersome solution, indicating the intended orientation. We have hereby demonstrated the first successful oriented insertion of a proton pump into an artificial asymmetric membrane.

Fusion Domains Guide the Oriented Insertion of Light-Driven Proton Pumps into Liposomes.

One major objective of synthetic biology is the bottom-up assembly of minimalistic nanocells consisting of lipid or polymer vesicles as architectural scaffolds and of membrane and soluble proteins as functional elements. However, there is no reliable method to orient membrane proteins reconstituted into vesicles. Here, we introduce a simple approach to orient the insertion of the light-driven proton pump proteorhodopsin (PR) into liposomes. To this end, we engineered red or green fluorescent proteins to the N- or C-terminus of PR, respectively. The fluorescent proteins optically identified the PR constructs and guided the insertion of PR into liposomes with the unoccupied terminal end facing inward. Using the PR constructs, we generated proton gradients across the vesicle membrane along predefined directions such as are required to power (bio)chemical processes in nanocells. Our approach may be adapted to direct the insertion of other membrane proteins into vesicles.

Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies.

This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail.

Engineering and Assembly of Protein Modules into Functional Molecular Systems.

Synthetic biology approaches range from the introduction of unique features into organisms to the assembly of isolated biomacromolecules or synthetic building blocks into artificial biological systems with biomimetic or completely novel functionalities. Simple molecular systems can be based on containers on the nanoscale that are equipped with tailored functional modules for various applications in healthcare, industry or biological and medical research. The concept, or vision, of assembling native or engineered proteins and/or synthetic components as functional modules into molecular systems is discussed. The main focus is laid on the engineering of energizing modules generating chemical energy, transport modules using this energy to translocate molecules between compartments of a molecular system, and catalytic modules (bio-)chemically processing the molecules. Further key aspects of this discourse are possible approaches for the assembly of simple nanofactories and their applications in biotechnology and medical health.

Engineering a Chemical Switch into the Light-driven Proton Pump Proteorhodopsin by Cysteine Mutagenesis and Thiol Modification.

For applications in synthetic biology, for example, the bottom-up assembly of biomolecular nanofactories, modules of specific and controllable functionalities are essential. Of fundamental importance in such systems are energizing modules, which are able to establish an electrochemical gradient across a vesicular membrane as an energy source for powering other modules. Light-driven proton pumps like proteorhodopsin (PR) are excellent candidates for efficient energy conversion. We have extended the versatility of PR by implementing an on/off switch based on reversible chemical modification of a site-specifically introduced cysteine residue. The position of this cysteine residue in PR was identified by structure-based cysteine mutagenesis combined with a proton-pumping assay using E. coli cells overexpressing PR and PR proteoliposomes. The identified PR mutant represents the first light-driven proton pump that can be chemically switched on/off depending on the requirements of the molecular system.