Folding of the α-helical membrane protein GlpF
Many membrane proteins form higher-ordered oligomers. However, the physiological impact of protein oligomerization is not obvious in many cases. All of the 13 aqua(glycerol)porins in humans form tetramers, and the structure of this class of proteins that mediate the flux of water and small polar molecules across biological membranes is highly conserved from bacteria to men. The structure and function of the tetrameric aquaglyceroporin GlpF have already been analyzed to great extent, making GlpF an ideal model for membrane protein folding studies.
We follow the folding and unfolding of wild-type GlpF and GlpF mutants using a variety of biochemical and biophysical methods including site-directed mutagenesis as well as tryptophan fluorescence studies, circular dichroism and stopped flow analyses. The combined results from these biochemical and biophysical analyzes allow us to define the pathways that are necessary for proper folding of α-helical membrane proteins.
- Trefz, M., Keller, R., Vogt, M. and Schneider, D. (2017) The GlpF residue Trp219 is part of an amino-acid cluster crucial for aquaglyceroporin oligomerization and function, Biochim. Biophys. Acta - Biomembranes, in press
- Klein, N., Neumann, J., O´Neil, J.D. and Schneider, D. (2015) Folding and stability of the aquaglyceroporin GlpF: Implications for human aqua(glycero)porin diseases, Biochim Biophys Acta – Biomembranes 1848, 622-633
Claudin 7 - a tight junction protein
Claudins are the major constituents of tight junctions, i.e. tight connections between adjacent cells. In humans, 27 claudin isoforms are expressed. Claudins are rather small proteins with four transmembrane helices that form stands to mediate cell adhesion. The protein forms complexes with other transmembrane and/or cytosolic proteins, like scaffold proteins, other claudins or occludins. The claudins stand function as paracellular barriers. Because of this, they can regulate cell polarity and paracelluar transport of ions and solutes. Most claudins have distinct charge selectivities. In some cancer types, claudin expression is modified, and thus claudins could be used as cancer cell markers.
Our current work focusses on the human Claudin 7, which is expressed in kidney, lung and prostate. Claudin 7 seems to prefer the transport of sodium ions through the paracellular barrier. In our in vitro and in cellulo analyses, we aim to define the forces guiding oligomerization and de-oligomerization of claudins finally resulting in the formation or the disassembly of tight junctions. In vitro, it forms extraordinary stable oligomers.
Small molecules affect the structure and properties of membranes
Besides defining the boundaries of cells and organelles, biomembranes are crucially involved in many cellular functions. Membrane constituents are involved in interactions with proteins and other external substances, and such interactions might trigger cellular processes.
Lipids, lipophilic compounds, as well as membrane integral and associated proteins do affect biological and biophysical membrane properties, but importantly also vice versa. We unravel the impacts defined molecules have on the structure and properties of membrane systems. These analyses do not only shine light on in vivo processes and how e.g. membrane binding of small molecules do affect the activity of membrane proteins, but also enables us to search for membrane modulates with defined features.
- Anbazhagan V., Munz C., Tome L. and Schneider D. (2010) Fluidizing the Membrane by a Local Anesthetic: Phenylethanol Affects Membrane Protein Oligomerization. J Mol Biol. 404, 773-777
Bacterial membrane proteins as drug targets
FACL6/mtFATP – an acyl-CoA synthetase from Mycobacterium tuberculosis
According to the world health organization (WHO), worldwide about every third person is infected with the human pathogen Mycobacterium tuberculosis. One of the greatest challenges in treating tuberculosis is the emerging drug-resistance of M. tuberculosis. A key reason for this is latent infections, since 85–95% of infections remain asymptomatic. During dormancy, triacylglycerol-containing lipid droplets are accumulated, supposable used as energy source by M. tuberculosis. These triacylglycerols originate from fatty acids derived from host lipids. Thus, to eventually modulate the uptake of host fatty acids in M. tuberculosis, it is fundamental to understand the process and the proteins involved. The fatty acid transporter FACL6/mtFATP might be crucial for accumulating triacylglycerols in the dormant phase by activating fatty acids within human cells. This makes FACL6/mtFATP an interesting candidate as novel drug target. Hence, we are currently studying the FACL6/mtFATP-mediated fatty acid activation in detail.
YqjA – a DedA protein family member from Escherichia coli
Members of the DedA/Tvp38 membrane protein family can be found in all domains of life. Moreover, in many sequenced bacterial genomes multiple genes for DedA protein family members are identified. Nevertheless, the function of this protein family is only poorly understood. However, it is known that in some pathogenic species DedA homologs are crucial for virulence, which makes them interesting candidates as novel drug targets. The two Escherichia coli DedA homologs YqjA and YghB are the currenetly best characterized DedA/Tvp38 protein family members, and all results thus far link the proteins´ functions to membrane processes. We are interested in determining the exact function of YqjA and its role in virulence to eventally identify modulates of the DedA activity.
- Keller, R., Schleppi, N., Weikum, J. and Schneider, D. (2015) Mutational analyses of YqjA, a Tvp38/DedA-protein of E. coli, FEBS Lett. 589, 842-848
- Keller, R. and Schneider, D. (2013) Homologs of the yeast Tvp38 vesicle-associated protein are conserved in chloroplasts and cyanobacteria. Front. Plant Sci. 4: 467
The bacterial ABC transporter BmrA
The family of ATP-binding cassette (ABC) transporters is one of the biggest known protein familiy. Family members can be found in prokaryotes as well as in eukaryotes. The transporters are integral membrane proteins, consisting of transmembrane and nucleotide-binding domains. With the energy of ATP-hydrolysis, substrates are transported across biomembranes, and the substrates can involve e.g. anti-cancer drugs used as therapeutics, which then contacts a chemo-therapy.
Modulating the activity of ABC transporters by small molecules could thus support certain therapies and could be used to treat various diseases.
Our current research focusses on the ABC-transporter BmrA, an ABC transporter from the bacterium Bacillus subtilis that shows a high sequence homology to human and other bacterial ABC transporters. Our analysis of this bacterial protein allows in-deep biochemical and biophysical analyses that will enable us to better understand the molecular details of ABC-transporter activities. This certainly is the basis for the next step: identifying ABC-transporter modulators.
- Neumann, J., Rose-Sperling, D. and Hellmich, U. A. (2017) Diverse relations between lipids and ABC transporters: an overview, Biochim. Biophys. Acta - Biomembranes 1859, 605-618