Day 3 :
Keynote Forum
Igor Sokolov
Tufts University, USA
Keynote: Atomic force microscopy for characterization of pericellular brush layer
Time : 10
Biography:
Igor Sokolov it is an expert in atomic force microscopy in studying cells and biological tissues. Being initially trained as a physicist, he received postdoctoral training in microbiology. He is the recipient of the E.L. Ginzton International Fellowship Award from Stanford University for his work on atomic force microscopy, he Graham Research Award (Clarkson University), Simon Greenberg Foundation Scholarship for study of aging skin, etc.. In 2000 he joined Clarkson University, where he achieved the title of full professor and served as director of the Nanoengineering and Biotechnology Laboratories Center. He has 150+ refereed publications, including such journals as Nature, Nature Nanotechnology, Advanced Materials, etc.. He holds 20+ patents (issued and pending). His current research focuses on nanomechanics of soft material, molecules and cells; atomic force microscopy; nanophotonics, and the studies towards understanding of nature of cancer, early detection of cancer based on altered biophysical properties; self-assembly
Abstract:
Statement of the Problem: The pericellular brush/coat (PB) is a brush-like layer that covers cell body of all eukaryotic and the majority of prokaryotic cells. The PB layer plays an important role in physics of cells. The changes in the PB layer have been implicated in the pathogenesis of many diseases, including cardio-vascular disorders, inflammation, and cancer. Nevertheless, the PB layer is rather poorly studied. The existing biochemical methods to study the pericellular coat are specific to a particular type of molecules (content of which is frequently unknown) and lack of spatial resolution.
Methodology & Theoretical Orientation: We describe two novel methods based on the use of atomic force microscopy (AFM) to study the PB layer. One method is based on the analysis of force curves recorded during cell indentation. The PB layer can be studied by processing these curves with so-called brush model. One can obtain physical characteristics of the PB layer, the grafting density and the brush size. The second method, Ringing mode is based on the analysis of the ringing signal recorded with the AFM sub-resonance tapping mode. One of the channels recorded in the ringing mode, the length of the grafted-to-the-cell-surface molecules, the size of the PB layer.
Findings: The first method can work with viable cells. It detects all molecules present in the PB layer without any presumptions of the biochemical methods. However, the spatial resolution is restricted in this method by the size of the appropriate AFM probe, which would be of the order of a micron. The problem of spatial resolution is solved in the second method, ringing mode. Although this method can be applied to both viable and fixed cells, it works the best on fixed cells dried in air. The lateral resolution can be as small as a few nanometers, Figure1.
Keynote Forum
Marek Cieplak
Polish Academy of Sciences, Poland
Keynote: Structural changes in proteins at fluid-fluid interfaces
Time : 11:05-11:50
Biography:
Marek Cieplak is the Head of Laboratory of Biological Physics, Institute of Physics, Polish Academy of Sciences in Warsaw, Poland. He completed his MS from Department
of Physics, University of Warsaw in 1973; PhD from Department of Physics, University of Pittsburgh in 1977; DSc from Department of Physics, University of Warsaw in
1984. He got his Professorial title in 1994. His fi elds of interest are condensed matter theory (spin waves, spin glasses, porous media, growth processes, atomic friction,
river networks, nanofl uidics, self-organized nanostructures) and biological physics (large conformational changes of biomolecules within coarse-grained models, especially
as induced by stretching, proteins with knots and slipknots, protein folding, dynamics of virus capsids and other multi-proteinic structures such as a cellulosome, interaction
of proteins with solids, proteins at air-water interface, modeling of proteasomes, inference of genetic networks from the microarray data).
Abstract:
We study the behavior of several proteins at the air–water and oil–water interfaces by all-atom molecular dynamics. Th e
proteins are found to change orientation and get distorted when pinned to the interface. Th is behavior is consistent with
the empirical way of introducing the interfaces in a coarse-grained model through a hydropathy related force. Proteins couple
to the oil-water interface stronger than to the air-water one. Th ey diff use slower at the oil-water interface but do not depin from
it, whereas depinning events are observed at the other interface. Th e reduction of the disulfi de bonds slows the diff usion down.
We use the model to study interfacial protein layers and demonstrate existence of glassy eff ects as evidenced by slowing down of
diff usion with increasing concentration of proteins. We also show that layers of two barley proteins, LTP1 and its ligand adduct
LTP1b, fl atten out at the interface and can make a continuous and dense fi lm that should be responsible for formation and stability
of foam in beer. Th e degree of the fl attening depends on the protein - the layers of LTP1b should be denser than those of LTP1 – as
well as on the presence of glycation and on the number of disulfi de bonds.
Keynote Forum
Guo-Ping Zhou
Gordon Life Science Institute, Boston, USA
Keynote: The Study of the Misfolding Mechanism of the Prion Protein by Incorporating the Wenxiang Diagrams into NMR Spectroscopy
Biography:
Dr. Zhou is a current Professor of Gordon Life Science Institute. He is also an Adjunct Professor of several academics in both USA and CHINA. He received his Ph.D in Biophysics from University of California at Davis, and completed his postdoctoral training at Stanford University and Harvard University, respectively. He has determined the 3D NMR structures of some important biomolecules, and successfully introduced the novel diagram approach to elucidate the mechanisms of the protein-biomolecule interactions, and protein misfolding diseases observed by NMR. His current research is focused on the molecular mechanism of Neural Cell Adhesion Molecule polysialylation using NMR and biophysical approaches. In addition, He has also edited some special issues on the fields of structural biology and medicinal chemistry for several influential scientific journals as an Editorial-Board Member and Guest Editor.
Abstract:
The conversion of a normal native-helix-rich prion protein (PrPc) to an abnormal polymeric ß-sheet-rich configuration (PrPsc) is a misfolding process. PrPsc is a disease associated fibril-forming isoform such as transmissible spongiform encephalopathies (TSEs) or prion diseases, a deadly disease occurred in both humans and many vertebrate animals. Our NMR studies have indicated that the misfolding process from PrPc to PrPsc is related to the unwinding and stability of the original α-helix structures in PrPc protein. Recently, we have also built up the wenxiang diagrams of all three helices (H1-H2-H3) of PrPc and observed that most hydrophobic residues of the all three helices (H1-H2-H3) in PrPc are distinctly distributed in one-half of the wenxiang diagram of each helix, and most hydrophilic residues are distributed in the other half of the wenxiang diagrams. Similarly, most residues formed salt bridges or ionic pairs in an-helical structure are close to each other in a wenxiang diagram plane. According to these features, the helix-helix interactions, stability of alpha-helical structure, as well as possible interactions between the helix and residues outside the helix (the residues in loops) can be quickly inferred and further verified incorporating NMR spectroscopy. Our results explain why H1 is the most stable helix, and H2 is the most unstable helix during the formation process of prion disease. Thus, the incorporation of the wenxiang diagrams into NMR may provide more insight on the molecular mechanisms of the protein misfolding diseases.
- Determination of 3D structures | Structural Biology in Cell Signalling
Location: Spreewald
Chair
Joachim Krebs
MPI for Biophysical Chemistry, Germany
Session Introduction
Peter B Stathopulos
University of Western Ontario, Canada
Title: Structural elements of stromal interaction molecule mediated store operated calcium entry regulation
Biography:
Peter B Stathopulos applies structural biology approaches to reveal molecular mechanisms driving calcium signaling processes in health and diseased states including
heart disease, cancer and immunodefi ciency. He integrates nuclear magnetic resonance spectroscopy and x-ray crystallography with a host of biophysical, chemical
biology and live cell methodologies to understand the relationship between structure and function of critical calcium signaling proteins. Ultimately, this structure-function
data is used for the rational identifi cation of new drug binding targets with the potential to modulate these pathways to maintain health or treat disease.
Abstract:
Calcium (Ca2+) is a universal signaling entity in eukaryotic cells mediating diverse processes such as the immune response,
hypertrophy, apoptosis, platelet aggregation and memory, to name a few. Th ese processes require a sustained elevation
of cytosolic Ca2+ levels which is facilitated by store operated Ca2+ entry (SOCE). SOCE is the process whereby endoplasmic
reticulum (ER) luminal Ca2+ depletion signals the opening of ion channels on the plasma membrane (PM) which facilitate
the movement of Ca2+ down the concentration gradient from the extracellular space into the cytosol. Th e principal molecules
that mediate SOCE include the ER resident stromal interaction molecule-1 (STIM1) and PM ORAI1 protein subunits which
assemble into a channel pore. Upon ER luminal Ca2+ depletion, STIM1 undergoes a destabilization coupled oligomerization
which leads to translocation of this Ca2+ sensor to ER-PM junctions where it couples to ORAI1 subunits and opens these PM
Ca2+ channels. Since the identifi cation of STIM1 and ORAI1 as the principal molecules driving SOCE, considerable progress
has been made elucidating their high-resolution structural mechanisms of action. Author will present available structural data
on the STIM1 Ca2+ sensing mechanism and how this regulator may complex to ORAI1 subunits. Th e coupling mechanism
revealed using soluble human STIM1 and ORAI1 fragments are congruent with the hexameric assembly elucidated in the D.
melanogaster crystal structure. Finally, author will present unpublished work showing how post translational modifi cations
within the luminal domain of STIM1 aff ects the structural mechanisms of Ca2+ sensing. Ultimately, the post-translation
modifi cation driven STIM1 structural and biophysical changes have implications in the agonist induced hypertrophic response
and pinpoint a new therapeutic target for heart disease.
Toshiya Senda
High Energy Accelerator Research Organization (KEK), Japan
Title: Automated systems for X-ray crystallography at Photon Factory
Biography:
Toshiya Senda has completed his PhD from Nagaoka University of Technology, Niigata, Japan in 1995. He was a Research Associate in Nagaoka University of Technology
during 1995-2001 and a Senior Researcher in Institute of Advanced Industrial Science and Technology during 2001-2012. Now, he is the Director/Professor of Structural
Biology Research Center of High Energy Accelerator Research Organization (KEK) in Japan. He was awarded the CrSJ (Crystallographic Society of Japan) award in 2014.
Abstract:
X-ray crystallography has been the most widely utilized method for 3D structure determination of biological macromolecules
at atomic resolution. Since modern biology is studied at the molecular level, the 3D structures of biological macromolecules
provide critical information for rational biological studies. X-ray crystallography is therefore becoming an essential tool for
molecular and cellular biology and is increasingly being adopted by non-crystallographers. To facilitate this movement, the
Structural Biology Research Center (SBRC) in KEK has developed several robotics and automation systems for crystallization,
data collection, and structure determination. For the initial crystallization screening, we have developed protein crystallization
system (PXS) that can automatically perform initial crystallization screenings in a sitting drop setting. Th e PXS possesses
an automated image acquisition function for crystallization droplets. Th e obtained images can be checked from outside the
KEK campus via a VPN connection. Moreover, the crystallization plate can be directly mounted to a specifi c goniometer at
BL-17A for in situ data collection. Our protein crystallography beamlines, BL-1A, 5A, 17A, AR-NW12A, and AR-NE3A,
are highly automated by the PReMo program. Once a cassette containing protein crystals is set to the system, a robot can
mount a crystal to the goniometer in a beamline; do the centering of the crystal and start data collection. Beginning in May
2018, we will provide a fully automated data collection service for users. In this service, the obtained diff raction data sets will
be automatically processed by a data-processing routine in PReMo. Th e processed data can then further be analyzed with
PReMo for structural determination using the SAD method. Although at present the structure determination following the
data processing must be performed manually, we are developing a new routine for automated structure determination. It is
of note that all these automated functions are based on a database system in PReMo, and thus data from crystallization to
structure determination can be treated in an integrated manner. Our service is expected to provide the basis of an intelligent
system for crystallography.
Grace E Stutzmann
The Chicago Medical School - Rosalind Franklin University, USA
Title: Calcium signaling in the CNS in health and neurodegenerative disease
Biography:
Grace E Stutzmann focuses on early mechanisms of neurodegenerative disorders. She received her PhD from New York University, Center for Neural Science and
completed her Postdoctoral fellowships at Yale University and UC Irvine. She is currently an Associate Professor at Rosalind Franklin University/The Chicago Medical
School and serves as the Director for the Center for Neurodegenerative Disease and Therapeutics. Her research investigates effects of calcium signaling dysregulations
associated with AD on neuronal physiology, synaptic transmission, and histopathology. She uses transgenic mice expressing human mutations that cause familial AD and
employs techniques such as in vitro electrophysiology and 2-photon calcium imaging to study activity in neurons, in addition to immuno-based assays, molecular biology
and behavioral approaches. Her research has shown that specifi c calcium-mediated signaling pathways are dysregulated in AD and over time, facilitate amyloid plaques
and tangles formation, interfere with memory encoding circuits, and eventually can kill the cell.
Abstract:
Statement of the Problem: A shared aspect among many neurodegenerative disorders is a limited understanding of cellular
disease mechanisms. In our research, we have found that dysregulated calcium channels play a central and early role in driving
diseases such as Alzheimer’s, Huntington’s and brain injury. Specifi cally, defects in intracellular calcium stores within the
endoplasmic reticulum (ER) and their resident release channels such as the ryanodine receptor (RyR) are upstream of the
major disease features such as protein aggregates and synaptic defi cits. By focusing upon the proximal calcium channelopathy,
rather than later-stage features, signifi cant advances in understanding disease etiology may be made.
Methodology & Th eoretical Orientation: Whole cell patch clamp electrophysiology combined with 2-photon calcium
imaging are used to record signaling from hippocampal neurons in acute brain slices from mouse models of Alzheimer’s
disease (AD) and human neurons transformed from AD patients. Immunoassays and qRT PCR probe protein and transcript
levels in mouse and human neurons.
Findings: In early stage AD mice, RyR-evoked calcium signals are signifi cantly greater than non-transgenic controls, while
calcium signaling though voltage-gated channels and NMDA receptors are not diff erent. Similar observations were made
in human neurons from AD patients. Additionally, RyR 2 message is increased in both the mouse and human AD neurons.
Pharmacologically normalizing RyR-calcium release restores RyR 2 levels in mice, as well as reduces many of the AD features
including beta amyloid, hyperphosphorylated tau and synaptic loss.
Conclusion & Signifi cance: Dysregulated ER calcium channel functioning is an early feature of AD in mouse models and
human samples and likely initiates a pathogenic cascade in the proximal disease stages. Because of the multiple and essential
roles of calcium signaling in neurons, alterations in RyR-channel properties can generate multiple downstream maladaptive
eff ects such as those seen in neurodegenerative disorders.
Jan B Parys
KU Leuven, Belgium
Title: Functional properties of the IP3 receptor at membrane contact sites
Biography:
Jan B Parys is a faculty member since 1997, ordinary Professor of Physiology since 2006 and Head of the Laboratory of Molecular and Cellular Signaling at the KU
Leuven, Belgium since 2013. His research always concerned the understanding of the mechanisms for Ca2+ homeostasis in general and the structure, function and
regulation of the inositol 1, 4, 5-trisphosphate receptor (IP3R), a central player in the initiation and propagation of intracellular Ca2+ signals, in particular. More recently, he
focused on the role of the IP3R in conjunction with other Ca2+-transporting proteins of the endoplasmic reticulum, the mitochondria and the lysosomes in cell survival and
cell death, and especially in the processes of apoptosis and autophagy. Since 2010 he is Secretary-General of the European Calcium Society (ECS) and was organizer
or co-organizer of multiple international workshops (2013) and meetings (2008, 2014, and 2016) of the ECS.
Abstract:
The inositol 1, 4, 5-trisphosphate (IP3) receptors (IP3Rs) are ubiquitously expressed Ca2+ release channels of the endoplasmic
reticulum (ER). Th e resulting Ca2+ signals are instrumental in the regulation of many intracellular processes. It is now
becoming increasingly clear that contact sites between ER and mitochondria or between ER and lysosomes is very important
for controlling mitochondrial energetics, autophagy and apoptosis. Anti-apoptotic Bcl-2 leads to increased cell survival by both
blocking pro-apoptotic family members like Bax and Bak and by inhibiting IP3R mediated Ca2+ transfer to the mitochondria.
As Bcl-2 is upregulated in a variety of cancers, including diff use large B-cell lymphoma (DLBCL) and chronic lymphocytic
leukemia (CLL), we developed in collaboration with Clark W Distelhorst (Case Western Reserve University, USA) BIRD-2, a
peptide tool that disrupts Bcl-2/IP3R interaction by binding to the Bcl-2 BH4 domain, increasing IP3 induced Ca2+ release and
triggering apoptosis of DLBCL cells and of primary CLL cells. Interestingly, DLBCL cell lines with high expression levels of
the high affi nity IP3R2 isoform, were the most sensitive to BIRD-2. In addition to high levels of IP3R2 expression, constitutive
IP3 signaling downstream of the tonically active B-cell receptor increases sensitivity towards BIRD-2. Under standard growth
conditions, this constitutive IP3 signaling fulfi lled a pro-survival role, since inhibition of phospholipase C using 2.5 μM U73122
caused cell death in various DLBCL cell lines and primary CLL cells, but at lower concentrations protected against BIRD-
2-induced Ca2+ release and apoptosis. Taken together, our data indicate that on the one hand, constitutive IP3 signaling is
necessary for cancer cell survival and proliferation but that on the other hand to survive this chronic signaling the cancer cells
are dependent on high Bcl-2 levels to prevent excessive Ca2+ release via IP3Rs. Disrupting Bcl-2/IP3R interaction switches
constitutive IP3 signaling to a pro-death signal and may therefore lead to a novel therapeutic approach
- Structural Enzymology | Computational Approaches | Recent Advancements in Structural Biology
Location: Spreewald
Session Introduction
Ray Unwalla
Pfizer Inc, USA
Title: Structure based approach to identify potent tissue selective androgen receptor modulators
Biography:
Ray Unwalla is a computational chemist at Pfi zer, USA working in the Infl ammation and Immunology Department. He has several years of drug discovery experience
and has expertise in the area of nuclear hormone receptors, kinases and GPCRs. He has an in depth knowledge of various computational tools and techniques to
provide insightful design hypothesis and specifi c compound proposals that test design hypothesis. He was involved in project teams that successfully delivered two drug
candidates i.e., Dutasteride for benign prostate hyperplasia, Duavee for hormone replacement therapy and six clinical candidates for the progesterone, ERα LXRβ, SARM,
JAK1 and JAK3 targets.
Abstract:
The steroids testosterone and dihydrotestosterone (DHT) are androgens that play an important role in the development and
maintenance of a variety of physiological responses such as male sexual function, bone density, muscle mass and strength.
Th e androgen receptor is a nuclear hormone receptor that is expressed in many tissues and is responsible for mediating the
actions of testosterone and DHT. Patients that have defects in the androgen receptor or have androgen defi ciencies can be
eff ectively treated with exogenous testosterone and other steroidal androgens as a hormone replacement therapy. Th e anabolic
eff ects of testosterone have shown benefi t in age related decline of bone density and muscle mass. However, the side eff ect profi le
of testosterone and other currently available anabolic steroids precludes their wide spread use and the chronic administration
of steroidal androgens is associated with potential serious side eff ects such as hepatotoxicity, prostate hypertrophy and cancer.
In addition, the oral bioavailability of testosterone is poor and the route of its administration is generally through topical
formulations. We will describe our eff orts to fi nd novel series of oral tissue selective androgen receptor modulators (SARM)
i.e., cyanopyrroles and indolines that selectively promote muscle growth while showing reduced androgenic eff ects on the
prostate and seminal vesicles. Using a docking approach, we were able to delineate the binding mode of these series and further
optimize the potency. An x-ray structure of a lead compound 7 bound to AR ligand binding domain revealed an interesting
electrostatically unfavorable interaction of the 3-fl uorophenol group with a carbonyl backbone of Leu704 residue. QM based
intermolecular potential calculation performed to understand the strength of this interaction along with ligand strain energy
calculations indicate a small energy penalty of ~0.6 kcal/mol paid by the ligand to adopt the bound state conformation
Biography:
R Holland Cheng is a Professor of Molecular and Cellular Biology at University of California. He received a Master degree in 1989 and a PhD degree in 1992 from Purdue
University. He has served as a Panel Reviewer for NIH programmed project grant, NIH Microbiology & Infectious Diseases Research Committee, NIH National Center
for Research Resources, Wellcome Trust, UK and Earth & Life Sciences Council, Netherlands (2002). He has actively served on the advisory board of the International
Microscopy Congress plus regional microscopy societies, American Biographical Institute, and International Virus Assembly Symposium. He is also a regular member of
the American Chemical Society, American Society for Microbiology and American Biophysical Society. He has published nearly fi fty papers in peer reviewed journals and
his research mainly focuses on proteome imaging of macromolecular systems.
Abstract:
Cryogenic electron microscopy (cryo-EM) provides a visible and quantitative approach to investigate the function of
structural proteins of viruses, both in assembly and disassembly. Th e fundamental biochemical properties of metastable
intermediates and stable mature form of viral capsids, responsible for their capacity to resist extreme pH and digestive enzyme
degradation, are revealed through cryo-EM. In this presentation, the structural functionality of Hepatitis E viral nanoparticles
(HEVNP), as a multimodal delivery platform, is assessed for the delivery of medicals or agents to target tumors through
both circulation and mucosal routes. Aided by multimodal imaging and integrative machine learning networks, the targeting,
tissue distribution, and payload packaging are investigated and demonstrated. Together, cryo-EM and cellular tomography are
exemplifi ed in the recent outcomes of unveiling the molecular mechanisms essential for platform vector design towards the
success of constructing a non-invasive, mucosal targeting system.
Biography:
Abstract:
A large number of viruses encode small proteins which are expressed in the infected cell to form ion and small molecules
conducting channels. Th ese proteins are also called viroporins. For some of the viruses these proteins are essential for
infectivity cycle and thus, they comprise interesting drug targets. From the structural and mechanistic point of view, they
comprise interesting tools to study membrane protein assembly and dynamics in particular when the outcome of the assembly
is an ion conducting system. Computational tools play an important part in this investigation. Using a 2D docking tool
structural models of polytopic membrane proteins such as p7 of hepatitis C virus (HCV) with two transmembrane domains
(TMDs) and E5 of human papilloma virus – 16 (HPV-16) with three TMDs are generated in a putative functional form.
From molecular dynamics (MD) simulations of these proteins embedded in a hydrated lipid bilayer ion selectivity of these
proteins is investigated also by calculating potential of mean force (PMF) profi les of ions along the conducting pathway.
Th e calculations support weak selectivity of these channel proteins, strengthening the quality of the modeled structures. In
addition, full correlation analysis (FCA) provides insight into the dynamic of these proteins. Th e individual subunits of the
protein assemblies show asymmetric behavior during equilibrium dynamics, allowing for proposing several models of gating.
Liliane Mouawad
Institut Curie, France
Title: Effects of sarcolipin on Ca2+ pump SERCA1a enzymatic cycle studied by normal mode analysis
Biography:
Liliane Mouawad was always interested in understanding the mechanism of action of proteins or protein assemblies. This understanding may be based on either molecular
simulations or on experiments like NMR. But her expertise is primarily in molecular dynamics simulations and more precisely in normal mode analysis (NMA). She
has developed several methods going from the calculation of normal modes of very large systems or of images, to the calculation of the pathway between two protein
conformations or to the prediction of the compactness of a calcium-binding protein. Recently, she was also involved in docking and virtual screening themes, where she
has acquired enough expertise to develop a new consensus methodology to overcome some issues observed in these approaches.
Abstract:
The Ca2+ pump SERCA1a is a P-type ATPase, localized in the sarcoplasmic reticulum membrane of striated muscle cells.
SERCA1a is involved in the contraction/relaxation process by fast pumping the cytoplasmic Ca2+ into the reticulum.
Th roughout its catalytic cycle, SERCA1a presents two major conformations: the E1 conformation where its Ca2+ channel is open
toward the cytoplasm and the E2 conformation where this channel is open toward the lumen. Th is conformational transition
is enhanced by ATP phosphorylation which occurs aft er Ca2+ binding by SERCA1a. Sarcolipin (SLN), a transmembrane helix
of 31 residues regulates SERCA1a by diminishing its affi nity to Ca2+. Th e mechanism of this regulation is not elucidated yet
despite the knowledge of the crystal structure (see fi gure). To decipher this mechanism, we performed normal mode analysis
(NMA), in the all-atom model on two systems, in the presence and the absence of SLN in a POPC membrane and one layer of
water for the soluble parts of the protein. Th is analysis showed that both systems are prone to go toward the conformation of
2Ca2+ E1 more easily than toward that of E2. However, both transitions seem more diffi cult in the presence of SLN, because of
some specifi c interactions with SERCA1a that result in additional fl uctuations of SERCA1a-SLN. A long-distance transmission
of information (over 35 Å) within the protein was also observed, explaining the phosphorylation diffi culty in the complex.
Th ese results provide new insights into the mechanism of the SERCA1a enzymatic cycle and its regulation by SLN.
Sangwook Wu
1Pukyong National University, South Korea
Title: Network analysis of the conformational change of c-Src, a tyrosine kinase, by molecular dynamics simulation
Biography:
Sangwook Wu received his BA degree in Biochemistry from Yonsei University, Korea in 1990. After working as a Scientist at Samsung Display Devices during 1995-1999,
he obtained his PhD in Theoretical Condensed Matter Physics from Iowa State University during 1999-2005. He joined the Computational Biophysics Lab at UNC-Chapel
Hill (Dr. Lee Pedersen) as postdoctoral research associate during 2005-2014. From 2014 to the present, he has been a faculty member at Pukyong National University in
Korea. His research interests are in the areas of computational dynamics of biological macromolecules.
Abstract:
Non-receptor tyrosine kinase c-Src plays a critical role in numerous cellular signaling pathways. Activation of c-Src
involves a change from its inactive to the active state accompanied by large-scale conformational change depending
on the phosphorylation state of two major phosphorylation sites, Tyr416 and Tyr527. A detailed mechanism for the entire
conformational transition of c-Src via phosphorylation control of Tyr416 and Tyr527 is still elusive. In this study, we investigated
the inactive to active conformational change of c-Src by targeted molecular dynamics simulation. Based on the simulation, we
proposed a dynamical scenario for the activation process of c-Src. A detailed study of the conformational transition pathway
based on network analysis suggests that Lys321 plays a key role in the c-Src activation process.
Session Introduction
Yuri L. Lyubchenko
University of Nebraska Medical Center, USA
Title: Nanoscale dynamics of protein-DNA complexes as revealed with high-speed AFM
Biography:
Yuri L Lyubchenko is a Professor of Pharmaceutical Sciences at the University of Nebraska Medical Center, USA. His research focuses on understanding
fundamental mechanisms underlying health and disease, which is a key for developing new and more effective diagnostics and medications. This primary basic
research allows him not only to 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 effi cacy at the molecular level.
Abstract:
High-speed atomic force microscopy (HS-AFM) is an advanced technique with numerous applications in biology,
particularly in molecular biophysics. Developed as a time-lapse AFM technique for direct imaging fully hydrated
biological molecules, HS-AFM is currently capable of visualizing the dynamics of biological molecules and their complexes
at a video-data acquisition rate. Spatial resolution at the nanometer level is another important characteristic of HS-AFM. Th is
presentation focuses on examples of primarily protein-DNA complexes to illustrate the high temporal and spatial resolution
capabilities of HS-AFM that have resulted in novel models and/or the functional mechanisms of these biological systems.
Biography:
Emmanuelle Bignon is a Postdoctoral researcher working in the Computational Biology Laboratory within the Danish Cancer Society Research Center, Copenhagen,
Denmark. Her research interests focus on biochemical phenomena implicated in diseases onset and development. She uses computational chemistry tools, i.e.,
all-atom molecular dynamics, quantum calculations, QM/MM(-MD) techniques, in order to investigate reactivity and dynamical behavior of DNA and disease
related proteins. Her Postdoctoral project, supported by the Alfred Benzon Foundation, deals with redox signaling mechanisms, especially S-nitrosylation (SNO)
of cysteines, a post-translational mutation which plays an important role in cancer cells. She also investigated the structure and reactivity of damaged DNA during
her PhD obtained in June 2017.
Abstract:
Nitric oxide plays an important role in the redox signaling pathway. Indeed, its reaction with cysteines, resulting in the
formation of S-nitrosothiols, has shown to be involved in a broad variety of biochemical and physiological processes in a
large variety of organisms. Large amounts of investigations have been led in order to understand the regulation mechanisms
driven by protein S-nitrosylation and their implications in cancer cells development. Th ousands of proteins have been proven
to undergo S-nitrosylation in vivo and the dysregulation of this process is implicated in various types of severe diseases,
including cancer onset, progression and treatment resistance. Th us, the understanding of S- nitrosylated proteins (SNOproteins)
structural behavior and reactivity is of utmost importance towards the development of new anti-cancer therapeutics.
Nowadays, there is a lack of information concerning the structural and electronic features of nitrosylated cysteine, with only few
NMR and x-ray structures reported for SNO proteins. In this framework, molecular modeling has been proven to be a useful
tool to obtain predictive structural and dynamical behavior of biomolecules. Th e S-nitrosocysteine (CysNO) non-canonical
amino acid exhibits a very complex chemistry, due to the presence of two antagonist resonance structures with very diff erent
electronic features. Th erefore, the accurate description of this moiety’s structure is highly challenging, and prediction of its
chemical properties has to be performed using high-level quantum methods. Unfortunately, such methods are strongly timeconsuming,
and their computational cost is prohibitive for the study of large system such as proteins. Force fi eld parameters
have been developed in order to perform classical molecular dynamics simulations of S-nitrosylated proteins to decipher their
dynamical features. Nevertheless, the accuracy of these parameters has not been extensively cross-validated and still need to be
tested. For this purpose, we performed extensive all-atom classical molecular dynamics simulations on proteins for which the
crystal structures harboring CysNO have been reported. Th e conformations sampled by all atom simulations with the two sets
of parameters have been confronted to the experimental structures in order to validate their effi ciency in reproducing CysNO
behavior. Th is checking is an important step in the design of accurate force fi eld parameters, which would allow computational
chemist to investigate the dynamical properties of S-nitrosylated proteins and their involvement in cancer cells mechanisms.
Manuela A A Ayee
University of Illinois at Chicago, USA
Title: Endothelial membrane stiffening under osmotic challenge
Biography:
Manuela A A Ayee is a Postdoctoral research fellow in the Department of Pulmonary and Critical Care Medicine at the University of Illinois at Chicago. Her current
research focuses on investigating the modulation of cellular biomechanics by oxidized lipids and sterols under hypercholesterolemic conditions. She obtained her
PhD in Chemical Engineering with a specialization in Computational Biomolecular Modeling and Interfacial Phenomena. Her broad research goals include combining
computational and experimental techniques to reveal the role of small molecules in pathogenesis and elucidating the molecular mechanisms underlying their effects
on membrane biomechanics and their subsequent modulation of cell biophysical properties. She will begin as a Professor of Chemical Engineering and Chemistry
at Dordt College, Iowa in 2018.
Abstract:
Cell volume regulation is a fundamental property of all mammalian cells. Numerous signaling pathways are known to be
activated by cell swelling and to contribute to cell volume homeostasis. Cellular biomechanics and membrane tension
have long been proposed to couple cell swelling to signaling pathways; however, the impact of swelling on these parameters
has yet to be fully elucidated. In this study, we utilize atomic force microscopy (AFM) under isotonic and hypotonic conditions
to measure the mechanical properties of human aortic endothelial membranes. From AFM force/displacement curves, we
obtain estimates of membrane elastic modulus, which refl ects the stiff ness of the sub-membrane cytoskeleton complex and the
force required for membrane tether formation, refl ecting membrane tension and membrane cytoskeleton attachment. We fi nd
that hypotonic swelling results in signifi cant stiff ening of the membrane region of endothelial cells, without a corresponding
change in membrane tension or membrane-cytoskeleton attachment. Furthermore, depolymerization of F-actin in the
cytoskeleton, which as expected results in a dramatic decrease in the cellular elastic modulus of both the membrane and the
deeper cytoskeleton, indicating a collapse of the cytoskeleton scaff old, does not abrogate swelling-induced stiff ening of the
membrane, instead this stiff ening is enhanced. We propose that the hypotonically induced membrane stiff ening should be
attributed to an increase in hydrostatic pressure that results from an infl ux of solutes and water into the cells. Most importantly,
our results suggest that increased hydrostatic pressure, rather than changes in membrane tension, could be responsible for
activating volume sensitive mechanisms in hypotonically swollen cells.
Shu-Fang Hsu
Institute of Biological Chemistry, Academia Sinica, Taiwan
Title: Hybrid methods reveal the structural architecture of PTPN3-p38 γ active-state complex
Biography:
Shu-Fang Hsu has her expertise in cell biology focusing on tyrosine phosphorylation-dependent cell signaling which coordinated by protein tyrosine kinases (PTKs)
and protein tyrosine phosphatases (PTPs). PTPs comprise a superfamily of enzymes that control a diverse array of signal transduction pathways, therefore exert
their biological functions including tumorigenicity. Her current interest is crystal structure study on PTPN3-p38γ complex for structure-based drug design as an
anticancer therapy for colon cancer.
Abstract:
PTPN3 (also known as PTPH1) cooperate to promote Ras-induced oncogenesis in human colorectal cancer. Comprehensive
structural information on the PTPN3-p38γ interaction is critical for structure-based drug design as a new means in anticancer
therapy.
Methodology & Th eoretical Orientation: In order to obtain the architecture features of PTPN3-p38γ active-state complex,
a hybrid method combining small-angle x-ray scattering (SAXS), chemical cross-linking coupled to mass spectrometry (CXMS),
hydrogen deuterium exchange mass spectrometry (HDX-MS), and x-ray crystallography were adopted.
Findings: To build the molecular architecture of PTPN3-p38γ active-state complex, the phosphatase domain of PTPN3 in its
substrate trapping mutant form was used to interact with the phosphorylated p38γ in vitro. Isothermal titration calorimetry
(iTC) analysis showed that PTPN3 binds to phosphorylated p38γ in a submicromolar affi nity, suggesting the formation
of a stable complex. CX-MS analysis unraveled the close proximity between the catalytic site of PTPN3 and the activation
loop of phospho-p38γ. HDX-MS results further demonstrated that the glutamic acid-containing loop (E-loop) and the
phosphotyrosine recognition loop (pY loop) of PTPN3 play a critical role in recruiting p38γ as a substrate during catalysis.
Preliminary crystals of PTPN3-p38γ active-state complex were obtained and the microseeding technique was applied to
optimize the crystal formation.
Conclusion & Signifi cance: Atomic structure of PTPN3-p38γ active-state complex may reveal the molecular features for the
design of new drug against the progression of colorectal cancer.
- Structural Biology | Sequence Analysis | Molecular Modelling
Location: Spreewald
Chair
Igor Sokolov
, Tufts University, USA
Session Introduction
Igor Sokolov
Tufts University, USA
Title: Quantitative and repeatable measurements of elastic moduli of cells
Biography:
Igor Sokolov is an expert in atomic force microscopy in studying cells and biological tissues. Being initially trained as a Physicist, he received Postdoctoral training
in Microbiology. He is the recipient of the E L Ginzton International Fellowship Award from Stanford University for his work on atomic force microscopy, he received
Graham Research Award (Clarkson University), Simon Greenberg Foundation Scholarship for the study of aging skin, etc., in 2000 he joined Clarkson University,
where he achieved the title of Full Professor and served as Director of the Nano-engineering and Biotechnology Laboratories Center. He has 150+ refereed
publications, including such journals as Nature, Nature Nanotechnology, Advanced Materials, etc., He holds 20+ patents. His current research focuses on nanomechanics
of soft material, molecules and cells; atomic force microscopy; nanophotonics and the studies towards understanding of nature of cancer, early detection
of cancer based on altered biophysical properties; self-assembly
Abstract:
Mechanics of eukaryotic cells at the nanoscale is a challenging problem due to complexity of cells. Besides fundamental
interest, such measurements are important because it has been demonstrated that the elastic modulus of cells can
correlate with various diseases and even aging. Atomic force microscopy (AFM) is the technique of use. At the same time, there
is a lot of confusion in the process of measurements of the elastic modulus of cells. In the talk, author will present the modern
overview of the topic, describe step-by-step how to do the measurements of the elastic modulus in quantitative manner that is
independent of the particular microscope, AFM probe, and mechanical model.
John B Bruning
The University of Adelaide, Australia
Title: The human sliding clamp as a therapeutic target
Biography:
John B Bruning received BSc from Texas A&M University in 1997. He began crystallography in the Laboratory of Yousif Shamoo at Rice University. During his
graduate studies he worked on the structural mechanism of the human sliding clamp and its interactions with DNA replication proteins. He received his PhD in 2005
and completed two successful Post-docs; the fi rst was at the Scripps Research Institute from 2005-2007 working on structural studies of nuclear receptors including
PPAR, RXR, ER and TR and his second Post-doc was with Jim Sacchettini in the Houston Medical Centre and as a part of the TB structural genomics consortium.
He received his fi rst faculty position at the University of Adelaide as a Lecturer in 2012. He was tenured in 2015 and promoted as Senior Lecturer in 2016. Due to
his continued collaboration with Scripps Research Institute, he was also appointed as Adjunct Professor of the Scripps Research Institute in 2016.
Abstract:
The human sliding clamp (also known as PCNA) controls access to DNA of many of the proteins involved in essential
processes such as DNA replication, DNA repair and cell cycle control. Proteins compete for interaction with the PCNA
surface by means of a short, conserved peptide sequence known as the PCNA-interacting protein motif (or PIP-box). Binding
to PCNA via the PIP box allows access to DNA. For example, the major replicative polymerase, pol delta, requires PCNA for
processive DNA synthesis, without interaction with PCNA the polymerase dissociates from DNA and is incapable of processive
DNA synthesis. As such, many groups have proposed the usefulness of PIP box mimetics for use as cancer therapeutics given
they would block upregulated PCNA form allowing interaction with pol delta and hence would inhibit DNA replication.
However, no peptide mimetics of PCNA have been forthcoming to date. Here we describe the design and synthesis of the fi rst
PCNA peptidomimetic. Our mimetic, ACR2, was designed through synthetic lactam chemistry to constrain the secondary
structure of the peptide for optimized binding to PCNA. NMR solution studies show that the wild type p21 peptide from which
ACR2 was designed adopts no defi ned secondary structure in solution, while our mimetic adopts a 310 helix in solution, which
has been shown in previous studies to be essential for PIP box binding to PCNA. Binding experiments determined a KD of 200
nM of ACR2 for PCNA, which is higher than the wild type peptide. A co-crystal structure of ACR2 bound to hPCNA revealed
the mechanism of interaction of this mimetic with PCNA.
Miki Senda
High Energy Accelerator Research Organization (KEK), Japan
Title: Crystallization strategy when no crystals are obtained in the initial screening
Biography:
Miki Senda has completed her PhD from Nagaoka University of Technology in 2008. She is an Assistant Professor of Structural Biology Research Center in High
Energy Accelerator Research Organization (KEK). She has several collaborations in which she has worked as an expert of protein crystallization and crystal quality
improvement. She received Oxford Cryosystems Low Temperature Prize at the 63rd Annual meeting of the American Crystallographic Association (ACA) in 2013.
Abstract:
Protein crystallography is an indispensable tool in the pharmaceutical and biochemical sciences. Refi nements of the protein
crystallography beam lines and their integrated programs for crystal structure analysis allow us to perform automatic or
semi-automatic structural determinations using well-diff racted crystals. However, the production of well-diff racted crystals
is still a bottleneck, even when using crystallization robots and common screening kits. Th e process of protein crystallization
does not follow a standardized, routine protocol, except in the case that good crystals are obtained at an initial crystallization
screening. In the more frequent case that no crystals or only poor crystals appear at the initial screening, there is no general
consensus regarding the next step. Nonetheless, even in the absence of well-diff racted crystals at an initial screening, it is still
possible to optimize the crystallization conditions based on the accumulated data from a wide range of protein-crystallization
attempts. Th e more such crystallization data are available, the more appropriate and effi cient optimization will be possible,
since the crystallization conditions diff er for each protein. Th erefore, at our laboratories, we are currently trying to accumulate
many experiences of protein crystallization and crystal quality improvement of poor crystals through collaboration with not
only academia but also with pharmaceutical companies. Here, we present our strategy for the effi cient generation of good
quality crystals and the successful application of our crystal quality improvement method to histone chaperone TAF-Ibeta,
the CagA oncoprotein from Helicobacter pylori and GTP sensor PI5P4Kbeta. We believe that our strategy will be applicable to
other proteins as well.
Thomas Prevenslik
QED Radiations Discovery Bay, China
Title: Protein folding and unfolding by quantum mechanics
Biography:
Thomas Prevenslik is a retired American living in Hong Kong and Berlin. Because classical physics does not work at the nanoscale, he has developed the theory of
QED radiation based on quantum mechanics. He developed the simple theory of QED based on the Planck law of QM. Differing from the complex QED by Feynman
and others, simple QED assumes any heat absorbed at the nanoscale having high surface-to-volume ratios place interior atoms under high EM confi nement that
by the Planck law of QM precludes the atoms from having the heat capacity to conserve heat by an increase in temperature. In the instant topic of protein folding
and unfolding by quantum mechanics, the atoms may only conserve heat by creating EM radiation that by removing electrons by the photoelectric effect charges
the atoms positive inducing Coulomb repulsion that enhances unfolding. On a picosecond time scale, the electrons recombine with charged atoms to place the
protein under van der Waals attraction that fold the protein. Driven by heat, protein folding, and unfolding is the consequence of fl uctuations between QM induced
charge and neutral states.
Abstract:
Proteins are sensitive to electrostatic charges from amino acid side chains that change with conformation. But molecular
dynamics (MD) simulations of protein folding and unfolding are based on classical force fi elds with electrostatic interaction
represented by fi xed point charges. Quantum mechanics (QM) modifi cation of point charges during conformational changes is
required but is impractical because of computational costs. Computation costs aside, even if point charges were continuously
updated, the eff ect on protein folding and unfolding would be insignifi cant compared to the more fundamental QM eff ect
of the Planck law on the heat capacity of atoms. In this regard, proteins are generally thought to unfold upon increasing
temperature based on the classical assumption the constituent atoms have heat capacity. But the Planck law requires the heat
capacity of the atom to vanish with conservation proceeding by creating EM radiation that by the photoelectric eff ect removes
electrons to positively charge the protein atoms. What this means is the heat thought to induce unfolding by increasing the
temperature of proteins is actually conserved by producing charge that unfolds the protein by Coulomb repulsion. To illustrate
QM induced charge, the MD simulation of folding and unfolding using for a simple 5-atom protein is illustrated in Figure 1.
Initially, the protein in the form of a semi-circle relaxes under L-J forces but does not unfold. L-J stands for Lennard-Jones.
Unfolding occurs upon applying QM induced repulsive positive charge (0.5-1 electron charges) on each atom. Folding back to
an intermediate cluster occurs by relaxing the protein with L-J forces alone without QM induced charge. Th e protein returns
to an inverted semi-circular shape by unfolding the cluster by applying the QM induced repulsive charge. How the protein
constantly modifi es QM induced charge is discussed.
- Structural Biology in Drug Design | Frontiers in Structural Biology
Location: Spreewald
Chair
Peter B Stathopulos,
University of Western Ontario, Canada
Session Introduction
Fabrice Gorrec
Fabrice Gorrec
Title: The Morpheus III protein crystallization screen: at the frontier of drug discovery
Biography:
Abstract:
The original Morpheus screen has proven to be a very effi cient protein crystallization screen in the long term for a broad
variety of protein samples and the fi rst follow-up screen, Morpheus II starts to have an impact for many research groups.
Th e main ideas behind the formulation of Morpheus III were the same as originally to increase the chances of crystal nucleation
and growth by integrating mixes of additives that can act as stabilizers, cross-linkers, etc. Follow systematic approaches to select
the reagents and formulate the screen, such as the integration of ligands that are highly represented in the PDB and a 3D grid
formulation. Consider pragmatic ways to facilitate structure solution. For example, the screening of cryoprotectants and fl ashfreezing
crystals are simplifi ed. Th e novelty in Morpheus III is the selection of small drug-like compounds as additives (average
MW=248 Da). To some extent, the approach can be compared to fragment-based lead discovery. Th e primarily aim however is
to obtain novel macromolecular crystals (with or without ligand observed in the structures). Th e fi nal formulation of the new
96-condition crystallization screen integrates 44 compounds overall, divided into 8 mixes of additives. Each mix of additives is
combined with 4 cryo-protected precipitant mixes and 3 buff er systems to form the 3D grid.
Vesa P Hytönen
1University of Tampere, Finland
Title: Steered molecular dynamics and single molecule atomic force microscopy to explore the mechanical response of talin
Biography:
Vesa P Hytönen is a Head of the Protein Dynamics research group in Faculty of Medicine and Life Sciences at the University of Tampere, Tampere, Finland. After
graduating PhD from the University of Jyväskylä, Jyväskylä, Finland at 2005, he conducted Postdoctoral training at ETH Zurich, Switzerland during 2005–2007. He then
continued as a Postdoctoral researcher at the University of Tampere and established independent research group at 2010. He is currently working as Associate Professor
at the University of Tampere. His research interests are mechanobiology, protein engineering and vaccine research and he has authored more than 100 scientifi c articles.
Abstract:
Talin is cytoplasmic protein connecting integrin receptors to actomyosin network. Th is linkage is essential for cell anchoring
and spreading and naturally also for development. Talin acts as a hub for molecular interactions in focal adhesions and
the interactions between talin and binding partners are regulated by mechanical signals. We study the mechanical response of
talin by steered molecular dynamics. Both constant force and constant velocity simulations in explicit water have been found
useful to explore the molecular features of talin. We have found talin rod subdomains to diff er from each other in terms of their
mechanical stability. Importantly, we have been able to compare and validate the results by using experimental data obtained
with single-molecule atomic force microscopy. Destabilizing point mutations applied on talin rod have been found to cause
signifi cant changes in cell spreading, migration and cellular traction force. Our recent studies focus on mechanically weakened
talin forms, intermediates of protein unfolding and engineering of unfolding-resistant talin forms.
Fabrice Gorrec,
MRC Laboratory of Molecular Biology, UK
Title: Automated protocols for macromolecular crystallization at the MRCLaboratory of Molecular Biology
Biography:
Fabrice Gorrec has participated in the development of innovative technologies applied to protein crystallization over 14 years, including microplates (e.g., TOPAZ®),
liquid-handlers (e.g., Dragonfl y®) and initial screens (e.g., MORPHEUS™). Since 2008, he is responsible for the crystallization facility at the MRC Laboratory of Molecular
Biology (MRC-LMB, Cambridge, UK) where he invented and developed the 96 condition Morpheus protein crystallization screens. He pursued his Master’s degree in
Molecular Biology, Biochemistry and Biophysics from University of Rennes, France.
Abstract:
When high quality crystals are obtained that diff ract x-rays, the crystal structure may be solved at near atomic resolution.
Th e conditions to crystallize proteins, DNAs, RNAs and their complexes can however not be predicted. Employing
a broad variety of conditions is a way to increase the yield of quality diff raction crystals. Two fully automated systems have
been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization
screening against 1,920 initial conditions by vapor diff usion in nano liter droplets. Semi-automated protocols have also
been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that
potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefl y
discussed. Taken together, they enable convenient and highly effi cient macromolecular crystallization in a multi-user facility,
while giving the users control over key parameters of their experiments.
Raphael Taiwo Aruleba
University of Zululand, South Africa
Title: Development of new and effective anti-schistosomal drugs using antimicrobial peptides as lead compounds: A computer-aided drug design study
Biography:
Raphael Taiwo Aruleba is an Advocator for human and renders selfl ess service especially in the fi eld of science to humanity. Growing up he envisioned making positive
impact on many people as possible, which ultimately led him to the discovery of anti-microbial peptides that can be used in tackling Schistosomiasis using various
bioinformatic techniques. He also used ULBP2 to study cancer cells and Ganoderma lucidum to inhibit the growth of Plasmodium berghei in mice.
Abstract:
With the exponential increase in the prevalence of schistosomiasis, Praziquantel (PZQ) remains the only eff ective drug
in the anti-schistosomal arsenal. However, no signifi cant approaches have been made in recent years in the discovery
of new anti-schistosomal drugs, even with widely-reported resistance of the schistosome worm to PZQ over very large foci.
Th erefore, it is imperative to develop a new drug against this debilitating disease using the broad-spectrum therapeutic potentials
of antimicrobial peptides (AMPs). AMPs are natural antibiotics produced by all living species; they have multifunctional
properties and are currently explored as a vital source for the development of new drugs. In this study, six putative AMPs
(TAK1-TAK6) were identifi ed to possess anti-schistosomal capabilities. Added to this, glycosyltransferase and axonemal
dynein intermediate chain schistosomal proteins were identifi ed using in silico methods as vital proteins for the survival of the
parasite in the host. Th e 3D structures of the AMPs and the proteins were modelled using the I-TASSER, while Patch Dock
was employed to ascertain the interaction between these schistosome proteins and the AMPs. Results obtained show the
putative AMPs have good binding affi nity to the schistosomal proteins. So, TAK3 and TAK6 showed highest binding affi nities
to glycosyltransferase and axonemal dynein intermediate chain respectively. On the whole, all generated AMPs are potential
therapeutics target that could be further developed as drug candidates in the fi ght against schistosomiasis and could as well
prove eff ective against PZQ resistant schistosome strains.
Patrick Carl
Bruker Biospin, Germany
Title: Probing structures, interactions and rearrangements by EPR
Biography:
Electron Paramagnetic Resonance (EPR) is a powerful technique used to explore biological macromolecular structures.
Using intrinsic or extrinsic EPR species, the specifi cs of structural topology, protein-protein/protein-RNA interactions and
structural rearrangements are observed. Continuous wave EPR is used to probe the local environment to obtain information
such as pH, viscosity, rotational correlation time, and hydrophobicity. Th is information can easily be obtained using a user
friendly benchtop spectrometer, the EMXnano. Pulse EPR opens the possibility to probe more specifi cally the interactions in
the biological structures. Double Electron Electron Resonance (DEER) permits the direct distance determination from 2 nm
up to 10 nm. Th e DEER technique is not size limited and aides in the determination of multidomain protein and nucleic acid
structures.