UNDERSTANDING MOVEMENT AND MECHANISM IN MOLECULAR MACHINES
COST ACTION CM1306
2014 - 2018
Structural biology has seen tremendous progress in the elucidation of static atomic structures of increasingly complex biomolecular systems, many of which are potential targets for future molecular medicine and biotechnology applications. However, understanding of biomolecular mechanism and function also requires knowledge of dynamics, both locally at active sites and more globally in terms of long range domain movements. Recent advances in the development of such methods make it timely to establish an EU network to bring together the spectroscopic and structural biology communities that will enable integration of the static and dynamic aspects of important biomolecules.
This COST Action has promoted the collaborations necessary to achieve this integration, and has disseminated knowledge and target research that has translated into new understanding of complex biochemical processes.
AIMS & OBJECTIVES
The main objective of the Action was to bring together European researchers at the forefront of methodological developments in structural biology, spectroscopy and simulation in order to stimulate the development of these methods to the point where they can be routinely applied to resolve structure-dynamics-function relationships in complex biomacromolecular assemblies. More specifically:
· Improve dissemination and interactions between European spectroscopic and structural biology communities.
These groups are often tackling similar biological problems from different angles and hence share a common challenge. Although there was some coordination on the national scale, as well as pockets of pair-wise collaborations between countries, these groups predominantly acted independently and hence as yet did not benefit from one another.
· Improve transnational access to advanced and expensive equipment as well as expertise that currently exists in only a few specialised labs across Europe or in centralised facilities.
This Action has made broadly available the knowledge and equipment scattered over these labs and created added value to the large amount of national funding already received.
· Provide training and mentoring to ESRs.
Using Short-Term Scientific Missions (STSMs), Training Schools and Workshops we enabled ESRs to travel between world-leading European groups in the area of biomolecular structure, function and dynamics. This is a growing area of science, and ESRs trained in this area will have the opportunity to move into careers across biomedicine, biotechnology and fundamental bioscience.
STRUCTURE
Participants of this COST Action are organised into five Working Groups:
Working Group 1 – Methodology development in time-resolved measurements
Working Group 2 – Imaging and in situ structure determination
Working Group 3 – Methodology development in biomolecular spectroscopy
Working Group 4 – Methodology development in biomolecular simulation
Working Group 5 – Applications and Dissemination
Our faculty members have participated in Working Groups 3, 4 and 5.
WORKING GROUP THREE: METHODOLOGY DEVELOPMENT IN BIOMOLECULAR SPECTROSCOPY
Working group chair – Claus Seidel (Germany)
A wide range of spectroscopic techniques (UV, visible, near-IR, vibrational mir-IR, Raman and magnetic resonance techniques) have long been used successfully to study biological molecules with suitable chromophores in solution as well as, in a subset of systems, the crystal. The ability to correlate structure with both function and dynamics requires that these techniques be applied in a time resolved manner and, indeed, many of these spectroscopies are amenable to fast time resolved data collection if initiation and repetition methods are available as fast transient events. It is this initiation that is often the limiting factor. Where reversible photochemistry is not possible, other possible strategies are needed, both to initiate transients – for example by rapid mixing in jets or stopped-flow, and to deal with irreversible systems – for example by sample exchange methods. Long range structural constraints imposed by nanometer distance measurements are available through labelling methods in conjunction with both EPR and FRET. These are presently limited in accuracy by type of label, their mobilities and in maximum distance estimates as a result of machine sensitivities. Thus far they have also not yet been adapted to routinely perform time resolved experiments.
Working group objectives:
1) To address many of these limiting factors by stimulating exchanges of information between spectroscopic instrument makers and those with expertise in new methods of transient initiation/repetition; Furthermore, to develop, and provide access to, methods of transient initiation/repetition, specifically addressing issues of improved and wider applicability of photochemistry, non-photochemical means of transient initiation and strategies to deal with irreversible systems.
2) To create and update a comprehensive database of the unique instrumentation in Europe; And to create links between technologists and users with specific biological questions in order to provide access opportunities for applications to genuine biological problems.
3) To train new researchers in these areas through Workshops and Training Schools.
4) To develop new and more rigid labels to improve distance accuracies in EPR/FRET measurements; to extract multiple distance information from multiply labelled samples.
5) To improve EPR spectrometer performance including accessing RT distance measurements, novel pulse techniques and accessing longer distances as well as orientations.
6) Development of novel approaches to performing time-resolved distance measurements both in solution and ultimately in single crystals.
WORKING GROUP FOUR: METHODOLOGY DEVELOPMENT IN BIOMOLECULAR SIMULATION
Working group chair – Thomas Stockner (Austria)
The shortfall in known structures relative to the database of known sequences continues to increase. However, the number of folds that proteins of typical size can adopt seems to be limited and has been estimated to be in the range of 10-15,000. Homology modeling can be used to develop models for those protein sequences for which no structure is available. This task becomes increasingly difficult with increasing sequence divergence. Simulations can provide precise predictions of possible structures and their dynamics. However, independent validation of such Molecular Dynamics (MD) predictions are essential if these computational techniques are to have a major impact and contribution to understanding of macromolecular functions. Direct comparison between simulations and experiments has proven difficult, since data are often determined on different time and size scales, and simulations typically look at patches of nanometer size for sub microsecond times. However, MD simulation times and size are now beginning to extend into ranges where direct comparison and experimental testing becomes feasible. This Working Group will be a forum for theoreticians addressing method development to interact with those who will provide experimental data that can be used to test/verify in silico predictions; substantial overlap with WG1, WG3 and WG5 should facilitate these collaborative efforts. Synergistic effects of this information exchange will also help the experimental groups gain an understanding of the various theoretical methods and their applicability.
Working group objectives:
1) Exchange of information, expertise and theoretical background of computational methods with groups in WG1/2/3/5.
2) Address the limitations of current structure and complex prediction methods.
3) Develop methods and explore new possibilities for comparison between simulation and experiments.
4) Organize training and workshops for experimentalists and theoreticians.
5) Presentation at international meetings and publication of results in high impact journals.
6) Promote parameterisation of experimental probes (spin & fluorescent) for their use in all atomic and coarse grained MD simulations.
7) Identify geometric parameters that can be extracted from experimental data and incorporated as restraints in homology modelling, docking and conformational dynamics.
8) Stimulate identification and testing of experimentally measured parameters that can be directly compared to simulations (WG1/2/3/5).
9) Promote investigations into large scale molecular motions.
WORKING GROUP FIVE: APPLICATIONS AND DISSEMINATION
Working group chair – Manuela Pereira (Portugal)
This WG has provided a forum for dissemination and application of the techniques developed in WGs1-4. In this WG, a firm two-way link with biological research groups was established that enabled both the provision of model systems for methods development in WGs1-4 as well as high impact, and especially medically relevant, biological systems for which a high resolution structural and dynamic description was needed. These relevant systems included membrane transporters including multidrug resistance (MDR) transporters, membrane receptors (G protein coupled receptors, GPCRs), which are the target of many administered drugs as well as molecular machines and large macromolecular complexes. This WG has also provided a link to chemical and medicinal chemical researchers were able to make use of such dynamic structural information for the development of new drugs and biotechnological tools. The following systems have been identified as excellent candidates for one or more of the methodological approaches detailed in this Action: membrane transporters, GPCRs, virus assembly/disassembly, proton-coupled electron transfer, and large macromolecular machines. The key questions addressed were: a) the mechanism of membrane transport including the conformational rearrangement of the transport cycle as well as substrate and inhibitor binding to the transporters, b) the mechanism and the dynamics of signal transduction of GPCRs and the regulation of G-proteins, c) the role of RNA and interacting proteins for virus assembly/disassembly, the site of drug action and the proteins and conformations involved, d) the movement of domains, their regulation and coupling mechanisms in proton-coupled electron transfer, as well as e) subunit movement and sequence of events in catalysis and force generation of large molecular machines (e.g. molecular motors, FAS, PKS, actin cytoskeleton and ribosome).
Working group objectives:
1) To exchange information with the technologists and ask specific key biological questions for which new experimental approaches are required and need to be developed.
2) To educate researchers from WGs1-4 in Training Schools and Workshops hence improving their fundamental understanding of the systems under study.
3) Dissemination of results in conferences and in high impact journals.
4) To provide high quality samples for method development groups of WG1-3.
5) To apply the novel techniques (developed in WG1-4) to gain insights which were not achievable before.
6) To develop a specific understanding at the molecular level of structures, dynamics and conformational changes inherent in the function of these molecular machines.
PUBLISHED WORK BY OUR FACULTY
2018
Georgia I. Mitrou, Giorgos K. Sakkas, Konstantina P. Poulianiti, Aggeliki Karioti, Konstantinos Tepetes, Grigorios Christodoulidis, Giannis Giakas, Ioannis Stefanidis, Michael A. Geeves, Yiannis Koutedakis, Christina Karatzaferi (2018). Evidence of functional deficits at the single muscle fiber level in experimentally-induced renal insufficiency, Journal of Biomechanics 82 259-265
2017
This Project has received funding from:
