Recent advances in coarse-grain modeling allow us to zoom out from individual atoms and molecules to supramolecular complexes and subcellular compartments that contain tens of millions of particles and capture the complexity of the crowded environment of real cell membranes. Here I will describe the state-of-the-art of modelling cell membrane processes with the coarse-grain Martini model developed in our lab. I will illustrate the power of the model by providing a few in-depth examples of large-scale simulations, including the exchange of electron carriers in the photosystem II complex embedded in the thylakoid membrane, and the lateral organization of lipids and proteins in a realistic plasma membrane model.

Our results provide a molecular glimpse of the complexity of lipid-protein interactions, with po-27 tentially far reaching implications for the overall organization of the cell membrane.

Computational enzyme design holds great promise for providing new biocatalysts for synthetic chemistry. A strategy to design small mutant libraries of complementary enantioselective epoxide hydrolase variants for the production of highly enantioenriched (S,S)-diols and (R,R)-diols is developed. Key features of this strategy (CASCO, catalytic selectivity by computational design) are the design of mutations that favor binding of the substrate in a predefined orientation, the introduction of steric hindrance to prevent unwanted substrate binding modes, and ranking of designs by high-throughput molecular dynamics simulations. Using this strategy we obtained highly stereoselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.Recentsuccesses with de novo computational protein design (CPD) [1] and redesign of enzyme active sites [2] suggest that computational methods can be used for controlling enzyme selectivity, which is an important goal in industrial biocatalyst development. Enantioselectivity engineering is often pursued by directed evolution, in most cases with the use of large mutant libraries. [3] Although important successes have been reported, [3] further development could benefit from improved predictability of mutant properties and a higher abundance of beneficial substitutions in mutant libraries. We have recently explored the use of computational methods for the design of small mutant libraries harboring thermostable enzyme variants. [4] In the work reported herein, we demonstrate the use of computational library design for engineering enzyme stereoselectivity.The computational strategy that we explored is to place a substrate in the enzyme active site in an orientation required for the desired enantioselective conversion and to redesign the active site to stabilize that geometry. Protein sequences forming predefined substrate-binding sites or sites with shape complementarity to transition-state models can be generated by CPD methods such as implemented in RosettaDesign [5] after which laboratory screening is carried out to identify improved variants. [6] In the approach that we termed CASCO (Scheme 1), high-throughput molecular dynamics simulations that predict relative reactivity and thereby enantioselectivity replace most of the experimental screening. The strategy was tested with a challenging case of biocatalytic relevance: the design of a pair of enantiocomplementary epoxide hydrolases for the enantioselective transformation of cyclopentene oxide (1 a) to yield either (R,R)-or (S,S)-cyclopentane-1,2-diol (1 b) (Scheme 2). As the stereochemical outcome of this asymmetric conversion is controlled by regioselectivity of water attack, accurate placement of the substrate in the active site is essential. This is highly challenging because epoxide 1 a lacks bulky or polar groups that could position the substrate in a defin...

The ESCRT pathway mediates a series of important cellular membrane remodeling and fission events, including cytokinetic abscission. During these processes, ESCRT-III family proteins, including CHMP1B and IST1, form filaments that appear to constrict membranes and facilitate fission. Here, we report the first structure of ''open'' and assembled ESCRT-III proteins. Nearatomic resolution electron cryomicroscopy reveals that filaments comprise a copolymeric assembly of an open conformation inner strand and, unexpectedly, a closed conformation outer strand.

No abstract