Scientific Program

Conference Series Ltd invites all the participants across the globe to attend 9th International Conference on Structural Biology Zurich, Switzerland.

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Day 2 :

Keynote Forum

Timothy A. Cross

Florida State Univ. & National High Magnetic Field Lab, USA

Keynote: Membrane Protein Structure, Dynamics & Function: Oriented Sample and Magic Angle Sample Spinning Solid State NMR
Structural Biology 2017 International Conference Keynote Speaker Timothy A. Cross photo
Biography:

Timothy Cross has more that 30 years of experience characterizing membrane proteins in lipid bilayer environments using solid state NMR of liquid crystalline bilayer preparations of peptides and proteins. This has brought to light a fundamental understanding of membrane protein biophysics that has led to detailed functional characterizations of membrane channels – in particular the gramicidin monovalent cation channel and the influenza A M2 proton channel. In both systems the unique features of the membrane environment play crucial roles in the functional mechanisms and kinetics of ion conductance. Is this understanding of the influence of membrane and lipid environments through the use of solid state NMR that has driven his research at the frontier of membrane protein biophysics.

Abstract:

Statement of the Problem: Unlike water soluble proteins that have a relatively homogeneous environment, membrane proteins exist in a dramatically heterogeneous environment. The result is protein structure that is stabilized by a different balance of molecular interactions for the membrane embedded portion of the protein compared to the water soluble or membrane interfacial regions of the protein. The result is a need to model the membrane environment as closely as possible to that of the native environment for structural, dynamic and functional characterizations. Methodology: Biological solid state NMR provides a unique opportunity to model the membrane environment with liquid crystalline lipid bilayers and a wide variety of lipids. The samples can be prepared either as liposomes for magic angle sample spinning (MAS) or as uniformly oriented samples (OS) for the spectroscopy. The former provides solution like spectra for both distance and isotropic chemical shift restraints, while oriented samples provide absolute restraints that restrain the atomic sites in the protein structure to the bilayer normal. In addition to structural restraints it is possible to characterize the protein’s dynamics and kinetic rates. Findings, Conclusion & Significance: The structure, dynamics and kinetics associated with the M2 proton channel from influenza A have been characterized yielding a unique mechanism for proton transport by this important drug target. In addition, the cholesterol binding to M2 has been found to stabilize the amphipathic helix in the lipid interface an essential feature for this protein’s functional role in viral budding. Recent structural studies of the CrgA protein from Mycobacterium tuberculosis  have characterized a dimeric structure stabilized primarily by intermolecular b-sheet in the membrane interfacial region. The protein is part of the cell division apparatus and appears to play a role in recruiting multiple proteins to the divisome, potentially through its transmembrane domain.

Keynote Forum

Wladek Minor

University of Virginia, USA

Keynote: Reproducibility in Biomedical Sciences - Big Data Perspective
Structural Biology 2017 International Conference Keynote Speaker Wladek Minor photo
Biography:

Wladek Minor is a Harrison Distinguished Professor of Molecular Physiology and Biological Physics at University of Virginia. He is an expert in structural biology and data mining. He is an author of over 190 papers that attracted over 37,000 of citations. His Relative Citations Ratio is above 560.He trained over 80 scientists that currently pursue career in academia, government and industry.

Abstract:

Experimental reproducibility is the cornerstone of scientific research, upon which all progress rests. The veracity of scientific publications is crucial because subsequent lines of investigation rely on previous knowledge. Several recent systematic surveys of academic results published in biomedical journals reveal that a large fraction of representative sets of studies in a variety of fields cannot be reproduced in another laboratory. Big Data approach and especially NIH Big Data to Knowledge (BD2K) program is coming to the rescue.

The goal of the presented research is to provide the biomedical community with a strategy to increase the reproducibility of reported results for a wide range of experiments by building a set of “best practices”, culled by extensive data harvesting and curation combined with experimental verification of the parameters crucial for reproducibility. Experimental verification assisted by the automatic/semi-automatic harvesting of data from laboratory equipment into the already developed sophisticated laboratory information management system (LIMS) will be presented. This data in, information out paradigm will be discussed. 

Keynote Forum

Yuri L. Lyubchenko

University of Nebraska Medical Center, USA

Keynote: Protein-protein interaction and amyloid cascade hypothesis for Alzheimer’s disease
Structural Biology 2017 International Conference Keynote Speaker Yuri L. Lyubchenko photo
Biography:

Yuri L. Lyubchenko is Professor of Pharmaceutical Sciences at the University of Nebraska Medical Center, Omaha, NE, USA. His research focuses on understanding fundamental mechanisms underlying health and disease, which are key to developing new and more effective diagnostics and medications. This primarily basic research allows him not only identify new drug targets for small molecule drugs, it also leads to development of the nanotools and methods to discover novel approaches for diagnostic, treatment and disease prevention and to more rapidly determine their efficacy at the molecular level.

Abstract:

The amyloid cascade hypothesis is currently considered as the main model for a vast number of neurodegenerative diseases including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Numerous studies have shown that amyloidogenic proteins are capable of spontaneous assembly into aggregates, and eventually form fibrillar structures found in amyloid or amyloid‐like deposits. However, there is a serious complication with translating current knowledge on amyloid aggregation in vitro to understand the aggregation process in vivo. If the critical concentration for the spontaneous aggregation of Aβ peptide in vitro is in the micromolar range, physiological concentrations of Aβ are in the low nanomolar range making impossible amyloids to assemble. We have discovered a novel on-surface aggregation pathway that allows for spontaneous assembly of amyloid beta peptides at the physiological concentration range. Our combined experimental and computer modeling approaches demonstrate that the on-surface aggregation is a dynamic process, so the assembled aggregate can dissociate from the surface to the bulk solution. As a result, the dissociated oligomers can play roles of seeds for aggregation in the bulk solution, or start a neurotoxic effect such as phosphorylation of the tau protein to initiate its misfolding and aggregation. Both processes lead to neurodegeneration. Importantly, in the vast majority of cases, we found that aggregates formed on the surface are oligomers, which are considered to be the most neurotoxic amyloid aggregates. Therefore, we posit that on-surface aggregation is the mechanism by which neurotoxic amyloid aggregates are produced under physiological conditions. A change in membrane properties leading to an increase in affinity of amyloid proteins to the membrane surface facilitates the assembly of stable oligomers. The proposed model is a significant departure from the current model as it directs the development of treatments and preventions towards approaches that control the cell membranes properties and composition preventing the on-surface aggregation process.   

  • Track 9: Structural Biology in Complexity Arenas
Speaker
Biography:

Charlie Carter is an X-ray crystallographer who studies the origin, evolution, and structural biology of aminoacyl-tRNA synthetases. His research group introduced the use of Urzymes—highly conserved structural cores that retain large fractions of the transition-state stabilization free enregies of full length enzymes—as experimental models of ancestral enzymes. 

Abstract:

Enzyme mechanisms, especially those that couple NTP hydrolysis to mechanical work and information, use sophisticated dynamic networks to transduce active-site chemistry into domain motions that change binding affinities [1]. We measured and cross-validated the energetics of such networks in B. stearothermophilus Tryptophanyl-tRNA synthetase (TrpRS) using both multi-mutant and modular  thermodynamic cycles [2]. Coordinated domain motions develop shear in a core packing motif conserved in >125 different protein superfamilies [2]. Multi-dimensional combinatorial mutagenesis showed that four side chains from this “molecular switch” move coordinately with the active-site Mg2+ ion in the transition state for amino acid activation [3]. A modular thermodynamic cycle consisting of full-length TrpRS, the Urzyme, and the Urzyme plus each of the two domains deleted in the Urzyme [4]gives similar energetics. These complementary experiments establish that catalysis and specificity in full-length TrpRS are both coupled by 5 kcal/mole to:  (i) the core packing region where domain movement generates shear, and (ii) the simultaneous motion of the two domains relative to the Urzyme. Theory shows that the minimum action path algorithm estimates thermodynamically meaningful contributions of domain movement to kinetic rates [5]. Correlations between those parameters, the experimental rates, and structural variations induced in the combinatorial mutants confirm that these estimates are realistic. These results validate our previous conclusion that catalysis by Mg2+ ion is coupled to the overall domain motion (3). Computational free energy surfaces demonstrate that TrpRS catalytic domain motion itself is endergonic but is driven thermodynamically by PPi release [5,6]. Comparison of the impact of combinatorial mutagenesis on pre-steady state and steady-state rates confirm that dynamic active-site pre-organization endows TrpRS with the elusive conditionality of NTP utilization on domain motion.           

Speaker
Biography:

The Buck laboratory studies two receptor families responsible for cell guidance and positional maintenance (Plexins and Ephrins), both with key involvement in cardiovascular and neuronal development and disease, esp. cancer. We use a wide range of structural biology (solution NMR / x-ray crystallography) and protein biophysical tools (CD, fluorescence spectroscopy, ITC and SPR) in a problem oriented approach. Part of the laboratory also pursues computational modeling and molecular dynamics to provide additional perspective on the problems, provide new insights into the experimental data and to suggest further studies.  Small GTPases and their interaction with the plexin receptor cytoplasmic domains has been a major focus of the laboratory and recently we have become very interested in protein-membrane interactions; both the transmembrane regions of the receptors as well as the transient interactions of receptor and GTPase domains with membranes

Abstract:

It is now recognized that protein-protein interactions in solution are often dynamic, especially if the binding affinities are only moderately strong. Dynamics originate in part from the interconversion between structures of the protein complex, e.g. one bound state that is in equilibrium with one or several alternate configurations. We determined the structure of such a complex using NMR restraints [1] and saw the transitions between different configurations in microsecond length all-atom molecular dynamics simulations [2]. Recently, we also studied the dissociation process of mutant complexes which had a weakened primary interaction interface. Those simulations suggested that there is no single dissociation pathway, but that the separation first involves transitions to binding interfaces with fewer/weaker contacts [3].

Comparison is made between experimental NMR relaxation measurements on the ps-ns as well as µs-ms timescale with the microsecond all atom simulations, also in the context of new simulations of the protein association process. The functional significance of the protein complex alternate states and their dynamics are discussed.

In a second part of the presentation, we consider a second system involving transient interactions; this time between K-Ras and the lipid bilayer of the plasma membrane [4]. Our recent simulations the full length GTPase at different membranes reveal the underlying rules of the interactions, emphasizing electrostatic contacts but also protein topology [5].  Again, simulations are compared with NMR experiments, carried out at model systems for the membrane.

Christophe Guyeux

Université de Bourgogne Franche-Comté, France

Title: On two ways to predict the protein folding process over a chaotic model

Time :

Speaker
Biography:

Guyeux has a record of about 120 scholarly publications. Since 2010, he published 43 articles in peer-reviewed international journals (as a co-author, including the top-ranked ones in the areas of computer science and interdisciplinary applications, such as AIP Chaos, Plos One, or Clinical Infectious Diseases). He is co-author of 2 book chapters and 2 scientific monograms. He is also author of 4 software patents, 53 articles that appeared in proceeding of peer-reviewed international conferences. His topics of research encompasses bioinformatics, discrete dynamical systems, and information security.

Abstract:

In our first theoretical studies about folded self-avoiding walks, we have raised raises several questions regarding the protein structure prediction problem and the current ways to solve it. In one category of PSP software, the protein is supposed to be synthesized first as a straight line of amino acids, and then this line of a.a. is folded out until reaching a conformation that optimizes a given scoring function. The second category consider that, as the protein is already in the aqueous solvent, it does not wait the end of the synthesis to take its 3D conformation. So they consider SAWs whose number of steps increases until the number of amino acids of the targeted protein and, at each step, the current walk is stretched (one amino acid is added to the protein) in such a way that the pivot k  placed in the position that optimizes the scoring function. We have proven that the two sets of possible conformations are different. So these two kinds of PSP software cannot predict the same kind of conformations.

We have proven too that the folding process G in the 2D model is chaotic according to Devaney. A consequence of this theorem is that this process is highly sensitive to its initial condition. If the 2D model can accurately describe the natural process, then this theorem implies that even a minute difference on an intermediate conformation of the protein, in forces that act in the folding process, or in the position of an atom, can lead to enormous differences in its final conformation. In particular, it seems very difficult to predict, in this 2D model, the structure of a given protein by using the knowledge of the structure of similar proteins. Let us remark that the whole 3D folding process with real torsion angles is obviously more complex. And finally, that chaos refers to our incapacity to make good prediction, it does not mean that the biological process is a random one.

Beat Vögeli

University of Colorado at Denver, USA

Title: Functional protein conformation networks probed by NMR nanorulers

Time :

Speaker
Biography:

Beat Vögeli has his expertise in nuclear magnetic resonance (NMR) spectroscopy of biomacromolecules. He develops methodology for the elucidation of conformation and communication networks within and between proteins and nucleic acids. He received his Ph.D. degree at the ETH Zürich in the group of Konstantin Pervushin. After a postdoctoral stay at the National Institutes of Health, Bethesda USA, in the group of Ad Bax, he returned to ETH Zürich to become Oberassistant in the group of Roland Riek and Privatdozent. He is currently an assistant professor at the University of Colorado at Denver in the Department of Biochemistry and Molecular Genetics.

Abstract:

Beat Vögeli has his expertise in nuclear magnetic resonance (NMR) spectroscopy of biomacromolecules. He develops methodology for the elucidation of conformation and communication networks within and between proteins and nucleic acids. He received his Ph.D. degree at the ETH Zürich in the group of Konstantin Pervushin. After a postdoctoral stay at the National Institutes of Health, Bethesda USA, in the group of Ad Bax, he returned to ETH Zürich to become Oberassistant in the group of Roland Riek and Privatdozent. He is currently an assistant professor at the University of Colorado at Denver in the Department of Biochemistry and Molecular Genetics.

Speaker
Biography:

Marta obtained Ph.D. in 2007 in nanomaterials from Cranfield University. In 2011, she joined Structural Biology group led by Alaistair Lawson at UCB. For the past five years she has been developing expertise in small molecule drug discovery using a fragment based approach. She had also been involved in the assay development for a novel antibody-enable drug discovery approach. Prior to joining UCB she was awarded a Postdoctoral Fellowship at the Institute of Food Research in Norwich to study the mono and multilayer films used to encapsulate active ingredients for a controlled and site-specific delivery within the GIT. During her time at IFR she worked on numerous projects including a commercial project for Pfizer focused on AFM imaging of proteins and polysaccharides constituting vaccines. She has also been part of the team under the leadership of Claudio Nicolletii investigating the outcome of consumption of the probiotic L. casei Shirota in subjects with Seasonal Allergic Rhinitis. 

Abstract:

Protein-protein interactions (PPIs) are of critical importance in the majority of biological cellular processes including DNA repair, immune, and allergic responses. Despite their therapeutic relevance, PPIs are intrinsically challenging targets due to complexity of interactions, assay tractability and the lack of well-defined binding pockets at the interacting surfaces.  

In the quest for small molecule drug candidates targeting PPIs, there have been a number of different approaches adopted, which include: use of existing lead or drugs, natural products, high-throughput screening and more recently established powerful fragment-based drug discovery.

At UCB we have integrated the fragment-based methodology with biological and structural information obtained from antibody-validated protein targets, to develop specific small molecule inhibitors of PPIs. An ensemble of biophysical methods (i.e. SPR, ITC, FRET, MS and ligand-based NMR), corroborated by functional data, were employed to identify and validate fragment hits that constituted the starting point for our PPI inhibitor drug discovery programs. We have also employed antibodies as research tools to hold target proteins in biologically active conformations, aiding the discovery of new small molecules for challenging targets. By holding the target protein in biologically relevant conformations, new sites (in particular allosteric sites), which would otherwise be inaccessible, may become available for binding. The ability to capture the target protein in a specific conformation with high affinity for a significantly long time opens the possibility for a small-fragment molecule screening.

Speaker
Biography:

Shin-ichi Tate has got his Ph.D. degree from the University of Tokyo in 1993. He experienced visiting researcher at ETH (Prof. K. Wühtrich) and NIH (Dr. A. Bax). He has been a professor in the department of the mathematical and life sciences at Hiroshima since 2006. He is now the director of the Research Center for the Mathematics on Chromatin live Dynamics (RcMcd), and the dean of the school of science in Hiroshima University.

 

Abstract:

The functionally relevant inter-domain communication between the domains linked by intrinsically disordered region (IDR) was explored by NMR in combination with small angle X-ray scattering (SAXS). Based on the ensemble structure analyses and the numerical simulations to reproduce the chemical shift changes along with the substrate concentration, we have demonstrated how the domains cooperate to enhance the protein function through the substantially dynamic spatial allocation of the domains.

Pin1, a proline cis/trans isomerase, comprises two domains linked by 10-residue IDR; one is the substrate biding domain to recognize pSer/pThr-Pro motif and the other is the enzyme domain that rotates the Pro peptide bond in the motif. The enzyme domain shows very limited affinity to the substrate, but its binding ability was enhanced by two orders of magnitude in the presence of the substrate binding domain linked by IDR; in which the inter-domain ‘fly-casting’ mechanism plays to keep the substrate bound to Pin1 by tossing and receiving the substrate between the domains, once the substrate in bound to either one of the domains. A new functional aspect of IDR will be addressed.

 

Biography:

Enrica Bordignon is an associate professor at the Ruhr University of Bochum, where she leads an EPR laboratory dedicated to the study of membrane proteins. Approximately 50 papers in protein research by EPR methods. Research interests: understanding the mechanism of action of proteins by EPR. 

Abstract:

ABC exporters pump substrates across the membrane by coupling ATP- driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF and OF) orientations. Understanding their cycle has potential for medical applications because they are involved in multidrug resistance of cancer cells.

Site-directed spin labeling electron paramagnetic resonance (EPR), and in particular the dipolar spectroscopy technique called DEER (or PELDOR) was used to investigate the conformational transition of the ABC heterodimeric exporter TM287/288 from the hyperthermophile T. maritima. The analysis revealed that with nucleotides the transporter exists in an equilibrium between the IF and OF states. ATP binding without hydrolysis was sufficient to partially populate the OF state, and an almost complete conformational shift was observed when nucleotides were trapped in a pre- or post-hydrolytic state. At physiological temperature and without nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric ABC exporters from their homodimeric counterparts. Our data suggest hydrolysis-independent partial closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis. A unified mechanism is established, which reconciles the available information for heterodimeric ABC exporters1.

Biography:

Jaydeb Chakrabarti is a theoretical physicist trained in condensed matter physics. He looks into Statics and Dynamics of soft matter systems including bio-macromolecules. The theoretical methods used in these studies are: (1) Computer simulations based on Molecular Dynamics, Monte Carlo and Brownian Dynamics; (2) Mean field calculations based on classical density functional theories. The goal of his researches is to relate macroscopic properties to microscopic motions.

Abstract:

Statement of the Problem: The microscopic basis of connection between protein conformation and function is a fundamental challenge. Recent experiments show the importance of conformational changes in providing stability to protein complexes. The changes in conformational state have both enthalpy and entropic components. It has been possible to quantify the entropy changes due conformational changes from Nuclear magnetic Resonance data. Here we show microscopic calculation of both the enthalpy and entropy contribution while protein conformations change from the equilibrium distribution of the dihedral angles of proteins. We have shown that the free-energetically destabilized and entropically disordered residues in a given conformation compared to a reference conformation act as binding residue in the giv en conformation. Here we show that this principle can: (1) ascertain ligand binding of a protein in different conformations; (2) supplement the structure of missing fragments in a protein; and (3) serve as a guideline for allosteric changes. All these applications point to tune protein in-silico which would help to design functional materials with protein as building blocks.

Speaker
Biography:

Fabio C. L. Almeida has his expertise in protein structure and dynamics by nuclear magnetic resonance (NMR). He solved the structure of several proteins by NMR. He has important contribution in the structure and dynamics of plant defensins. Fabio and his group showed that despite the conserved folding, defensins display a wide variation in dynamics, which enabled the mapping of binding regions and description of the mechanism of membrane recognition. The group also showed that dynamics are also the key for the understanding of the mechanism of membrane recognition of antimicrobial peptides. Pre-existent order in flexible peptides permits discrimination between the regions of specific and non-specific binding. Fabio´s group also described the structure and dynamics of the water cavity of thioredoxin, which is an essential structural element for catalysis. Fabio is the director of the National Center of NMR (CNRMN, http://cnrmn.bioqmed.ufrj.br) and president of the Brazilian NMR Association (AUREMN).

Abstract:

Proteins are dynamic entities able to move in a wide range of timescales that goes from picoseconds to seconds. Motions that occur in microseconds to seconds define biologically relevant events that are frequently involved in binding, allostery and catalysis1,2. In our laboratory, we used relaxation parameter, relaxation dispersion experiments and molecular dynamic simulation to correlate conformational equilibrium with molecular recognition and catalysis.

Dengue and Zika are major arthropod-borne human viral disease, for which no specific treatment is available. The flavivirus capsid protein is the trigger of virus assembly. Capsid proteins are located at the cytoplasm bound to lipid droplets (LD). Binding to LDs are essential for virus assembly3,4. We previously showed that the positively charged N-terminal region of Dengue virus capsid protein prompts the interaction with negatively charged LDs, after which a conformational rearrangement enables the access of the central hydrophobic patch to the LD surface5. We also showed the participation of the intrinsically disordered region in binding and possible regulation of capsid assembly6.

We probed the structure and dynamics of Dengue virus and Zika virus capsid proteins (DENVC and ZkC) by nuclear magnetic resonance. They bind lipid droplets (LD) in the cytoplasm, which mediates virus assembly in an unknown way. We showed that the dynamics of the capsid protein is intrinsically involved in the mechanism of LD and RNA binding and virus assembly. We also measured binding to nucleic acids and probed the assembly using small angle x-ray scattering and negative staining electron microscopy. The understanding of the participation of the intrinsically disordered N-terminal region and its dynamics helped us propose a mechanism for Dengue and Zika virus assembly and to develop a peptide with the potential to block virus assembly.

ACKNOLEDGEMENTS: FAPERJ, CAPES, CNPq, INBEB-CNPq.

  • Track 10: GPCRs & Signalling Biology
Biography:

Francis Millett received his B.S in Chemistry from the University of Wisconsin in 1965, his Ph.D. in Chemical Physics from Columbia University in 1970, and was an NIH Postdoctoral Fellow at California Institute of Technology from 1970-1972.  He joined the faculty of the University of Arkansas in 1972, and is now a Distinguished Professor.  He developed, together with Bill Durham, the ruthenium photoreduction method which made it possible to measure the kinetics of key steps in electron transfer during mitochondrial oxidative phosphorylation.  He has directed collaborative, multidisciplinary research which combines rapid kinetics methods, site-directed mutagenesis, X-ray crystallography, and NMR to investigate protein structure-function relationships.

Abstract:

The electron transfer reactions within wild-type Rhodobacter  sphaeroides  cytochrome bc1 (cyt bc1) were studied using a ruthenium dimer to rapidly photooxidize cyt c1.  It was found that when cyt bH was initially reduced before the reaction, photooxidation of cyt c1 led to bifurcated reduction of both the iron-sulfur protein and cyt bL by QH2 in the Qo site, followed by re-oxidation of two equivalents of cyt bL and cyt bH.  It was proposed that the newly formed ubiquinone diffused through the hydrophobic cavity linking the Qo site of the reactive monomer A to the Qi site of the other monomer B, leading to oxidation of cyt bH in monomer B  followed by oxidation of cyt bL in monomer A by cross-monomer electron transfer.  Addition of one equivalent of the Qi site inhibitor antimycin to the cyt bc1 dimer had very little effect on any of the electron transfer reactions, while addition of a second equivalent completely inhibited re-oxidation of cyt bL and cyt bH.  It was also found that addition of one equivalent of the Qo site inhibitor stigmatellin to the cyt bc1 dimer completely inhibited all electron transfer reactions in both monomers of the dimer.  These experiments are consistent with a half-of-the-sites mechanism in which only one monomer of the dimer is active at a time, implying monomer-monomer interactions.  The rapid electron transfer reaction from the ISP to cyt c1 was found to be greatly decreased by viscosity, indicating a multi-step diffusional mechanism as the iron-sulfur protein rotates from the b state to the c1 state.    

Speaker
Biography:

Leonas Valkunas is the author of more than 350 international publications, head of the department of Theoretical Physics at Vilnius university and head of the Department of Molecular Compounds Physics at the Center for Physical Sciences and Technology in Vilnius. He is involved in studies of primary processes of photosynthesis such as excitation dynamics and photoinduced  charge separation in various photosynthetic systems based on the spectroscopic data and using various theoretical modelling approaches.

Abstract:

Fucoxanthin–chlorophyll protein (FCP) is the key molecular complex performing the light-harvesting function in diatoms, which, being a major group of algae, are responsible for up to one quarter of the total primary production on Earth. These photosynthetic organisms contain an unusually large amount of the carotenoid fucoxanthin, which absorbs the light in the blue–green spectral region and transfers the captured excitation energy to the FCP bound chlorophylls. Due to the large number of fucoxanthins, the excitation energy transfer cascades in these complexes are particularly tangled. Energy transfer processes and coherent phenomena in the fucoxanthin–chlorophyll protein complex, which is responsible for the light harvesting function in marine algae diatoms, were investigated at 77 K by using two-dimensional electronic spectroscopy. Experiments performed on femtosecond and picosecond timescales led to separation of spectral dynamics, witnessing evolutions of coherence and population states of the system in the spectral region of Qy transitions of chlorophylls a and c. Analysis of the coherence dynamics allowed us to identify chlorophyll (Chl) a and fucoxanthin intramolecular vibrations dominating over the first few picoseconds. Closer inspection of the spectral region of the Qy transition of Chl c revealed previously not identified, mutually non-interacting chlorophyll c states participating in femtosecond or picosecond energy transfer to the Chl a molecules. Consideration of separated coherent and incoherent dynamics allowed us to hypothesize the vibrations-assisted coherent energy transfer between Chl c and Chl a and the overall spatial arrangement of chlorophyll molecules.

Jianmin Cui

Washington University in St. Louis, USA

Title: PIP2 modulation of KCNQ1 channels
Speaker
Biography:

Jianmin Cui is a professor on the Spencer T. Olin Endowment at Washington University in St. Louis, in the Department of Biomedical Engineering. He received Ph.D. in Physiology and Biophysics from State University of New York at Stony Brook and a post-doctoral training at Stanford University. He was an assistant professor of Biomedical Engineering at Case Western Reserve University before moving to St. Louis. His research interests include BK-type calcium-activated potassium channels and IKs channels.

Abstract:

Voltage-gated ion channels generate dynamic ionic currents that are vital to the physiological functions of many tissues. These proteins contain separate voltage-sensing domains, which detect changes in transmembrane voltage, and pore domains, which conduct ions. Coupling of voltage sensing and pore opening is critical to the channel function and has been modeled as a protein–protein interaction between the two domains. However, our data show that coupling in Kv7.1 channels requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP2. This result is due to loss of coupling because PIP2 was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP2-dependent coupling at the interface between the voltage-sensing domain and the pore domain. This site is actually a conserved lipid-binding site among different K+ channels, suggesting that lipids play an important role in coupling in many ion channels. To further investigate the mechanism of PIP2 mediated VSD-pore coupling, we identified a compound that mimics PIP2 structure and function as a molecular probe. This compound was identified using an in silico screening approach based on molecular docking of a library of compounds to the PIP2 binding site in a homology model of the Kv7.1 channel. Our results show that this compound can substitute PIP2 in activating the Kv7.1 channel.  

Speaker
Biography:

Igor Barsukov has expertise in structural biology, primarily using NMR spectroscopy and X-ray crystallography. The main focus of his research has been on the structure and function of integrin-mediated cell-matrix adhesions, where he directed full structural analysis of the key adhesion proteins talin, leading to the currently widely used model of stretch-dependent talin activation. He is currently extending the model of talin functionality to include competitive, talin based, interaction networks. Recently he identified interactions between neuronal scaffold proteins Shank3 and Ras-family GTPase that regulate integrin activity and have implications for control of synaptic plasticity.

Abstract:

Statement of the Problem: Cell-matrix adhesion requires assembly of large multi-protein complexes linked to the cyto-domains on the integrin adhesion receptors. These complexes dynamically change in response to the adhesion forces and environmental signals. Mechano-sensitive adaptor protein talin couples the force and adhesion signaling by acting as a hub for numerous, often competitive, interactions. The molecular mechanism that controls the co-ordination of the interactions in time and space is currently not understood. The aim of this study is to define the interactions between talin and Rho-GAP Deleted in Liver Cancer 1 (DLC1) that regulates adhesion forces. Methodology: Structure of the talin/DLC1 complex was solved by X-ray crystallography and the interactions between the proteins analysed by NMR spectroscopy. Fluorescent imaging was used to define the interactions within the adhesion complexes and cancer cell lines were employed to characterise the effect of the interaction on the biological activity. Findings: We defined the atomic details of the talin/DLC1 interactions and used this information to identify signalling protein paxillin as a talin ligand. Based on the structural information we designed a range of talin mutants that modulate the interactions and demonstrated that the mutations reduce DLC1 signalling in adhesion. Conclusion & Significance: Talin recognises LD motifs in DLC1 and paxillin through a set of well-defined charge interactions. These interactions are similar to other LD motif interactions previously identified in signalling pathways. These interactions are also similar to the interactions between talin, RIAM and vinculin that were previously not assigned to the LD motif family. Together, the relatively weak LD motif interactions within the adhesion complex create a protein network that could dynamically respond to the adhesion signals.

Joachim Krebs

Max Planck Institute for Biophysical Chemistry, Germany

Title: Calcium, Calmodulin and the Plasma Membrane Calcium Pump
Speaker
Biography:

Prof. RJP Williams at Oxford, UK. In 1977 he accepted a staff position at the Institute of Biochemistry at the Swiss Federal Institute of Technology (ETH) in Zürich, Switzerland. He was lecturing different courses in biochemistry and biophysics, and was leading a Lab working on the structure-function relationship of calcium-binding and calcium-transporting proteins. After retirement from the ETH he continued his research as a consultant of the Lab of Prof. Christian Griesinger at the MPI in Göttingen, Germany. He has authored, co-authored and edited numerous articles in international journals. Recently he edited a book on “Calcium: A matter of life or death” published in 2007. He is at the Editorial Board of BBA Molecular Cell Research and Archives of Biochemistry and Biophysics.

Abstract:

Calcium is the third most abundant metal in nature and a versatile carrier of many signals within and outside the cell. Due to its peculiar coordination chemistry calcium is highly flexible as a ligand which enables it to regulate many important aspects of cellular activity. Calcium can fulfill its many different functions insite and out of the cell due to an integrated network of calcium channels, exchangers and pumps. In this presentation I will give an overview on our studies of calcium binding proteins, their interaction with protein targets resulting in specific modulations  of protein-protein interactions. This will be demonstrated by the interaction of the calcium binding protein calmodulin with one of its targets, the plasma membrane calcium pump, an important regulator of calcium homeostasis of the cell.

Speaker
Biography:

Irena Levitan has done her PhD and is a Professor of Medicine and Adjunct Professor of Bioengineering at the University of Illinois at Chicago. Her current research focuses on cholesterol regulation of ion channels and cellular biomechanics. Her group has provided the first comprehensive structural insights into cholesterol regulation of K+ channels and the cross-talk between cholesterol and other regulators of these channels. She was named a Guyton Distinguished Lecturer by the Association Chairs of Departments of Physiology for her quantitative and biophysical work on cholesterol modulation of ion channels and how this can affect integrated organ function. She is an author of more than 70 publications and a leading Editor of Cholesterol Regulation of Ion Channels and Receptors (Wiley, 2012) and Vascular Ion Channels in Physiology and Disease (Springer, 2016).

Abstract:

Cholesterol is known to play a major role in regulating the function of multiple membrane proteins including a growing number of ion channels. Our studies focus on inwardly-rectifying K+ (Kir) channels that are ubiquitously expressed in mammalian cells and are known to play major role in membrane excitability and shear stress sensation. In this study, we have shown that Kir channels are suppressed by loading the cells with cholesterol and enhanced by cholesterol depletion. A series of studies revealed that cholesterol interacts with the channels directly by stabilizing them in a long-lived closed “silent” state and that multiple structural features of the channels are essential for conferring their cholesterol sensitivity. Using a combined computational-experimental approach, we show that cholesterol may bind to two nonannular regions that form hydrophobic pockets between the transmembrane helices of the adjacent subunits of the channel. The location of the binding regions suggests that, cholesterol modulates channel function by affecting the hinging motion at the center of the porelining transmembrane helix that underlies channel gating. In addition, we identified a series of residues in the C and N-terminus of the channel. These are critical for conferring cholesterol sensitivity to the channels, but are not part of the binding sites. These residues form a distinct cytosolic structure, a cholesterol sensitivity belt which surrounds the cytosolic pore of the channel in proximity to the transmembrane (TM) domain, and includes residues whose mutation results in abrogation of the channel’s cholesterol sensitivity. Further analysis identified a reversal residue chain comprised of residues that link one of the cholesterol sensitivity belt residues with a distant cytosolic residue that constitute a two-way molecular switch of the channel sensitivity to cholesterol. Further studies are needed to elucidate the connection between cholesterol binding and channel. 

Speaker
Biography:

Thomas Braun received his Ph.D. 2002 in biophysics from the Biozentrum, University of Basel, Switzerland. During his Ph.D. thesis he applied high-resolution electron microscopy and digital image processing to study the structure and function of membrane proteins. Subsequently, he worked on nanomechanical sensors to characterize the mechanics of membrane proteins at the Institute of Physics, University Basel and the CRANN, Trinity College Dublin, Ireland. He has been working at the Center for Cellular Imaging an NanoAnalytics (Biozentrum, University of Basel, Switzerland) since 2009 and is developing new methods for electron microscopy, single cell analysis and nanomechanical sensors for biological applications.

Abstract:

Cryo-electron microscopy (cryo-EM) sample preparation techniques ensure that biological specimens can be investigated at physiological conditions in the electron microscope.  However, these preparation methods suffer from extensive blotting steps leading to a massive loss of sample [1] and sometimes to partial denaturation of sensitive protein complexes. We have developed a simple method for the almost lossless conditioning and preparation of nanoliter-volumes of biological samples for EM [2, 3]. The method does not involve any blotting steps. A microcapillary is used to aspirate 3 to 20 nanoliters of sample, depending on the experiment (Figure 1A). The sample is applied (left) and spread (center) on the EM-grid. Real-time monitoring allows the thickness of the water film to be assessed and decreased to the optimum value prior to vitrification (right). We prepared cryo-EM grids of various samples, e.g., bacteriophages and soluble proteins as shown in Figure 1B&C, to demonstrate the usefulness and general applicability of the method. We also showed that high-resolution 3D structures can be calculated from single-particle preparations of a soluble protein [3]. In addition to cryo-EM grid preparation, the versatile method allows nanoliter-sized sample volumes to be conditioned for EM, e.g., negatively stained with heavy metal salts or embedded in trehalose [2].

In addition, we combine the new sample preparation method with a single cell lysis device for adherent eukaryotic cells [4] and image the aspirated cell contents by TEM. To demonstrate the usefulness of this new “visual proteomics” approach we visualized the changes occurring in single cell proteomes upon heat shocking the cells [2]. Furthermore, we have developed a protein-fishing method based on a magnetic trap and photo-cleavable composite material, to ‘fish’ untagged proteins from cell lysate by antibodies. This allows target proteins to be isolated from approx. 40’000 cells in 90 min and analysed by EM [5]. 

Speaker
Biography:

Vesa P Hytönen is a Head of the Protein Dynamics research group in BioMediTech at the University of Tampere, Tampere, Finland. After graduating as a PhD from the University of Jyväskylä, Jyväskylä, Finland in 2005, he has conducted Post-doctoral training at ETH Zurich, Zürich, Switzerland from 2005-2007. He then continued as a Post-doctoral researcher at the University of Tampere and established independent research group in 2010. He is currently working as Associate Professor at the University of Tampere. His research interests are mechanobiology, protein engineering and vaccine research and authored more than 100 scientific articles.

Abstract:

Talin is a central adhesion protein linking β-integrin cytosolic domains to actin fibers. It participates in the transmission of mechanical signals between extracellular matrix and cell cytoskeleton. Talin rod domain consists of a series of mechanically vulnerable α-helical subdomains containing binding sites for other adhesion proteins such as vinculin, actin and RIAM. Force induced unfolding of these rod subdomains has been proposed to act as a cellular mechanosensor, but so far evidence linking their mechanical stability and cellular response has been lacking. We show that stepwise mechanical destabilization of talin rod subdomain increases talin and vinculin accumulation into cell-matrix adhesions and decreases cell migration rate. In addition, mechanical destabilization of talin subdomain was found to decrease cellular traction force generation and to promote the formation of adhesions on fibronectin over vitronectin. Experiments with truncated talin forms confirmed the mechanosensory role of the talin subdomain and excluded the possibility that the observed effects are caused solely by the release of talin autoinhibition. We demonstrate that by modulating the mechanical stability of an individual talin rod sub-domain, it is possible to affect traction force generation, ECM sensing and consequently highly coordinated processes such as cell migration. Our results suggest that talin acts as a mechanosensor and is responsible for controlling the cellular processes dependent on mechanical signals and cellular mechanosensing.

Speaker
Biography:

Arfaxad Reyes has his expertise in structure and stability of G-protein coupled receptors and passion for improving and creating new drug discovery platforms that greatly contribute in the development of more selective drugs with minor side effects. His studies about biased agonism in Galanin receptors help to understand the relationship between conformational structure of the receptor and its corresponding physiological effect induced by a specific ligand. Recently he and co-workers were able to develop a highly selective agonist for Galanin receptor 2 with anxiolytic effect in vivo (Arfaxad Reyes-Alcaraz et al Sci. Rep 2016) which was the base to discover how different ligand structures induce different conformations on the structure of Galanin receptors. This work greatly contribute to understand the relationship between structure and function of Galanin receptors.

Abstract:

Statement of the Problem: G protein coupled receptors (GPCRs) also known as seven-transmembrane receptors and are the largest family of cell-surface receptors that communicate extracellular stimuli to the cell interior (1). It is now accepted that chemically distinct ligands bind to the same GPCR and can stabilize the receptor in multiple active conformations, which results in differential activation of cell signaling pathways and, eventually, in different physiologic outcomes a phenomenon known as biased agonism (2). Biased agonism can be exploited to design drugs that selectively activate signaling pathways, leading to the desired physiologic effects while on target side effects elicited by activation of other signaling pathways via the same receptor subtype (3). Methodology & Theoretical Orientation: The aim of this study was to stablish a relationship between conformational changes in Galanin receptors and their signaling properties in living cells, for that purpose we develop a structural complementation assay based on NanoBit technology and a series of conformational fluorescein arsenical hairpin (FIASH) bioluminescence resonance energy transfer (BRET) biosensors to monitor structural changes β-arrestin 2 induced by the binding with each Galanin receptor. Findings: Here we show that Galanin receptors impose different conformational signatures in β-arrestin, moreover structurally different ligands activating the same receptor imposed different conformations in β-arrestin 2 producing biased signaling. Conclusion & Significance: Our data provide definite evidence that a receptor activated by structurally different ligands can adopt multiple active conformations. Moreover, this finding also demonstrates that functionally specific structural Galanin receptor conformations can indeed be translated to downstream effectors producing a different physiological response. 

Speaker
Biography:

As being a computational biophysicist, my research has focused on understanding underlying molecular mechanisms of biologically important problems and also providing mechanistic insight at the molecular level. In particular, I have been working with GPCRs and their interacting partners which are responsible for cellular signaling. In order to complement relevant experimental studies one needs to access long time-scales and big system sizes which are beyond the classic MD simulations. In this respect, my expertise in doing long-time MD simulations and application of enhanced sampling techniques such as accelerated MD, metadynamics, and steered MD which provides a good fit. I work in close collaboration with medicinal chemists to direct them for effective molecular designs. In addition, I am also responsible for testing the efficacy of these molecules in silico before transferring them to either in vitro or in vivo studies. Recently, I have been awarded an international COST (European Cooperation in Science and Technology) grant which is based on developing heterobivalent molecules capable of binding more than one target for treatment of symptoms of Parkinson’s disease. 

Abstract:

Arrestins (Arrs) are a family of four proteins (Arr1- 4) which mediate G-protein-coupled receptor (GPCR) desensitization and internalization by coupling to active and phosphorylated receptor. Recently, they have also been shown to mediate GPCR-independent signaling pathways. The specific functions of Arrs (desensitization vs. G-protein-independent signaling) can be regulated by differential phosphorylation of the receptor, which is known as the phosphorylation barcode. The molecular mechanism responsible for formation of a high-affinity complex between an Arr subtype and a GPCR having a certain phosphorylation pattern remains elusive but is crucial for directing the subtype towards a specific functional role, and hence paves the way for development of safer therapeutics with fewer side-effects. As a first step in that direction, we have started with elucidating the activation mechanism of Arr subtypes by carrying out comparative molecular dynamics (MD) studies of the two members of the family, namely Arr1 and Arr3, which exhibit the largest differences in terms of phosphorylation selectivity. In addition, we also modeled and simulated Arr1-R175E mutant, which is known to be constitutively active, and compared it to Arr1 and Arr3 to detect activation-related rearrangements. We found novel structural elements that had not been considered before as determinants for activation and can be targeted with drugs for functional modulation. The emerging model also proposes that activation of Arr1-R175E is connected to perturbation of the well-known region, namely, the polar-core, whereas no changes were observed in that region in Arr3 in spite of the presence of other activation-related changes. With that, we could propose a structural model to explain the molecular mechanism responsible for markedly reduced selectivity of Arr3 towards phosphorylated GPCRs. Finally, knowledge achieved in this study can also be utilized to modulate Arr binding to GPCRs under disease conditions such as otozomal dominant disorders and congestive heart failure.

  • Workshop by NanoTemper Technologies GmbH

Session Introduction

Tobias Pflüger

NanoTemper Technologies GmbH, Germany

Title: Solutions for studying protein complexes in structural biology and drug development
Biography:

Tobias Pflüger studied Chemistry at the Albert-Ludwigs-University in Freiburg with an emphasis on biochemistry. He received his PhD in Structural Biology investigating the structure and function of membrane proteins involved in cellular signalling cascades. In late 2015, Tobias joined NanoTemper Technologies as an application specialist.

Abstract:

Key to understanding the functional role of target proteins is elucidating the structure-function relationship between two proteins or protein complexes. Current analysis is often complex, time-consuming, and lacks data quality.

One of the goals of structural biology is to bring together multiple disciplines and methodologies as a means to better characterize proteins and protein complexes. For highly utilized techniques such as cryo-EM, X-ray crystallography and NMR, having the ability to identify and quantify binding affinities as well as analyze conformational and colloidal properties, enables researchers to gain a better understanding of the functional properties of protein targets.

During this workshop, you will learn about practical solutions to monitor and optimize protein stability and quality.  An overview of the various analytical and biophysical methods commonly used by structural biologist will be discussed. We will share case studies demonstrating the benefits of understanding conformational and colloidal properties of protein complexes and how to use this information for further downstream analysis. Finally, we will share examples of protein-small molecule and protein-protein interactions and novel methodologies that assist researchers in making better decisions.