Erik Freier

Proton transfer via a transient linear water-molecule chain in a membrane protein

High-resolution protein ground-state structures of proton pumps and channels have revealed internal protein-bound water molecules. Their possible active involvement in protein function has recently come into focus. An illustration of the formation of a protonated protein-bound water cluster that is actively involved in proton transfer was described for the membrane protein bacteriorhodopsin (bR) [Garczarek F, Gerwert K (2006) Nature 439:109–112]. Here we show through a combination of time-resolved FTIR spectroscopy and molecular dynamics simulations that three protein-bound water molecules are rearranged by a protein conformational change that resulted in a transient Grotthuss-type proton-transfer chain extending through a hydrophobic protein region of bR. This transient linear water chain facilitates proton transfer at an intermediate conformation only, thereby directing proton transfer within the protein. The rearrangement of protein-bound water molecules that we describe, from inactive positions in the ground state to an active chain in an intermediate state, appears to be energetically favored relative to transient incorporation of water molecules from the bulk. Our discovery provides insight into proton-transfer mechanisms through hydrophobic core regions of ubiquitous membrane spanning proteins such as G-protein coupled receptors or cytochrome C oxidases.

In Channelrhodopsin-2 Glu-90 Is Crucial for Ion Selectivity and Is Deprotonated during the Photocycle

Background: Channelrhodopsin-2 is a light-gated ion channel extensively used in optogenetics. Results: Glu-90 is deprotonated in the open state and is crucial for ion selectivity. Conclusion: Protonation change of Glu-90 is part of the opening/closing of the conductive pore, and the functional protein unit is assumed to be the monomer. Significance: Understanding the gating mechanism is necessary for optimizing this optogenetic tool. The light-activated microbial ion channel channelrhodopsin-2 (ChR2) is a powerful tool to study cellular processes with high spatiotemporal resolution in the emerging field of optogenetics. To customize the channel properties for optogenetic experiments, a detailed understanding of its molecular reaction mechanism is essential. Here, Glu-90, a key residue involved in the gating and selectivity mechanism of the ion channel is characterized in detail. The deprotonation of Glu-90 during the photocycle is elucidated by time-resolved FTIR spectroscopy, which seems to be part of the opening mechanism of the conductive pore. Furthermore, Glu-90 is crucial to ion selectivity as also revealed by mutation of this residue combined with voltage clamp experiments. By dynamic homology modeling, we further hypothesized that the conductive pore is flanked by Glu-90 and located between helices A, B, C, and G.

The role of protein-bound water molecules in microbial rhodopsins ☆

Protein-bound internal water molecules are essential features of the structure and function of microbial rhodopsins. Besides structural stabilization, they act as proton conductors and even proton storage sites. Currently, the most understood model system exhibiting such features is bacteriorhodopsin (bR). During the last 20 years, the importance of water molecules for proton transport has been revealed through this protein. It has been shown that water molecules are as essential as amino acids for proton transport and biological function. In this review, we present an overview of the historical development of this research on bR. We furthermore summarize the recently discovered protein-bound water features associated with proton transport. Specifically, we discuss a pentameric water/amino acid arrangement close to the protonated Schiff base as central proton-binding site, a protonated water cluster as proton storage site at the proton-release site, and a transient linear water chain at the proton uptake site. We highlight how protein conformational changes reposition or reorient internal water molecules, thereby guiding proton transport. Last, we compare the water positions in bR with those in other microbial rhodopsins to elucidate how protein-bound water molecules guide the function of microbial rhodopsins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Directional Proton Transfer in Membrane Proteins Achieved through Protonated Protein-Bound Water Molecules: A Proton Diode†

The key function of energy-transducing membrane proteins is the creation of a proton gradient by directional proton transfer. The role of protein-bound water molecules herein is not fully understood, as X-ray diffraction analysis has resolved the positions of oxygen, but not of hydrogen atoms in such protein–water complexes. Here we show, now timeresolved at atomic resolution, how a membrane protein achieves directional proton transfer via protein-bound water molecules in contrast to random proton transfer in liquid water. A combination of X-ray structure analysis, timeresolved FTIR spectroscopy, and molecular dynamics (MD) simulations elucidates how directionality is achieved. Using the proton-pump bacteriorhodopsin as the paradigm, we show how controlled conformational changes of few amino acid residues rearrange preordered water molecules and induce directional proton transfer. This mechanism is analogous to an electronic diode: a “proton diode”. According to the chemiosmotic theory, the creation of a proton gradient in photosynthesis and oxidative phosphorylation by means of directional proton transfer is the key step for energy transduction in living cells. ATPases use this proton gradient to produce ATP, the fuel for life. In contrast to this directional mechanism in proteins, proton transfer in liquid water is random. Bacteriorhodopsin (bR), a protein that belongs to the microbial rhodopsin family, achieves this directional proton transfer by a light-driven protonpumping mechanism. Like other microbial rhodopsins, bR exhibits a structural motif of seven transmembrane a-helices and a retinal chromophore covalently bound to a lysine through a protonated Schiff base. The light-induced retinal isomerization from all-trans in the ground state (BR) to the 13-cis conformer drives bR through a photocycle with intermediates named J, K, L, M, N, and O in order of their appearance. During the L to M transition, the protonated Schiff base (C=NH), the central proton-binding site, deprotonates and protonates its counterion Asp85 (step 1 in Figure 1a). Protonation of Asp85 breaks its salt bridge to Arg82, which then moves towards Glu194/Glu204 (step 2). The orientation of Arg82 depends on the protonation state of Asp85. The arginine movement destabilizes a protonated water cluster between Arg82, Glu194, and Glu204 (step 3 in Figure 1a), and a proton is released to the bulk. However, the detailed nature of the proton-release group is still under debate. QM/MM simulations of the protonrelease group propose a shared proton between Glu194 and Glu204, a Zundel cation with two water molecules (H5O2 ), or an asymmetric Eigen cation of four water molecules (H9O4 ). From time-resolved FTIR experiments with site-directed mutations around the protonated water cluster and H/D-exchange experiments we have concluded that the proton-release group forms a protonated water cluster, most likely an asymmetric Eigen ion as shown in Figure 1a in purple. Glu194 and Glu204 are clearly deprotonated in the bR ground state. This experimental result was recently confirmed by L renz-Fonfr a et al. Nevertheless, the exact nature of the protonated water cluster and the release mechanism has still to be determined. Here, we used X-ray structure analysis to determine the positions of the water oxygen atoms and FTIR difference spectroscopy to determine the dynamics of the corresponding water hydrogen atoms. The proton release to the bulk in the L [*] Dr. S. Wolf, E. Freier, Dr. M. Potschies, Prof. Dr. E. Hofmann, Prof. Dr. K. Gerwert Lehrstuhl f r Biophysik, Ruhr-University Bochum Universit tsstrasse 150, 44780 Bochum (Germany) Fax: (+49)234-321-4238 E-mail: gerwert@bph.rub.de Homepage: http://www.bph.rub.de Dr. S. Wolf, Prof. Dr. K. Gerwert Department of Biophysics CAS–Max-Planck Partner Institute for Computational Biology Shanghai Institutes for Biological Sciences 320 Yue Yang Road, 200031 Shanghai (P.R. China) [] Current address: Lehrstuhl f r Biophysik, Universit t Konstanz (Germany) [] These authors contributed equally to this work.

How Does a Membrane Protein Achieve a Vectorial Proton Transfer Via Water Molecules

We present a detailed mechanism for the proton transfer from a protein-bound protonated water cluster to the bulk water directed by protein side chains in the membrane protein bacteriorhodopsin. We use a combined approach of time-resolved Fourier transform infrared spectroscopy, molecular dynamics simulations, and X-ray structure analysis to elucidate the functional role of a hydrogen bond between Ser193 and Glu204. These two residues seal the internal protonated water cluster from the bulk water and the protein surface. During the photocycle of bacteriorhodopsin, a transient protonation of Glu204 leads to a breaking of this hydrogen bond. This breaking opens the gate to the extracellular bulk water, leading to a subsequent proton release from the protonated water cluster. We show in detail how the protein achieves vectorial proton transfer via protonated water clusters in contrast to random proton transfer in liquid water.

Nanoscale distinction of membrane patches - a TERS study of Halobacterium salinarum

The structural organization of cellular membranes has an essential influence on their functionality. The membrane surfaces currently are considered to consist of various distinct patches, which play an important role in many processes, however, not all parameters such as size and distribution are fully determined. In this study, purple membrane (PM) patches isolated from Halobacterium salinarum were investigated in a first step using TERS (tip-enhanced Raman spectroscopy). The characteristic Raman modes of the resonantly enhanced component of the purple membrane lattice, the retinal moiety of bacteriorhodopsin, were found to be suitable as PM markers. In a subsequent experiment a single Halobacterium salinarum was investigated with TERS. By means of the PM marker bands it was feasible to identify and localize PM patches on the bacterial surface. The size of these areas was determined to be a few hundred nanometers.

Raman fiber-optical method for colon cancer detection: Cross-validation and outlier identification approach

Endoscopy plays a major role in early recognition of cancer which is not externally accessible and therewith in increasing the survival rate. Raman spectroscopic fiber-optical approaches can help to decrease the impact on the patient, increase objectivity in tissue characterization, reduce expenses and provide a significant time advantage in endoscopy. In gastroenterology an early recognition of malign and precursor lesions is relevant. Instantaneous and precise differentiation between adenomas as precursor lesions for cancer and hyperplastic polyps on the one hand and between high and low-risk alterations on the other hand is important. Raman fiber-optical measurements of colon biopsy samples taken during colonoscopy were carried out during a clinical study, and samples of adenocarcinoma (22), tubular adenomas (141), hyperplastic polyps (79) and normal tissue (101) from 151 patients were analyzed. This allows us to focus on the bioinformatic analysis and to set stage for Raman endoscopic measurements. Since spectral differences between normal and cancerous biopsy samples are small, special care has to be taken in data analysis. Using a leave-one-patient-out cross-validation scheme, three different outlier identification methods were investigated to decrease the influence of systematic errors, like a residual risk in misplacement of the sample and spectral dilution of marker bands (esp. cancerous tissue) and therewith optimize the experimental design. Furthermore other validations methods like leave-one-sample-out and leave-one-spectrum-out cross-validation schemes were compared with leave-one-patient-out cross-validation. High-risk lesions were differentiated from low-risk lesions with a sensitivity of 79%, specificity of 74% and an accuracy of 77%, cancer and normal tissue with a sensitivity of 79%, specificity of 83% and an accuracy of 81%. Additionally applied outlier identification enabled us to improve the recognition of neoplastic biopsy samples.