Dynamics and Driving Forces of Macromolecular Complexes

Many functions in living cells are governed by macromolecular complexes. To fully describe the underlying mechanisms, they have to be understood at atomic level. The present study combines data obtained by X-ray crystallography and cryo-electron microscopy (cryo-EM) with molecular dynamics (MD) simulations. Two functions of macromolecular complexes, the downregulation of neurotransmitter release by the SNARE protein complex under oxidative stress and the translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis, were investigated. First, the hypothesis that oxidation of two cysteines on linker of the SNARE protein SNAP-25B and consequent disulfide bond formation shortens this linker sufficiently to hinder complex formation was tested. For this purpose, MD simulations of the SNARE complex with and without the disulfide bond were compared. Disulfide bond formation lead to conformational changes of the linker and of three central hydrophobic layers necessary to form the SNARE complex. Previously, mutations of residues contributing to these layers have been shown to reduce neurotransmitter release, suggesting that the stability of these layers is crucial for complex formation. The results from the simulations agree with the hypothesis that disulfide bond formation leads to a destabilization of the SNARE complex thus rendering it dysfunctional. This mechanism is interpreted as a chemomechanical regulation to shut down neurotransmitter release under oxidative stress, which has been linked to neurodegenerative diseases. In a second part I investigated the ribosome, where after peptide bond formation, bound tRNAs translocate by more than 7 nm to adjacent binding sites (A and P to P and E), accompanied by large-scale conformational motions (L1-stalk, intersubunit rotation) of the ribosome. By combining existing cryo-EM reconstructions of translocation intermediates with high resolution crystal structures, we obtained 13 near-atomic resolution structures. Subsequently, MD simulations of were carried out for each intermediate state. The obtained dynamics within these states allowed to estimate transition rates between states for motions of the L1-stalk, tRNAs and intersubunit rotations. Rapid motions of the L1-stalk and the small (30S) subunit on sub-microsecond timescales were revealed, whereas tRNA motions were seen to be rate-limiting for most transitions. By calculating the interaction free energy between L1-stalk and tRNA, molecular forces were derived showing that the L1-stalk is actively pulling the tRNA from P to E binding site, thereby overcoming barriers for the tRNA motion. Further, ribosomal proteins L5 and L16 guide the tRNAs by 'sliding' and 'stepping' mechanisms involving key protein-tRNA contacts. This explains how tRNA binding affinity is kept sufficiently constant to allow rapid translocation despite large-scale displacements. Translocation is accompanied by rotations of the 30S ribosomal subunit of more than 20 degrees relative to the large (50S) subunit. For each translocation intermediate, the affinity of the two subunits with each other must be finely tuned enabling such conformational flexibility while maintaining integrity of the ribosomal complex. Analyzing the trajectories at residue level reveals two classes of intersubunit contact interactions: i) persistent residue contacts which are independent of 30S rotation and primarily located close to the axis of rotation. ii) contacts that are formed and ruptured depending on the rotation angle, seen mainly on the periphery. Strikingly, also these rotation specific contacts substantially contribute to the overall stability of the ribosomal assembly and are expected to maintain a constant interaction energy with low barriers for rotation. The simulations reveal that upon removal of tRNAs peripheral contacts are weakened and, in turn, intersubunit rotation angles decrease, in agreement with cryo-EM analysis of tRNA depleted ribosomes. The identified mechanisms for lowering free energy barriers and for fine-tuning affinities might have developed similarly in other macromolecular complexes

Om bygningsarkæologien i arkitektfagets uddannelse og praksis

Building archaeology in education and practiceof the architectural professionBy Lars Nicolai BockThis article discusses how building archaeology relates to the architectural profession and the education of architects – mostly in a Danish context. The text is based on the authors experience as an assistant professor at the Aarhus School of Architecture as well as from his professional life. Building archaeology is a discipline practiced by a number of professions, most of these academic. One can speak of a situation where professional roles and their relation to field objects are rather unclear and need to be discussed. The different professions – that is for instance architects, archeologists, historians, art historians, conservators etc. – deal with objects that are different and alike. This situation calls for clarification and cooperation. The goal for a discussion could be an optimized education, practice and use and of the different professions and a more intense interdisciplinary and multidisciplinary use of the many special competencies. In Denmark, traditions coming from the academy of art have had a strong influence on the education of architects. This tradition, to a certain degree, rules out the more research-minded approach to for instance conservation and preservation. In the field of conservation and preservation there is a very strong need for knowledge as background for architectural projects. Knowledge about a historic building and its different periods is the background and prerequisite for defining both values and cultural significance. An architectural project that is not based on conscious and transparent valuation of the object – both as a whole and in details – will often loose the historic and architectural significance of the building. There is an increasing need for optimized skills, more knowledge as well in the field of research as in the field of design dealing with historic structures. There is a need for better educations, cooperation, interdisciplinary and multidisciplinary practice in the series of professions that deals with building archeology, conservation and preservation of historic buildings in the future

BiQ Analyzer HT: locus-specific analysis of DNA methylation by high-throughput bisulfite sequencing

Bisulfite sequencing is a widely used method for measuring DNA methylation in eukaryotic genomes. The assay provides single-base pair resolution and, given sufficient sequencing depth, its quantitative accuracy is excellent. High-throughput sequencing of bisulfite-converted DNA can be applied either genome wide or targeted to a defined set of genomic loci (e.g. using locus-specific PCR primers or DNA capture probes). Here, we describe BiQ Analyzer HT (http://biq-analyzer-ht.bioinf.mpi-inf.mpg.de/), a user-friendly software tool that supports locus-specific analysis and visualization of high-throughput bisulfite sequencing data. The software facilitates the shift from time-consuming clonal bisulfite sequencing to the more quantitative and cost-efficient use of high-throughput sequencing for studying locus-specific DNA methylation patterns. In addition, it is useful for locus-specific visualization of genome-wide bisulfite sequencing data

GNSS processing at CODE: status report

Since May 2003, the Center for Orbit Determination in Europe (CODE), one of the analysis centers of the International GNSS Service, has generated GPS and GLONASS products in a rigorous combined multi-system processing scheme, which promises the best possible consistency of the orbits of both systems. The resulting products, in particular the satellite orbits and clocks, are easily accessible by the user community. In the first part of this article, we focus on the generation of the combined global products at CODE, where we put emphasis not only on accuracy, but also on completeness. We study the impact of GLONASS on the CODE products, and the benefit of using them. Last, but not least, we introduce AGNES (Automated GNSS Network for Switzerland), a regional tracking network of small extensions (roughly 400km East-West, 200km North-South), which consequently tracks all GNSS satellites and analyzes their measurements using the CODE product