Our research activities lie at the interface of the three scientific core disciplines biology, chemistry and physics with a particular focus on the biophysics of membrane proteins and membrane-mimetic systems. Membrane proteins are involved in numerous biological processes, play key roles in cellular communication and transport, represent the majority of drug targets, and increasingly find use in biotechnology. However, membrane proteins are utterly challenging research objects for biophysical, structural, and functional investigations, as they usually need to be extracted from their complex cellular membrane environment to become tractable in vitro. After extraction, membrane proteins depend on membrane mimics, which should reproduce the most important features of their natural membrane environment in order to retain their native structures and functions. Our efforts are devoted to the three major lines of research described below.
Membrane-mimetic nanodiscs on the basis of amphiphilic copolymers
Detergents are traditionally used for extracting and solubilising membrane proteins. In the highly dynamic environment of a detergent micelle, however, many membrane proteins tend to denature irreversibly. Some styrene/maleic acid (SMA) copolymers enable a fundamentally new approach for investigating membrane proteins, as they obviate the use of conventional detergents. These polymers can extract proteins and surrounding lipids directly from cellular membranes to form nanosized discs, where the polymer wraps around a lipid-bilayer patch. Such nanodiscs are readily amenable to optical and nuclear magnetic resonance (NMR) spectroscopy, which sets them apart from traditional bilayer systems such as vesicles. We combine these spectroscopic methods with concepts from solution thermodynamics to rationalise polymer/lipid interactions and thus fully exploit the potential of SMA for membrane-protein research.
Recently, we could demonstrate that another copolymer named diisobutylene/maleic acid (DIBMA) is equally capable of accommodating membrane proteins and lipids in native nanodiscs, thus rendering them amenable to biophysical, structural and functional scrutiny. The major advantage of this new polymer lies in the fact that it is compatible with optical spectroscopy in the ultraviolet range, does not disturb the order, dynamics, and hydration of the extracted membrane fragment, and tolerates elevated concentrations of metal ions often required for membrane-protein activity.
Grethen et al. J Membr. Biol. 2018, in press; Oluwole et al. Langmuir 2017, 33, 14378–14388; Grethen et al. Sci. Rep. 2017, 7, 11517; Cuevas Arenas et al. Sci. Rep. 2017, 7, 45875; Oluwole et al. Angew. Chem. Int. Ed. 2017, 56, 1919; Cuevas Arenas et al. Nanoscale 2016, 8, 15016; Vargas et al. Nanoscale 2015, 7, 20685
Fluorinated surfactants for membrane-protein research
A different approach towards building mild membrane mimics relies on fluorinated surfactants. Owing to the weak affinity of fluorocarbons for hydrocarbons and to the larger volume of the former, fluorosurfactants are less destabilising because they do not compete with native protein/protein and protein/lipid (or cofactor) interactions. For the same reason, however, fluorosurfactants are widely thought to be unable to extract proteins and lipids directly from biological membranes. Hence, conventional detergents are still required for initial solubilisation, and fluorinated surfactants come into play only at a later stage of the purification process, at which point labile proteins may have suffered irreversible damage. Contrary to this paradigm, we have recently demonstrated that the poor miscibility of fluorocarbons and hydrocarbons at the macroscale does not necessarily apply at the nanoscale. Rather, the chemistry of the polar headgroup of the surfactant plays a decisive role: A zwitterionic phosphocholine moiety, as found in many natural phospholipids, confers the expected “membranophobic” properties to a fluorosurfactant. Combination of the same fluorocarbon chain with a neutral maltose headgroup, by contrast, results in a surfactant that solubilises lipid vesicles and biological membranes in a manner reminiscent of conventional detergents without compromising membrane order, dynamics, and hydration at subsolubilising concentrations. Owing to this mild and unusual mode of solubilisation, this compound outperforms lipophobic fluorosurfactants in chaperoning the functional refolding of membrane proteins.
We are capitalising on this discovery to develop and apply fluorosurfactants that (i) display favourable physicochemical properties such as small and well-defined micelles amenable to membrane-protein crystallogenesis, (ii) partition into, translocate across, and solubilise membranes in a rapid, thermodynamically controlled manner, (iii) extract proteins directly from cellular membranes without requiring harsher detergents, and (iv) provide these proteins with a stabilising membrane-mimetic environment that preserves their native structures and functions for extended periods of time. This interdisciplinary project is accomplished together with Prof. Grégory Durand (Avignon), Prof. Eva Pebay-Peyroula, Prof. Christine Ebel, Dr. Cécile Breyton (Grenoble), and Dr. Annette Meister (Halle) within an international consortium funded by Deutsche Forschungsgemeinschaft (DFG) and Agence Nationale de la Recherche (ANR).
Frotscher et al. Anal. Chem. 2017, 89, 3245; Vargas et al. Nanoscale 2015, 7, 2068; Frotscher et al. Angew. Chem. Int. Ed. 2015, 54, 5069; Abla et al. J. Colloid Interface Sci. 2015, 445, 127
Membrane-protein folding and function and protein/ligand interactions
The third major line of research focusses on the interactions of proteins with small-molecule ligands, lipid membranes, and other proteins. We are particularly interested in noncanonical membrane proteins involved in the regulation of bacterial biofilms. During biofilm formation, cells that otherwise are motile and solitary communicate with each other to coordinate the production of extracellular matrix components. Biofilms are of major concern in freshwater supply and many medical applications but also provide new opportunities for industrial-scale protein production and biotransformation. In Bacillus and other Gram-positive bacteria, biofilm formation is preceded by cellular potassium ion release and can be induced by ionophores, but the endogenous mechanism that triggers potassium ion release remains unclear. Still, it is known that two hitherto sparsely characterised membrane proteins are essential for biofilm formation. We combine the membrane mimics developed in the above projects with spectroscopic methods to elucidate the structure, dynamics, and functions of these proteins to understand the mechanism of biofilm regulation at the molecular level.
Moreover, we have studied numerous proteins, protein/liquid interactions, and protein/membrane interactions by various calorimetric, spectroscopic, and hyphenated chromatographic techniques and have developed new methods and protocols for this purpose.
Frotscher et al. J. Phys. Chem. Lett. 2018, in press; Brautigam et al. Nat. Protoc. 2016, 11, 882; Scheidt et al. Biophys. J. 2015, 109, 586; Broecker et al. J. Am. Chem. Soc. 2014, 136, 13761; Fiedler et al. Anal. Chem. 2013, 85, 1868; Keller et al. Anal. Chem. 2012, 84, 5066; Kemmer and Keller Nat. Protoc. 2010, 5, 267–281