Final Report Summary - BIOL-TRANSP-COMPUT (Mechanisms of transport across biological membranes) Project IRG 276920 aimed to contribute to our understanding of how biological transporters work. To this end, computational biophysics methods were used.The specific systems proposed for study in the project are the SecA protein motor, the AHA2 plasma membrane proton pump, and a homology model of channelrhodopsin-2. These are biological systems of great interest to modern biophysics. SecA is the motor protein of the bacterial Sec protein secretion system; description of how the bacterial Sec machinery works has the potential to contribute to our understanding of the general physical-chemical principles of protein secretion, and have applications in drug design. AHA2 is a member of the P-type ATPase family of cation transporters, which includes potential drug targets. Channelrhodopsin-2, a light-driven cation channel from the family of microbial rhodopsins, is used in neurobiology applications, and provides an excellent model system to understand the architecture of channel vs. pump proteins. Conformational dynamics and interactions with the environment are essential for the functioning of membrane transporters. To understand the conformational dynamics of SecA, within the Marie Curie project extensive all-atom simulations of SecA in bulk water have been carried out. These simulations provided valuable insights into the motions of SecA from three different bacteria, at room temperature, in fluid water environments. Important observations from work on SecA include the tight coupling of protein and water interactions to the nucleotide-binding state, or the atomic-detail view of the structure and dynamics of the protein region where the chemical reaction takes place (Milenkovic and Bondar, manuscript in preparation). This information is valuable for considerations of the reaction coordinate of the motor.Proton transfer across long distances involves protein sidechains and water molecules that act as intermediate proton carriers or affect the reaction indirectly – for example, by helping control the geometry and proton affinity of the proton/donor acceptor groups. In the case of the AHA2 proton pump, the path followed by the protons and the role of discrete waters are poorly understood. Work carried out within the Marie Curie project highlighted the accessibility to water of the primary proton donor/acceptor group, and couplings of structure and dynamics to the protonation state (Guerra and Bondar 2014). An important limitation in understanding how channelrhodopsin-2 works is the lack of high-resolution three-dimensional structures of wild-type channelrhodopsin variants. To derive structural information on channelrhodopsins, in the framework of the project sequence analyses were performed (Nack et al 2012, del Val et al 2014a) and homology models of several channelrhodopsin variants were derived (del Val et al 2014a). The homology models provide starting coordinate sets that can help interpret experimental data, or be used for all-atom simulations to dissect conformational dynamics and study reaction mechanisms. The work on homology models of channelrhodopsins was augmented by systematic investigations of mutants of a homologue protein, the bacteriorhodopsin proton pump (del Val et al 2014b). An intriguing suggestion from this work is that a carboxylate/hydroxyl inter-helical hydrogen-bonding motif may help couple the dynamics of protein helical segments. In previous work (del Val et al 2012), the presence of motifs of hydroxyl-group sidechains along a transmembrane helix was associated with altered local structure and dynamics.The reaction cycles of retinal proteins such as channelrhodopsin or bacteriorhodopsin start with the photoisomerization of the retinal chromophore. A key question concerns the relaxation path of the retinal polyene chain. In work using combined quantum mechanics/molecular mechanics (Wolter et al 2014), it was shown that the relaxation path of the retinal chain in bacteriorhodopsin is largely determined by the internal torsional energetics of the retinal, and shaped by hydrogen bonding with surrounding protein and water groups.Hydrogen-bonding interactions are particularly important for the conformational dynamics of a transporter. Aspects pertaining to hydrogen bonding were addressed in several works published during the Marie Curie project. In ref. (Bondar and White 2012), the role of dynamical hydrogen-bonding networks in membrane protein function was addressed, and the hypothesis put forward that dynamical hydrogen bonds can be important for the conformations a protein samples during its reaction cycle. In ref. (Bondar and Dau 2012), a systematic analysis of hydrogen bonds in photosystem II was performed; this analysis allowed the authors to identify a carboxylate pair that may be protonated. Knowledge of groups that may store protons is essential for understanding the mechanism of action of proton-coupled biological systems such as photosystem II or channelrhodopsin.ReferencesBondar A-N, White SH. Hydrogen bond dynamics in membrane protein function. Biochimica et Biophysica Acta (Biomembranes) 1818: 942-950 (2012). Bondar A-N, Dau H. Extended protein/water H-bond networks in photosynthetic water oxidation. Biochimica et Biophysica Acta (Bioenergetics) 1817:1177-1190 (2012). del Val C, White SH, Bondar A-N. Ser/Thr motifs in transmembrane proteins: conservation patterns and effects on local protein structure and dynamics. Journal of Membrane Biology 245:717-730 (2012).del Val C, Bondar L, Bondar A-N. Coupling between water dynamics and inter-helical hydrogen bonds in a proton transporter. Journal of Structural Biology 186:95-111 (2014a).del Val C, Royuela-Flor J, Milenkovic S, Bondar A-N. Channelrhodopsins: a bioinformatics perspective. Biochimica et Biophysica Acta (Bioenergetics) 1837:643-655 (2014b). Guerra F, Bondar A-N. Dynamics of the plasma membrane proton pump. Journal of Membrane Biology, doi: 10.1007/s00232-014-9732-2 (2014).Wolter T, Elstner M, Fischer S, Smith JC, Bondar A-N. Mechanism by which untwisting of retinal leads to productive bacteriorhodopsin photocycle states. Journal of Physical Chemistry B, In press, doi: 10.1021/jp505818r (2014).Contact InformationHost: Prof. Dr. Joachim HeberleDepartment of Physics, Freie Universitaet BerlinArnimallee 14, D-14195 Berlin-Dahlem, Germanywww.physik.fu-berlin.de/en/einrichtungen/ag/ag-heberle/index.htmlFellow: Prof. Dr. Ana-Nicoleta BondarDepartment of Physics, Freie Universitaet BerlinArnimallee 14, D-14195 Berlin-Dahlem, Germanywww.physik.fu-berlin.de/en/einrichtungen/ag/ag-bondar/index.html
