Research Projects: Proteorhodopsin, Diacylglycerolkinase, Multidrug Efflux Pumps, GPCRs, Solid-state NMR & DNP

Proteorhodopsin: A ubiquitous membrane protein of marine bacterial origin

Proteorhodopsin (PR) is a 7 TM retinal proteins found to high abundance in marine bacteria. Members of the PR family are colour tuned to their environment. The light-driven proton pumping activity of the green PR depends on environmental factors such as pH, which could indicate an involvement in both energy production as well as sensing/regulation.  PR assembles into  a 'donut-shaped' haxameric complex (Klyszejnko et al. 2007). This is in contrast to the well known bR trimer.

We are using solid-state NMR in combination with different isotope labelling schemes and site-directed mutagenesis to understand the functional mechanism of PR. We have been able to characterize chromophore and Schiff base in ground state PR (Pfleger et al. 2008 & 2009), we have found an unusual His-Asp cluster which explains the high pKa of PR's proton acceptor (Hempelmann et al. 2011) and we were able to characterize the molecular dynamics within lipid bilayers (Yang et al. 2011).

Recent papers:

• Hempelmann, F., Hölper, S., Verhoefen, M.K., Wörner, A., Köhler, T., Fiedler, S.A., Pfleger, N., Wachtveitl., S., Glaubitz. C. (2011) The His75-Asp97 Cluster in Green Proteorhodopsin. J. Am. Chem. Soc., Epub ahead of print,

• Yang, J., Aslimovska, L., Glaubitz, C. (2011) Molecular Dynamics of Green Proteorhodopsin in Lipid Bilayers by Solid-state NMR., J. Am. Chem. Soc.,  Epub ahead of print.

• Pfleger, N., Woerner, A.C., Yang, J., Shastri, S., Hellmich, .A., Aslimovska, L., Maier, M.S.M., Glaubitz, C. (2009) Solid-state NMR and functional studies on proteorhodopsin, BBA Bioenergetics, 1787, 697-705

• Klyszejnko, A.L., Shastri, S., Mari, S.A., Grubmüller, H., Müller, D.J., Glaubitz, C. (2008) Folding and Assembly of Proteorhodopsin. JMB 376, 35-41.

• Pfleger, N., Lorch, M., Wörner, A., Shastri, S., Glaubitz, C. (2008) Characterisation of Schiff base and Chromophore in Green Proteorhodopsin by Solid-State NMR. J. Biomol. NMR 40, 15-21. 

• Shastri, S., Vonck, J., Haase, W., Pfleger, N, Kuehlbrandt, W., Glaubitz, C. (2007) Proteorhodopsin: Characterisation of 2D Crystals by Electron Microscopy and Solid-State NMR. BBA Biomem. 1768, 3012-3019.

Diacylglycerolkinase: How membrane bound enzymes regulate lipid signalling

Lipids are not only the main structural element of biological membranes and an important reservoir for energy storage but they also play an important role in transmembrane signalling. Enzymes modifying those lipids often catalyse reactions taking place in both the aqueous and the membrane phase by involving soluble and lipophilic substrates. These enzymes are usually transmembrane or membrane associated proteins. E. coli diacylglycerolkinase (DGK) is an integral membrane protein converting diacylglcerol (DGK) into phospatidic acid (PA) under ATP hydrolysis. Both lipids play important roles in different signalling pathways, which are controlled by the activity of DGK.  Unfortunately, the hydrophobic character of lipids and membrane proteins causes a significant challenge for their analysis preventing a detailed mechanistic analysis of enzymes such as DGK. A solution is offered by solid-state NMR, which enables the simultaneous detection of ATP turnover in the aqueous phase and PA generation in the lipid phase. Based on this method, we were able for the first time to resolve the kinetics of a membrane bound kinase using time-resolved MAS-NMR. Our data allow a detailed insight into the functional mechanism of DGK and demonstrate a promising methodological approach for further studies towards resolving the molecular details of lipid signalling.

Recent papers:

• Ullrich, S.J., Hellmich, U.A., Ullrich, S., Glaubitz, C. (2011) Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS -NMR. Nature Chem. Biol. Epub ahead of print.

• Lorch, M., Fahem, S., Kaiser, C., Weber, I., Mason, A.J., Bowie, J., Glaubitz, C. (2005) How to prepare membrane proteins for solid-state NMR - A case study on the alpha-helical membrane protein DGK from E.coli, ChemBioChem 6, 1693-1700

Understanding Multidrug Transport Proteins

Multidrug resistance is an important problem in cancer chemotherapy and in the treatment of infectious diseases. The most distinct mechanism for multidrug resistance is based on secondary and primary active transport proteins which extrude a wide range of antibiotics out of the cell.

The mechanism by which they recognize and transport drugs remains to be resolved. We use solid-state NMR in combination with other spectroscopic and biochemical methods to resolve the structure-function relationship of these proteins directly within the lipid bilayer. We study both ABC transporters (LmrA) and secondary drug-proton antiporters (EmrE, TBsmr, Hsmr). We are interested in key events and structural changes during the transport cycle, in characterising the properties of the drug binding pockets, and investigate the role of lipids and oligomerisation for protein activity.

The key problem of multidrug efflux:

The unusual broad substrate specificity of multidrug efflux pumps and biochemical hints for membrane embedded binding site(s) of these transporters raise the question, whether the membrane could act as a drug selectivity filter. Therefore, an extensive study on the interaction between typical multidrug transporter substrates with model membranes has been carried out. The location profile of these molecules across the membrane was determined by ¹H MAS NMR. We have developed data analysis tools which allow extensive, semi-automatic drug-membrane screens. Although structurally rather diverse, all tested substances are found to have their highest concentration between the phosphate of the lipid headgroup and the upper segments of the lipid hydrocarbon chains (Siarheyeva et al., 2006).

EmrE: Alternating Access and Occluded States

EmrE and other proteins from the SMR family require pmf to drive substrate transport across the membrane. In order to investigate their transport activity in vitro, we have developed a fluorescence based assay in which a pH gradient is generated through co-reconstitution with the light-driven proton pump bacteriorhodopsin. Sample illumination activates bR and excites ethidium bromide fluorescence.

The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. Our data show the existence of an occluded state during the transport cycle for both EmrE and TBsmr (Basting et al. 2008).

Solid-State NMR reveals the formation of an asymmetric EmrE dimer:

Using cell free expression in combination with ¹³C double quantum filtering methods, we were able to selectively observe highly conserved and essential residue Glu-14 in EmrE, functionally reconstituted in E.coli lipids. For E14, two distinct sets of chemical shifts were observed which indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changed and altered line shapes were observed, demonstrating substrate coordination by both E14s in the dimer. Our data show directly that during the exchange reaction E14 of each protomer is involved in substrate binding and coordination (Lehner et al. 2008).

ABC Transporter LmrA:

LmrA, a 590-amino acid ABC-multidrug-transporter native to L. lactis, is a bacterial homologue of the human P-glycoprotein (Pgp). This primary efflux pump is active as a homodimer. Each of its monomers comprises of a six helical transmembrane domain (TMD) and one nucleotide binding domain (NBD), following the general architecture of ABC-transporters. we have for the first time applied solid-state NMR to full length LmrA in order to probe ist dynamic during the catalytic cycle (Siarheyeva et al. 2007). Using ³¹P-MAS NMR, ATP turnover by LmrA has been observed.

Recent papers:

• Lehner, I., Basting, D.,  Meyer, B., Haase, W., Manolikas, T., Kaiser, C., Karas, M., Glaubitz, C. (2008) The key residue for substrate transport in the EmrE dimer is asymmetric. J. Biol. Chem. 283, 3281-3288.

• Basting, D., Lorch, M., Lehner, I., Glaubitz, C. (2008) Transport Cycle Intermediate in Small Multidrug Resistance Protein is revealed by Substrate Fluorescence. FASEB J 22, 365-373

• Siarheyeva, A., Lopez, J.J., Lehner, I., Hellmich, U.A., van Veen, H., Glaubitz, C. (2007) Probing the Molecular Dynamics of the ABC Multidrug Transporter LmrA by Deuterium Solid-State NMR. Biochemistry 46, 3075-83.

• Siarheyeva, A., Lopez, J.J., Glaubitz, C. (2006) Localization of multidrug transporter substrates within model membranes. Biochemistry 45, 6203-11.

• Hellmich, U.A., Haase, W., Velamakanni, S., van Veen, H., Glaubitz, C., (2008) Caught in the Act: ATP hydrolysis of an ABC-Multidrug transporter followed by real-time Magic Angle Spinning NMR, Febs Letters 582, 3557-62

G-Protein coupled Receptors: Understanding Structure and Function

G protein-coupled receptors (GPCRs) are responsible for a large number of physiological processes, such as sensory transduction, mediation of hormonal activity and cell to cell communication. GPCRs are membrane proteins with seven transmembrane helices and are the target of some 50% of today’s modern drugs.  So far, two high resolution GPCR structures (rhodopsin, beta adrenergic receptor) are available. Pharmacological research and rational drug design aimed at GPCRs can be based on homology models derived from these structures but additional data are needed. This limitation could be potentially overcome by determining the structures of bound agonists, which activate GPCRs, and using these as structural templates for drug-design. Solid-state NMR is ideally suited to obtain such structural constraints. In a recent study, we have determined the backbone structure of the neuropeptide bradykinin bound to the human G-protein coupled bradykinin subtype 2 receptor by solid state NMR (Lopez et al. 2007). 13C chemical shift based torsion angle constraints were used for structure calculation which revealed an elongated conformation with an alpha-helical turn at the N-terminus and a beta-turn at the C-terminus with an  average backbone RMSD value of 0.5Å

Recent papers:

• Lopez, J.J., Shukla, A.K., Reinhart, C., Schwalbe, H., Michel, H., Glaubitz, C. (2007) The structure of bradykinin bound to the human GPCR bradykinin-2 as determined by solid-state NMR. Angew. Chem. Int. Ed.47, 1668-1671.
• Gieldon, A., Lopez. J.J., Glaubitz, C., Schwalbe, H. (2008) A Molecular Dynamics Study of the Human Bradykinin - Bradykinin B2 Receptor Complex Supported by Solid-State NMR Spectrocopy Data, ChemBioChem 9, 2487-2497

Solid-State NMR: Improving Methodology

Solid-state NMR spectroscopy is at the heart of the above mentioned projects. Technical and methodological improvements are needed to use this powerful technique to its full potential. We mainly use MAS NMR but also investigate the possibility of utilising uniformly aligned samples (static oriented and MAOSS NMR). The intrinsic signal-to-noise problem is addressed by dynamic nuclear polarisation and FAST-NMR methods which allow to make efficient use of data and instrument time. Concepts for 3D structure determination are explored in collaboration with the Peter Güntert lab. 

Figure: Basic principles of the RELOAD experiments (left, Lopez et al. 2009), graphical representation of different orientation distribution functions in ordered samples (right).  

Recent papers:

• Lopez, J.J., Kaiser, C., Asami, S., Glaubitz, C. (2009) Higher sensitivity through selective 13C excitation in solid-state NMR spectroscopy. J.Am.Chem.Soc. 131, 15970-15971

• Lopez, J.J., Kaiser*, C., Shastri, S., Glaubitz, C. (2008) Double quantum filtering homonuclear MAS NMR correlation spectra: A tool for membrane protein studies, J. Biomol. NMR 41, 97-10

• Kaiser, C., Lopez, J.J., Bermel, W., Glaubitz, C. (2007) Dual Transormation of Homonuclear Solid-State NMR Spectra - an Option to Decrease Measuring Time, BBA Biomem. 1768, 3107-3115

• Lopez, J.J., Mason, A.J., Kaiser, C., Glaubitz, C. (2007) Separated Local Field NMR Experiments on Oriented Samples Rotating at the Magic Angle. J. Biomol. NMR, 37, 97-111.

cwDNP supported MAS-NMR

cwDNP is used to enhance the sensitivity of low temperature MAS-NMR by 20-80 fold. A non commercial system has been established in the lab and is used for membrane protein studies. Data below by L. Reggie et al. (2010) unpublished. 

geändert am 23. März 2011  E-Mail: WebmasterKobylka@chemie.uni-frankfurt.de