Podcasts about conformational

References Mol Informatics. 2012 Feb;31(2):114-22. Nat Struct Mol Biol. 2017 Jun; 24(6):544–552 Guerra: chemical thermodynamics lectures --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.30.518485v1?rss=1 Authors: Duechs, M., Blazevic, D., Rechtsteiner, P., Kenny, C., Lamla, T., Low, S., Savistchenko, J., Neumann, M., Melki, R., Schoenberger, T., Stierstorfer, B., Wyatt, D., Igney, F., Ciossek, T. Abstract: Prion-like transmission of pathology in alpha-synucleinopathies like Parkinsons disease or multiple system atrophy is increasingly recognized as one potential mechanism to address disease progression. Active and passive immunotherapies targeting insoluble, aggregated alpha-synuclein are already being actively explored in the clinic with mixed outcomes so far. Here, we report the identification of 306C7B3, a highly selective, aggregate-specific alpha-synuclein antibody with picomolar affinity devoid of binding to the monomeric, physiologic protein. 306C7B3 binding is Ser129-phosphorylation independent and shows high affinity to several different aggregated alpha-synuclein polymorphs, increasing the likelihood that it can also bind to the pathological seeds assumed to drive disease progression in patients. In support of this, highly selective binding to pathological aggregates in postmortem brains of MSA patients was demonstrated, with no staining in samples from other human neurodegenerative diseases. To achieve CNS exposure of 306C7B3, an Adeno-Associated Virus (AAV) based approach driving expression of the secreted antibody within the brain of (Thy-1)-[A30P]-h-alpha-Synuclein mice was used. Widespread central transduction after intrastriatal inoculation was ensured by using the AAV2HBKO serotype, with transduction being spread to areas far away from the inoculation site. Treatment of (Thy-1)-[A30P]-h-alpha-Synuclein mice at the age of 12 months demonstrated significantly increased survival, with 306C7B3 concentration reaching 3.9 nM in the cerebrospinal fluid. These results suggest that AAV-mediated expression of 306C7B3 has great potential as a disease-modifying therapy for alpha-synucleinopathies as it ensures CNS exposure of the antibody, thereby mitigating the selective permeability of the blood-brain barrier. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.01.506163v1?rss=1 Authors: Cottone, G., Chiodo, L., Maragliano, L., Popoff, M.-R., Rasetti-Escargueil, C., Lemichez, E., Malliavin, T. E. Abstract: Although the botulinum neurotoxins (BoNTs) are among the most toxic compounds found in nature, their molecular mechanism of action is far from being elucidated. A key event is the conformational transition due to the acidification of the interior of synaptic vesicles, and leading to the translocation of the BoNT catalytic domain into the neuronal cytosol. To investigate these conformational variations, homology modelling and atomistic simulations are combined to explore the internal dynamics of the subtypes BoNT/A1, the most-used in medical applications, and BoNT/E1, the most kinetically efficient. This first simulation study of di-chain BoNTs in closed and open states includes the effects of neutral and acidic pH. The conformational mobility is driven by domains displacements; the ganglioside binding site in the receptor binding domain, the translocation domain (HCNT) switch and the belt helix visit multiple conformations depending on the primary sequence and on the pH. Fluctuations of the belt helix are observed for closed conformations of the toxins and at acidic pH, and patches of more accessible residues appear in the same conditions in the core translocation domain HCNT. These findings suggest that during translocation, the larger mobility of belt could be transmitted to HCNT, leading to a favorable interaction of HCNT residues with the non-polar membrane environment. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

This episode is also available as a blog post: http://biopatrika.com/2021/06/03/interview-conformational-flexibility-and-structural-variability-of-sars-cov2-s-protein/

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.390500v1?rss=1 Authors: Hou, Q., Stringer, B., Waury, K., Capel, H., Haydarlou, R., Abeln, S., Heringa, J., Feenstra, K. A. Abstract: Motivation: Antibodies play an important role in clinical research and biotechnology, with their specificity determined by the interaction with the antigen's epitope region, as a special type of protein-protein interaction (PPI) interface. The ubiquitous availability of sequence data, allows us to predicting epitopes from sequence in order to focus time-consuming wet-lab experiments onto the most promising epitope regions. Here, we extend our previously developed sequence-based predictors for homodimer and heterodimer PPI interfaces to predict epitope residues that have the potential to bind an antibody. Results: We collected and curated a high quality epitope dataset from the SAbDaB database. Our generic PPI heterodimer predictor obtained an AUC-ROC of 0.666 when evaluated on the epitope test set. We then trained a random forest model specifically on the epitope dataset, reaching AUC 0.694. Further training on the combined heterodimer and epitope datasets, improves our final predictor to AUC 0.703 on the epitope test set. This is better than the best state-of-the-art sequence-based epitope predictor BepiPred-2.0. On one solved antibody-antigen structure of the COVID19 virus spike RNA binding domain, our predictor reaches AUC 0.778. We added the SeRenDIP-CE Conformational Epitope predictors to our webserver, which is simple to use and only requires a single antigen sequence as input, which will help make the method immediately applicable in a wide range of biomedical and biomolecular research. Availability: Webserver, source code and datasets are available at www.ibi.vu.nl/programs/serendipwww/ Contact: k.a.feenstra@vu.nl Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.16.385187v1?rss=1 Authors: Oganesyan, I., Lento, C., Tandon, A., Wilson, D. J. Abstract: Both normal and pathological functions of -synuclein (SN), an abundant protein in the central and peripheral nervous system, have been linked to its interaction with membrane lipid bilayers. The ability to characterize structural transitions of SN upon membrane complexation will clarify molecular mechanisms associated with SN-linked pathologies, including Parkinsons disease (PD), Multiple Systems Atrophy and other synucleinopathies. In this work, Time-Resolved ElectroSpray Ionization Hydrogen/ Deuterium Exchange Mass Spectrometry (TRESI-HDX-MS) was employed to acquire a detailed picture of SNs conformational transitions as it undergoes complexation with nanodisc membrane mimics. Using this approach, SN interactions with DMPC nanodiscs were shown to be rapid exchanging and to have a little impact on the SN conformational ensemble. Interactions with nanodiscs containing lipids known to promote amyloidogenesis (e.g., POPG), on the other hand, were observed to induce substantial and specific changes in the SN conformational ensemble. Ultimately, we identify a region corresponding residues 19-28 and 45-57 of the SN sequence that is uniquely impacted by interactions with amyloidogenic lipid membranes and may therefore play a critical role in pathogenic aggregation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.06.370916v1?rss=1 Authors: Qin, B., Craven, G. B., Hou, P., Lu, X., Child, E. S., Morgan, R. M. L., Armstrong, A., Mann, D. J., Cui, S. Abstract: RNA viruses are critically dependent upon virally encoded proteases that cleave the viral polyproteins into functional mature proteins. Many of these proteases are structurally conserved with an essential catalytic cysteine and this offers the opportunity to irreversibly inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that selectively target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we effectively re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold as well as similar catalytic-triad. We demonstrate that the hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. In addition, we provide the first demonstration that targeting the active site cysteine of Mpro can also have profound allosteric effects, distorting secondary structures required for formation of the active dimeric unit of Mpro. These new data provide novel mechanistic insights into the design of EV71 3Cpro and SARS-CoV-2 Mpro inhibitors and identify acrylamide-tagged pharmacophores for elaboration into more selective agents of therapeutic potential. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.30.362590v1?rss=1 Authors: Gonzalez Buitron, M., Tunque Cahui, R. R., Garcia Rios, E., Hirsh, L., Fornasari, M. S., Parisi, G., Palopoli, N. Abstract: Conformational changes in RNA native ensembles are central to fulfill many of their biological roles. Systematic knowledge of the extent and possible modulators of this conformational diversity is desirable to better understand the relationship between RNA dynamics and function. We have developed CoDNaS-RNA as the first database of conformational diversity in RNA molecules. Known RNA structures are retrieved and clustered to identify alternative conformers of each molecule. Pairwise structural comparisons within each cluster allows to measure the variability of the molecule. Additional data on structural features, molecular interactions and functional annotations are provided. CoDNaS-RNA is implemented as a public resource that can be of much interest for computational and bench scientists alike. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.11.335299v1?rss=1 Authors: Gobeil, S., Janowska, K., McDowell, S., Mansouri, K., Parks, R., Manne, K., Stalls, V., Kopp, M., Henderson, R., Edwards, R. J., Haynes, B. F., Acharya, P. Abstract: The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we analyze the D614G mutation in the context of a soluble S ectodomain construct. Cryo-EM structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the conformational dynamics of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on the RBD dynamics triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational dynamics and allostery, and have implications for vaccine design. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.06.327965v1?rss=1 Authors: Mazumder, A., Wang, A., Uhm, H., Ebright, R. H., Kapanidis, A. N. Abstract: The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Forster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ~ 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes >1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.318220v1?rss=1 Authors: Nahass, G. R., Sun, Y., Xu, Y., Batchelor, M., Reilly, M., Benilova, I., Kedia, N., Spehar, K., Sobott, F., Sessions, R. B., Caughey, B., Radford, S. E., Jat, P., Collinge, J., Bieschke, J. Abstract: Alpha-synuclein (-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson's disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (A{beta}) and -syn, prompting our inquiry in its mechanism of action. Here we test the effects of Brazilin on both seeded and unseeded -syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of -syn by a unique mechanism that is distinct from other polyphenols and is also distinct from its effect on A{beta}. Brazilin preserves the natively unfolded state of -syn by stabilizing the compact conformation of the -syn monomer over the aggregation-competent extended conformation. Molecular docking of Brazilin shows the molecule to interact both with unfolded -syn monomers and with the cross-{beta} sheet structure of -syn fibrils. Brazilin eliminates seeding competence of -syn assemblies from Parkinson's disease patient brain tissue, and treatment of pre-formed fibril assemblies with Brazilin significantly reduces their toxicity in primary neurons. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinson's Disease. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.318543v1?rss=1 Authors: Croll, T. I., Read, R. J. Abstract: When building atomic models into weak and/or low-resolution density, a common strategy is to restrain its conformation to that of a higher-resolution model of the same or similar sequence. When doing so, it is important to avoid over-restraining to the reference model in the face of disagreement with the experimental data. The most common strategy for this is the use of "top-out" potentials. These act like simple harmonic restraints within a defined range, but gradually weaken when the deviation between the model and reference grows larger than a defined transition point. In each current implementation, the rate at which the potential flattens beyond the transition region follows a fixed form - although the form chosen varies between implementations. A restraint potential with a tuneable rate of flattening would provide greater flexibility to encode the confidence in any given restraint. Here we describe two new such potentials: a Cartesian distance restraint derived from a recent generalisation of common loss functions, and a periodic torsion restraint based on a renormalisation of the von Mises distribution. Further, we describe their implementation as user-adjustable/switchable restraints in ISOLDE, and demonstrate their use in some real-world examples. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.22.306647v1?rss=1 Authors: Wilkinson, M. E., Fica, S. M., Galej, W. P., Nagai, K. Abstract: The catalytic spliceosome exists in equilibrium between the branching (B*/C) and exon ligation (C*/P) conformations. Here we present the electron cryo-microscopy reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 [A] resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. In the Ci conformation, the exon-ligation factors Prp18 and Slu7 are already bound before ATP hydrolysis by Prp16, which destabilises the branching conformation. Biochemical assays suggest these pre-bound factors prime C complex for conversion to C* by Prp16. A complete model of the Prp19-complex (NTC) shows how branching factors Yju2 and Isy1 bind the NTC before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain associated with the C* complex to promote exon ligation. Our results explain how Prp16 action modulates dynamic binding of step-specific factors to alternatively stabilise the C or C* conformation and establish equilibrium of the catalytic spliceosome. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.20.305425v1?rss=1 Authors: Fernandes, D. D., Neale, C., Gomes, G.-N. W., Li, Y., Malik, A., Pandey, A., Orazietti, A., Wang, X., Ye, L., Prosser, S., Gradinaru, C. C. Abstract: G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A Receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Forster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes [≥] 3 ms. Local conformational dynamics around residue 229 on TM6 was measured using Fluorescence Correlation Spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 s) and slow (200-300 s) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288, and Y290) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.18.302638v1?rss=1 Authors: Bonilla, S., Sherlock, M., MacFadden, A., Kieft, J. S. Abstract: Structured RNA elements are essential for biology and are ubiquitously used by viruses to control diverse infection-critical processes. Single-stranded RNA viruses often encode multiple processes into a single RNA element to maximize the functional capacity of their compact genomes. These important and elegant 'multifunctional' RNAs are hypothesized to use programmed structural changes to coordinate several, sometimes opposing, functions. However, detailed molecular mechanisms of such elements are largely mysterious because of the difficulty of solving dynamic RNA structures. We exploited recent advances in cryo-EM to directly visualize the architecture and infer conformational dynamics of the brome mosaic virus tRNA-like structure (BMV TLS), a multifunctional tRNA mimic that participates in replication, translation, and encapsidation of viral RNAs and that has eluded structure determination for decades. We found that although BMV TLS is aminoacylated by cellular tyrosyl-synthetase (TyrRS), its primary conformation is incompatible with TyrRS binding. Rather, the RNA is preorganized for replication, positioning the replicase promoter and the initiation site in close proximity to each other. An alternative 'tyrosylation-ready' conformation requires repositioning of a conformationally dynamic structural domain and this change must induce additional rearrangements that disrupt the 'replication-ready' configuration. These results demonstrate how programmed RNA dynamics can evolve to coordinate interactions with diverse cellular and viral proteins and thus organize multiple functions on a single RNA platform. Our results support the paradigm that RNA structures are inherently dynamic conformational ensembles, which enables multifunctionality. This work also highlights the emerging power of cryo-EM to dissect the dynamic conformational landscape of small discrete functional RNAs. Furthermore, we anticipate our method of rapidly mapping RNA domains within cryo-EM maps to be broadly useful. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.13.286047v1?rss=1 Authors: Adams, S., Purkiss, A. G., Knowles, P. P., Nans, A., Briggs, D. C., Borg, A., Earl, C. P., Goodman, K. M., Nawrotek, A., Borg, A. J., McIntosh, P. B., Houghton, F. M., Kjaer, S., McDonald, N. Q. Abstract: RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFR co-receptors recognise and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2-CLD3. Comparison with a cryo-EM structure of a ligand-engaged zebrafish RETECD-GDNF-GFR1 complex indicates conformational changes within a clade-specific CLD3 loop adjacent to co-receptor. Our observations indicate RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFR co-receptors, while its rigid arm recognises a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualise linear arrays of RETECD-GDNF-GFR1 suggesting a conserved contact stabilises higher-order species. Our study reveals ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.08.288019v1?rss=1 Authors: Paydar, M., Kwok, B. H. Abstract: Microtubules, protein polymers of /{beta}-tubulin dimers, form the structural framework for many essential cellular processes including cell shape formation, intracellular transport, and segregation of chromosomes during cell division. It is known that tubulin-GTP hydrolysis is closely associated with microtubule polymerization dynamics. However, the precise roles of GTP hydrolysis in tubulin polymerization and microtubule depolymerization, and how it is initiated are still not clearly defined. We report here that tubulin-GTP hydrolysis can be triggered by conformational change induced by the depolymerizing kinesin-13 proteins or by the stabilizing chemical agent paclitaxel. We provide biochemical evidence that conformational change precedes tubulin-GTP hydrolysis, confirming this process is mechanically driven and structurally directional. Furthermore, we quantitatively measure the average size of the presumptive stabilizing "GTP cap" at growing microtubule ends. Together, our findings provide the molecular basis for tubulin-GTP hydrolysis and its role in microtubule polymerization and depolymerization. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.05.284026v1?rss=1 Authors: Wang, Q., Asarnow, D. E., Ding, K., Zhang, Y., Ma, Y., Cheng, Y., Beachy, P. A. Abstract: The DISP1 protein, related to the NPC1 and PTCH1 cholesterol transporters and to H+-driven transporters of the RND family, enables tissue patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly-localized sites of embryonic expression. A 2.5 [A] resolution cryo-EM structure of DISP1 revealed three Na+ ions coordinated within a channel that traverses its transmembrane domain. This channel and Na+-binding are disrupted in DISP1-NNN (3.4 [A] resolution), a variant with isosteric substitutions for three intramembrane aspartates that each coordinate and neutralize charge for one of the three Na+ ions. DISP1-NNN and other variants that singly disrupt the Na+ sites retain binding to the lipid-modified Hedgehog protein, but most fail to export Hedgehog. The Sonic hedgehog signal (ShhN) interacts with the DISP1 extracellular domains (2.7 [A] complex structure), including an unusual extended DISP1 arm, at a location well above the membrane. Variability analysis reveals a dynamic series of DISP1 conformations, only a restricted subset of which appear to bind ShhN. The bound and unbound DISP1 conformations display distinct Na+ site occupancies; these differences, in conjunction with the unusual locations of certain lipids bound to DISP1, suggest a mechanism by which transmembrane Na+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane, thus enabling its tissue patterning activity. The Na+-coordinating residues resolved in our DISP1 structures are wholly or partly conserved in some of the other metazoan RND family members, such as PTCH1 and NPC1, suggesting the utilization of Na+ flux to power their conformationally-driven activities. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.01.278812v1?rss=1 Authors: Yang, Y. X., Li, P., Wang, P., Zhu, B. T. Abstract: Insulin receptor plays an important role in regulation of energy metabolism. Dysfunction of insulin receptor (IR) can lead to many disease states, such as diabetes mellitus. Deciphering the complex dynamic structure of human IR and its mechanism of activation would greatly aid in understanding IR-mediated signaling pathways and in particular, in designing new drugs (including nonpeptidal insulin analogs) to treat diabetes mellitus. Experimental evidence about IR structure has been gradually obtained by biologists over the past three decades. Based on the available experimental structures of IR in different states, here we employ molecular modeling approach to construct the full-length IR structures in different states and model its structural and conformational changes during insulin-induced IR activation. Several key possible intermediate states are constructed based on structural alignment, rotation and computational modeling. Based on the structures of the full-length IR in different states, it appears that there are two possible conformational transition pathways: one is symmetric, and the other one is asymmetric. Structural changes and motions of different domains of the full-length IR along the pathways are analyzed. The role of insulin binding to IR in facilitating the conformational transition of the receptor is modeled. Information and insights derived from our present structural modeling analyses may aid in understanding the complex dynamic, structural and conformational changes during the process of IR activation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.01.277863v1?rss=1 Authors: Weerasinghe, N. W., Habibi, Y., Uggowitzer, K. A., Thibodeaux, C. Abstract: Lanthipeptides are ribosomally-synthesized and post-translationally modified peptide (RiPP) natural products that are biosynthesized in a multistep maturation process by enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding, and that conformational sampling of the enzyme-peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These biophysical processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native nanoelectrospray ionization coupled to ion mobility mass spectrometry (nanoESI-IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its non-covalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme-peptide contacts. Moreover, gas phase ion mobility studies of the natively-folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme, and that the precursor peptide itself exhibits higher order structure in the mass spectrometer. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes nanoESI-IM-MS as a versatile approach for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.25.266742v1?rss=1 Authors: Li, H., Bai, L., You, Q., Jain, B. K., Graham, T. R., Kovach, A. Abstract: The P4 ATPases are a family of membrane enzymes that transport lipids across membrane bilayers. The structures of the class-1 phosphatidylserine flippases have been reported, revealing a substrate site on lumenal side. However, the cytosolic binding site has not been found, and the transport mechanisms of P4 ATPases in other classes are unknown. Here we report a structural and functional study on plasma-membrane localized, class-3 P4 ATPases Dnf1-Lem3 and Dnf2-Lem3. We captured substrate lipids on both the exoplasmic and cytosolic sides, and found that these two enzymes have similar structures. We found a helix-turn-helix motif in the cytosolic P domain that is unique to the class-3 enzymes and plays a crucial role in their function. Unexpectedly, Lem3 contributes to substrate binding near the cytosolic surface. Conformational transitions of these two class-3 enzymes match those of the class-1 enzymes, suggesting a conserved mechanism among all classes of P4 ATPases. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.20.230268v1?rss=1 Authors: Brown, C. J., Verma, C. S., Lane, D. P., Lama, D. Abstract: Intrinsically disordered regions (IDRs) in proteins can regulate their activity by facilitating protein-protein interactions (PPIs) as exemplified in the recruitment of the eukaryotic translation initiation factor 4E (eIF4E) protein by the protein eIF4G. Deregulation of this PPI module is central to a broad spectrum of cancer related malignancies and its targeted inhibition through bioactive peptides is a promising strategy for therapeutic intervention. We have employed a structure-guided approach to rationally develop peptide derivatives from the intrinsically disordered eIF4G scaffold by incorporating non-natural amino acids that facilitates disorder-to-order transition. The conformational heterogeneity of these peptides and the degree of structural reorganization required to adopt the optimum mode of interaction with eIF4E underscores their differential binding affinities. The presence of a pre-structured local helical element in the ensemble of structures was instrumental in the efficient docking of the peptides on to the protein surface. These insights were exploited to further design features into the peptide to propagate bound-state conformations in solution which resulted in the generation of a potent eIF4E binder. The study illustrates the molecular basis of eIF4E recognition by a disordered epitope from eIF4G and its modulation to generate peptides that can potentially attenuate translation initiation in oncology. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.20.259572v1?rss=1 Authors: Zhang, Y.-W., Uchendu, S., Leone, V., Bradshaw, R. T., Sangwa, N., Forrest, L., Rudnick, G. Abstract: The human GlyT1 glycine transporter requires chloride for its function. However, the mechanism by which Cl- exerts its influence is unknown. To examine the role that Cl- plays in the transport cycle, we measured the effect of Cl- on both glycine binding and conformational changes. The ability of glycine to displace the high-affinity radioligand [3H]CHIBA-3007 required Na+ and was potentiated over 1000-fold by Cl-. We generated GlyT1b mutants containing reactive cysteine residues in either the extracellular or cytoplasmic permeation pathways and measured changes in the reactivity of those cysteine residues as indicators of conformational changes in response to ions and substrate. Na+ increased accessibility in the extracellular pathway and decreased it in the cytoplasmic pathway, consistent with stabilizing an outward-open conformation as observed in other members of this transporter family. In the presence of Na+, both glycine and Cl- independently shifted the conformation of GlyT1b toward an outward-closed conformation. Together, Na+, glycine and Cl- stabilize an inward-open conformation of GlyT1b. Our results suggest that Cl- acts by interacting with a conserved glutamine to allow formation of an ion pair that stabilizes the closed state of the extracellular pathway. Molecular dynamics simulations of a GlyT1 homologue suggest that this ion pair is formed more frequently as that pathway closes. Mutation of the glutamine blocks the effect of Cl-, and substituting it with residues of opposite charge lead to inverse conformations of the transporter. These results provide novel and unexpected insight into the role of Cl- in this family of transporters. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.18.256065v1?rss=1 Authors: Pedebos, C., Verli, H. Abstract: Oligosaccharyltransferases (OSTs) are enzymes that catalyze the transfer of a glycan chain to an acceptor protein. Their structure is composed by a transmembrane domain and a periplasmic / C-terminal domain, which can be divided into structural units. The Archaeoglobus fulgidus OST, AfAglB, has unique structural units with unknown functions. Here, we evaluate the stability role proposed for AfAglB units by employing molecular modelling and molecular dynamics simulations, to examine the effect of single and double deletions in the enzyme structure. Our results show a strong effect on the dynamics of the C-terminal domain for the mutated systems with increased fluctuations near the deleted areas. Conformational profile and stability are deeply affected, mainly in the double unit deletion, modifying the enzyme behavior and binding interfaces. Coordination at the catalytic site was not disrupted, indicating that the mutated enzymes could retain activity at some level. Hotspots of variation were identified and rationalized with previous data. Our data shows that structural units may provide stabilization interactions, contributing for integrity of the wild-type enzyme at high temperatures. By correlating our findings to structural units mutagenesis experimental data available, it was observed that structural units deletion can interfere with OSTs stability and dynamics but it is not directly related to catalysis. Instead, they may influence the OST structural integrity, and, potentially, thermostability. This work offers a basis for future experiments involving OSTs structural and functional characterization, as well as for protein engineering. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.11.243964v1?rss=1 Authors: Brotherton, D. H., Savva, C., Ragan, T., Linthwaite, V., Cann, M., Dale, N., Cameron, A. D. Abstract: CO2 is the inevitable by-product of oxidative metabolism. Many physiological processes such as breathing and cerebral blood flow are sensitive to CO2. Historically, the physiological actions of CO2 have been regarded as being mediated exclusively via changes in pH. Here, we change this consensus by showing that the gap junction protein Connexin26 (Cx26) acts as a receptor for CO2 showing sensitivity to modest changes in PCO2 around the physiological norm. Mass spectrometry analysis shows that CO2 carbamylates specific lysines on a regulatory loop of Cx26 at high, but not at low levels of PCO2. By means of high resolution cryo-EM, we have solved structures of Cx26 gap junctions at 1.9, 2.2 and 2.1 [A] for PCO2 of 90, 55 and 20 mmHg respectively, all at pH 7.4. Classification of the particles at each level of PCO2, shows the transmembrane helices and N-terminal helix flexing at the dynamic cytoplasmic side of the protein. Gating of Cx26 gap junctions by CO2 involves movements of the N-terminus to plug the channel at high PCO2. We therefore provide mechanistic detail for a new paradigm by which CO2 can directly control breathing8 and other key physiological functions. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.09.243030v1?rss=1 Authors: Tesei, G., Martins, J. M., Kunze, M. B. A., Wang, Y., Crehuet, R., Lindorff-Larsen, K. Abstract: Owing to their plasticity, intrinsically disordered and multidomain proteins require descriptions based on multiple conformations, thus calling for techniques and analysis tools that are capable of dealing with conformational ensembles rather than a single protein structure. Here, we introduce DEER-PREdict, a software to predict Double Electron-Electron Resonance distance distributions as well as Paramagnetic Relaxation Enhancement rates from ensembles of protein conformations. DEER-PREdict uses an established rotamer library approach to describe the paramagnetic probes which are bound covalently to the protein. DEER-PREdict has been designed to operate efficiently on large conformational ensembles, such as those generated by molecular dynamics simulation, to facilitate the validation or refinement of molecular models as well as the interpretation of experimental data. The performance and accuracy of the software is demonstrated with experimentally characterized protein systems: HIV-1 protease, T4 Lysozyme and Acyl-CoA-binding protein. DEER-PREdict is open source (GPLv3) and available at https://github.com/KULL-Centre/DEERpredict and as a Python PyPI package https://pypi.org/project/DEERPREdict. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.07.241463v1?rss=1 Authors: Tassis, K., Vietrov, R., Koning, M. d., de Boer, M., Gouridis, G., Cordes, T. Abstract: The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Foerster-resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the type-I ABC importer OpuA from Lactococcus lactis. Our studies include intradomain assays that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC and interdomain assays between SBDs or transmembrane domains. Using the methodology, we studied ligand-binding mechanisms as well as ATP and glycine betaine dependences of conformational changes. Our study expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.29.227363v1?rss=1 Authors: Palopoli, N., Marchetti, J., Monzon, A. M., Zea, D. J., Tosatto, S. C. E., Fornasari, M. S., Parisi, G. Abstract: Intrinsically disordered proteins (IDPs) lack stable tertiary structure under physiological conditions. The unique composition and complex dynamical behaviour of IDPs make them a challenge for structural biology and molecular evolution studies. Using NMR ensembles, we found that IDPs evolve under a strong site-specific evolutionary rate heterogeneity, mainly originated by different constraints derived from their inter-residue contacts. Evolutionary rate profiles correlate with the experimentally observed conformational diversity of the protein, allowing the description of different conformational patterns possibly related to their structure-function relationships. The correlation between evolutionary rates and contact information improves when structural information is taken not from any individual conformer or the whole ensemble, but from combining a limited number of conformers. Our results suggest that residue contacts in disordered regions constrain evolutionary rates to conserve the dynamic behaviour of the ensemble and that evolutionary rates can be used as a proxy for the conformational diversity of IDPs. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.26.219741v1?rss=1 Authors: Mansbach, R. A., Chakraborty, S., Nguyen, K., Montefiori, D., Korber, B., Gnanakaran, S. Abstract: The COVID-19 pandemic underwent a rapid transition with the emergence of a SARS-CoV-2 variant that carried the amino acid substitution D614G in the Spike protein that became globally prevalent. The G-form is both more infectious in vitro and associated with increased viral loads in infected people. To gain insight into the mechanism underlying these distinctive characteristics, we employed multiple replicas of microsecond all-atom simulations to probe the molecular-level impact of this substitution on Spike closed and open states. The open state enables Spike interactions with its human cellular receptor, ACE2. Here we show that changes in the inter-protomer energetics due to the D614G substitution favor a higher population of infection-capable (open) states. The inter-protomer interactions between S1 and S2 subunits in the open state of the D-form are asymmetric. This asymmetry is resolved in the G-form due to the release of tensile hydrogen bonds resulting in an increased population of open conformations. Thus, the increased infectivity of the G-form is likely due to a higher rate of profitable binding encounters with the host receptor. It is also predicted to be more neutralization sensitive due to enhanced exposure of the receptor binding domain, a key target region for neutralizing antibodies. Copy rights belong to original authors. Visit the link for more info

Distinguish between activated and deactivated proteins and how each form is reached.

In this episode I review “Influence People: Powerful Everyday Opportunities to Persuade That Are Lasting and Ethical,” written by Brian Ahearn. In addition to influencing people in general, information is provided for those who need to improve their sales cycle. His approach is very practical, laying out key principles and associated acronyms that can be used to practice sharpening you ability to influence people. His overall tone is about helping the read as an entire person, not just one aspect. The work is based on solid research. He boils the information down to 7 key shortcuts which basically are sound principles. They include: 1. Reciprocity - behave in a way that encourages others to relate to you 2. Liking - people want to do business with their friends or people they like 3. Authority - experienced or knowledgable - use it 4. Consensus - when no authority use how groups are moving in a given direction 5. Consistency - channel a person’s consistent behavior in a desirable direction. Be consistent yourself to develop trust. 6. Scarcity - people respond more to what they might lose than they do to what they might gain 7. Unity - people like to relate with people with whom they have a sense of belonging and with whom they may have common experiences Additional tools are presented: ▪ Compare and contrast - set the stage to make it easy for the other person to go in the direction you want, e.g., “This normally is $799 but since you are here I can give it to you at $599.” Another example is given with wine lists. When the wines are listed in decreasing price people buy more because they feel they are being practical by buying a good bottle but one not so expensive. When listed from lowest price to highest people buy substantially lower priced products because they are only seeing an increase in price rather than an opportunity to “save” ▪ Consistency vs authority. Consistency is driven by client history in terms of thoughts, feelings, and actions traditionally going in a specific direction. Authority is used when your expertise legitimately points the person being persuaded in the desired direction. This especially helps when the client is uncertain. ▪ Conformational bias. Present information that is ethical and honest but plays in the direction the other person wants to go. ▪ “Because I said so.” The word “because” allows people to be gracious and help. This works especially well when put in the form of a question, e.g., “Would you please get your report to me by Monday because I have to roll it into a larger report?” If they say “no” you can have a backup position of Wednesday. “How about Wednesday?” Usually people don’t want to say “no” twice in a row so with this approach you increase the odds of getting their report when you actually need it. They have a sense of reciprocity. Decision making and rationality are the next topics he presents. Most decision-making is essentially irrational, with some researchers believing >90% of our decision-making is driving by the unconscious. What you are exposed to and the order in which you are exposed sets the stage for how the decisions will flow. This gets back to the reality people respond more to concern about what they might lose compared to what they might gain. Several examples are given. Brian goes on to give about 15 tips for improving your bottom line. Case studies are then provided, some of which are fascinating and make it worth purchasing the book. This includes: - How Kodak went from having almost 100% of the image market to almost nothing - JCPenny losing 40% of it’s stock value by making changes that failed to take the customer’s wishes into consideration - How Bernie Madoff used the principles in this book unethically to swindle $65 billion - Why Starbucks is so pervasive even though they don’t advertise. What’s their secret? The etiquette for using social media is discussed. Use it to network and connect…don’t start selling right away! The book closes out with examples of how people behave in ways that show they are worth working with. The examples are quite good. Brian is is on LinkedIn and can also be contacted at brian.ahearn@influencepeople.biz. Need help dealing with complex situations? You can download CMC’s free e-book MINDSET – 5 SIMPLE WAYS TO LOOK AT COMPLEX PROBLEMS and learn how to find a simple vantage point from which you can resolve challenges. Your feedback is important. Choose from the following options: • place a review in iTunes, • click on “leave a comment” below, • send any comments along with your name and the show number to support@ctrchg.com, • go to Contact at Center for Managing Change and leave a message Listen to future episodes for our reply.

The Central Park jogger case was a criminal case based on the assault and rape of Trisha Meili, a white woman who was jogging in the park the night of April 19th, 1989. Four African American teenagers and one Hispanic teenager that were in the park that night were accused of the crime. The prosecution based their case on the alleged coerced testimony of the teenage boys. The 5 boys were convicted and received sentences ranging from 5-15 years. During the trial Donald Trump wrote and article calling for the death penalty, for criminals who commit these types of crimes. Today Matt and Tony are going to be breaking down the case and analyzing Trump’s influence on the effect of the outcome of the case.   Highlights: The alleged coerced testimonies by the teens Lead pipes and plumbers gear The teens were held without food and bathroom breaks Conformational bias Spinning the story to fit a variety of narratives   The Takeaway – The evidence was not strong enough to convict these kids.   Hollywood Improv Ticket Link: https://improv.com/hollywood/event/legally+insane/9370845/   Twitter: @mattritter1 @toekneesam   Website: www.cascademedia.com  

Breyers are the horse gateway drug, am I right? This week, we’re talking about how the barn brings us all together, no matter our age. Also why figure 8 bridles are cool and our favorite tricks for confirmationally-challenged horses. Listen in...

Conformational bias… We all have it or have had it… I’m for one sick of it and want to be more pragmatic about it all. But I still find myself getting angry because of it. Even with people who I know have others best interest at heart. I’ve been guilty of this, and I believe we've lost the forest for the trees!  This is a short podcast on confirmation bias and some strategies I believe will help you have better health and performance. I have the good doctor, Dr Phil Maffetone coming back on the show so we can dissect all these nutritional bias's and give you a simple path to better health and performance.  Enjoy. IN TODAY’S EPISODE, I DISCUSS: Confirmation bias What simple effective day to day nutrition looks like Does nutritional ketosis have a time and place? Benefits of metabolic flexibility Our obsession with health is making us unhealthy Listen & Subscribe on iTunes | Stitcher Radio SHOW LINKS: The below are IF you want to look into nutritional ketosis more. I don't believe you need to go "keto" as an endurance athlete. I do believe it has a time and place, but it is not a requirement unless you have some untoward things going on (metabolic disease, cancer, pre-diabetes, type II). But if you need education, Dominic D' Agostino is your man.  https://dominicdagostino.wordpress.com/ https://ketonutrition.org/ Join (for Free) our TRISPECIFIC CAFE https://www.facebook.com/TriSpecific http://www.instagram.com/trispecific SHOW SPONSORS: We have a number of options here at TS from the new Ironman and 70.3 Blueprints for those that just want a kick-ass plan and all the needed content to plug and play. Simply get it, follow the plan and advice and you can crush it. Go to trispecific.com/raceready TS LIFE membership for just $395 for the year is really the no-brainer. Once you sign up we send you a questionnaire and we set up your training in a training peaks account. We plug in the phases you need when you need them and take you towards your races and goals. You get all the supporting content in the member area and coaching support through the TS LIFE private FB group. Join the family today at http://www.trispecific.com/tslife / I’ll send you out our new Trucker cap too! And there is full coaching: Full coaching – it’s the full deal but we only take on a limited amount of folks per year. This is full commitment. But we will get you to your goals. We do play the long game. Some get there quicker and others take longer. If someone is selling 12 weeks to a 9 hour Ironman… You best be already in great shape and knocking on the door. Apply at http://www.trispecific.com/apply/ If you need to chat about which option is best for you. Fill in the coaching application at http://www.trispecific.com/apply and click I would like to discuss options. Get to Kona via TriSpecific’s plans, membership or coaching in 2018 and get yourself a FREE Ceepo frameset and other goodies.  PLEASE HELP US GROW… To subscribe to the podcast, please use the links below: Click Here to Subscribe via iTunes If you have a chance, please leave an honest rating and review on iTunes by clicking here. It will help the show and its ranking in iTunes immensely! We appreciate it! Enjoy the show!

Charge transfer in DNA cannot be understood without addressing the complex conformational flexibility, which occurs on a wide range of timescales. In order to reduce this complexity four dinucleotide models 1X consisting of benzophenone linked by a phosphodiester to one of the natural nucleosides X = A, G, T, C were studied in water and methanol. The theoretical work focuses on the dynamics and electronic structure of 1G. Predominant conformations in the two solvents were obtained by molecular dynamics simulations. 1G in MeOH adopts mainly an open geometry with a distance of 12-16 angstrom between the two aromatic parts. In H2O the two parts of 1G form primarily a stacked conformation yielding a distance of 5-6 angstrom. The low-lying excited states were investigated by electronic structure theory in a QM/MM environment for representative snapshots of the trajectories. Photo-induced intramolecular charge transfer in the S-1 state occurs exclusively in the stacked conformation. Ultrafast transient absorption spectroscopy with 1X reveals fast charge transfer from S-1 in both solvents with varying yields. Significant charge transfer from the T-1 state is only found for the nucleobases with the lowest oxidation potential: in H2O, charge transfer occurs with 3.2 x 10(9) s(-1) for 1A and 6.0 x 10(9) s(-1) for 1G. The reorganization energy remains nearly unchanged going from MeOH to the more polar H2O. The electronic coupling is rather low even for the stacked conformation with H-AB = 3 meV and explains the moderate charge transfer rates. The solvent controls the conformational distribution and therefore gates the charge transfer due to differences in distance and stacking.

Cyclo-octane, trans-cyclo-octene, and atripisomerism. Caroyophyllene and biphenyls.

Conformational analysis of 3,4,5 and 6-membered rings.

The conformational preferences of alikeness, aldehydes, ketones, esters and amides.

The factors responsible for the conformation of alkanes and heterosubstituted alkanes.

Historical background to conformational analysis, and Barton's contributions. The stereo electronic theory of conformational analysis.

The integrity of the genome displays a central role for all living organisms. Double strand breaks (DSBs) are probably the most cytotoxic and hazardous type of DNA lesion and are linked to cancerogenic chromosome aberrations in humans. To maintain genome stability, cells use various repair mechanisms, including homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. The Mre11:Rad50 (MR) complex plays a crucial role in DSB repair processes including DSB sensing and processing but also tethering of DNA ends. The complex consists of the evolutionarily conserved core of two Rad50 ATPases from which a long coiled-coil region protrudes and a dimer of the Mre11 nuclease. Even though various enzymatic and also structural functions of MR(N) could be determined, so far the molecular interplay of Rad50´s ATPase together with DNA binding and processing by Mre11 is rather unclear. The crystal structure of the bacterial MR complex in its nucleotide free state revealed an elongated conformation with accessible Mre11 nuclease sites in the center and a Rad50 monomer on each outer tip, thus suggesting conformational changes upon ATP and/or DNA binding. However, so far high resolution structures of MR in its ATP and/or DNA bound state are lacking. The aim of this work was to understand the ATP-dependent engagement-disengagement cycle of Rad50´s nucleotide binding domains (NBDs) and thereby the ATP-controlled interaction between Mre11 and Rad50. For this purpose high resolution crystal structures of the bacterial Thermotoga maritima (Tm) MR complex with engaged Rad50 NBDs were determined. Small angle x-ray scattering proved the conformation of the nucleotide bound complex in solution. DNA affinity was also analyzed to investigate MR´s DNA binding mechanism. ATP binding to TmRad50 induces a large structural change and surprisingly, the NBD dimer binds directly in the Mre11 DNA binding cleft, thereby blocking Mre11’s dsDNA binding sites. DNA binding studies show that MR does not entrap DNA in a ring-like structure and that within the complex Rad50 likely forms a dsDNA binding site in response to ATP, while the Mre11 nuclease module retains ssDNA binding ability. Finally, a possible mechanism for ATP dependent DNA tethering and DSB processing by MR is proposed.

Cyclo-octane, trans cyclo-octene, how to resolve it into configurational isomers, and atropisomerism in caryophyllene. Restricted rotation in biphenyls.

Conformational analysis of rings, and sugars.

Analysis of cyclopropane, cyclobutane, cyclopentane and cyclohexane, including ring-locking with large groups and anti-periplanar relationships of substituents.

Conformational analysis of alkyl substituted alkenes, aldehydes, ketones, esters and amides.

The conformations of alkanes, including butane, from the perspective of the three controlling influences in conformations.

The relevance of conformational analysis, definitions and introduction to the theory.

The phenomenon of atropisomeris, and restricted rotation in biphenyls

The conformation of cyclohexane and substituted cyclohexanes, including conformational locking. Trans-decalines, and anti-periplanar relationships, leading to steroids

The conformation of amides, cyclopropane, cyclubutanes, cyclopentanes and cyclohexanes.

The Gauche effect and its origins, dealing with heterosubstituted alkanes, alkenes, aldehydes, ketones and esters

A lecture covering the early history of and definitions in coformational analysis and introduction of the basic theoretical underpinnings of the topic.

Background: G. diazotrophicus and A. vinelandii are aerobic nitrogen-fixing bacteria. Although oxygen is essential for the survival of these organisms, it irreversibly inhibits nitrogenase, the complex responsible for nitrogen fixation. Both microorganisms deal with this paradox through compensatory mechanisms. In A. vinelandii a conformational protection mechanism occurs through the interaction between the nitrogenase complex and the FeSII protein. Previous studies suggested the existence of a similar system in G. diazotrophicus, but the putative protein involved was not yet described. This study intends to identify the protein coding gene in the recently sequenced genome of G. diazotrophicus and also provide detailed structural information of nitrogenase conformational protection in both organisms. Results: Genomic analysis of G. diazotrophicus sequences revealed a protein coding ORF (Gdia0615) enclosing a conserved "fer2" domain, typical of the ferredoxin family and found in A. vinelandii FeSII. Comparative models of both FeSII and Gdia0615 disclosed a conserved beta-grasp fold. Cysteine residues that coordinate the 2[Fe-S] cluster are in conserved positions towards the metallocluster. Analysis of solvent accessible residues and electrostatic surfaces unveiled an hydrophobic dimerization interface. Dimers assembled by molecular docking presented a stable behaviour and a proper accommodation of regions possibly involved in binding of FeSII to nitrogenase throughout molecular dynamics simulations in aqueous solution. Molecular modeling of the nitrogenase complex of G. diazotrophicus was performed and models were compared to the crystal structure of A. vinelandii nitrogenase. Docking experiments of FeSII and Gdia0615 with its corresponding nitrogenase complex pointed out in both systems a putative binding site presenting shape and charge complementarities at the Fe-protein/MoFe-protein complex interface. Conclusions: The identification of the putative FeSII coding gene in G. diazotrophicus genome represents a large step towards the understanding of the conformational protection mechanism of nitrogenase against oxygen. In addition, this is the first study regarding the structural complementarities of FeSII-nitrogenase interactions in diazotrophic bacteria. The combination of bioinformatic tools for genome analysis, comparative protein modeling, docking calculations and molecular dynamics provided a powerful strategy for the elucidation of molecular mechanisms and structural features of FeSII-nitrogenase interaction.

The vast majority of mitochondrial proteins are synthesized by the cytosolic ribosomes as precursor proteins which have to be transported into the organelle to reach their sites of function. The whole process of recognition, translocation, intra-mitochondrial sorting of and assembly of precursor proteins is achieved by the concerted action of different mitochondrial translocases. All proteins destined for the mitochondrial matrix and some inner membrane proteins are imported first by the TOM complex of the outer membrane and subsequently by the TIM23 complex of the inner membrane in an energy-driven process. The TIM23 complex was found to consist of ten components, conventionally divided into two sectors: membrane sector harbouring the translocation channel and the import motor on the matrix side of the membrane sector. In the first part of the present work, the two most recently discovered subunits of the TIM23 complex, Pam17 and Tim21 were characterized. A systematic characterization revealed that both of these non-essential subunits of the translocase are associated with Tim17-Tim23 core of the membrane sector of the TIM23 translocase. A functional connection between the two non-essential components was discovered. Results presented in this part showed that Pam17 and Tim21 modulate the functions of the TIM23 complex in an antagonistic manner. The second part of the work was directed towards understanding the motor sector of the translocase in terms of the regulated interaction between Tim44 and Ssc1. Previous studies on the Tim44:Ssc1 interaction were able to discern the steady-state properties of Tim44:Ssc1 interaction in organello and in vitro. However, due to the limitations of the techniques used, they were unable to shed light on the kinetics and dynamics of the process. The translocation event is a dynamic event with conformational cycling of the various components. Therefore, the kinetic components essential in defining the cycle of events in the motor sector were explored. A FRET based assay to analyze the Tim44:Ssc1 interaction in real time was developed. The same set of tools was also used to resolve the regions of the two proteins that determine their interaction. The substrate induced dissociation of Tim44:Ssc1 complex was found to be too slow to support a physiological rate of protein translocation. ATP-induced dissociation was observed to be fast enough to be physiologically relevant. The dissociation of Ssc1 from Tim44 occurred in a one step manner without Tim44 anchored conformational changes. Furthermore, peptide-array scanning of mitochondrial matrix proteins revealed that Ssc1 and Tim44 share complementary binding sites on the precursor proteins which could prevent backsliding of preproteins. The data support the Brownian ratchet model mediated translocation of preproteins into the mitochondrial matrix. The third part of the work aimed at dissecting the chaperone cycle of Ssc1 in the mitochondrial matrix, in terms of conformational changes and binding of co-chaperones. Using the FRET sensors developed, the inter-domain conformation and lid-base conformations of the PBD of Ssc1 could be investigated. Single particle FRET (SpFRET) analysis showed that in the ATP-bound form Ssc1 populates a homogeneous conformational state with respect to the inter-domain conformation and conformation of the lid to base of the PBD. On the contrary, in the ADP-bound state the conformation of the chaperone is heterogenous. Using the same sensors on bacterial homologue DnaK, specific differences in conformational distributions were observed. Furthermore, the active role of substrates in determining the inter-domain conformation and lid-closing was evident from the SpFRET based conformational analyses. Using ensemble time resolved FRET, the kinetics and dynamics of conformational changes along with binding of co-chaperones were explored. This provided a better understanding of the conformational dynamics of Ssc1 in the context of functional chaperone cycle in the mitochondrial matrix.

Conformational transitions in macromolecular complexes often involve the reorientation of leverlike structures. Using a simple theoretical model, we show that the rate of such transitions is drastically enhanced if the lever is bendable, e. g., at a localized hinge. Surprisingly, the transition is fastest with an intermediate flexibility of the hinge. In this intermediate regime, the transition rate is also least sensitive to the amount of "cargo" attached to the lever arm, which could be exploited by molecular motors. To explain this effect, we generalize the Kramers-Langer theory for multidimensional barrier crossing to configuration-dependent mobility matrices.

Molecular recognition, programmability, self-assembling capabilites and biocompatibility are unique features of DNA. The basic approach of DNA nanotechnology is to exploit these properties in order to fabricate novel materials and structures on the nanometer scale. This cumulative dissertation deals with three aspects of this young research area: fast analysis, autonomous control of functional structures, and biocompatible autonomous delivery systems for nanoscale objects. 1. At low temperatures and under favorable buffer conditions, two complementary DNA strands will form a double-helical structure in which the bases of the two strands are paired according to the Watson-Crick rules: adenine bases bind with thymine bases, guanine bases with cytosine bases. The melting temperature TM of a DNA duplex is defined as the temperature at which half of the double strands are separated into single strands. The melting temperature can be calculated for DNA strands of known sequences under standard conditions. However, it has to be determined experimentally for strands of unknown sequences and for applications under extreme buffer conditions. A method for fast and reliable determination of DNA melting temperatures has been developed. Stable gradients of the denaturing agent formamide were generated by means of diffusion in a microfluidic setup. Formamide lowers the melting temperature of DNA and a given formamide concentration can be mapped to a corresponding virtual temperature along the formamide gradient. Differences in the length of complementary sequences of only one nucleotide as well as a single nucleotide mismatch can be detected with this method, which is of great interest for the detection of sequence mutations or variations such as single nucleotide polymorphisms (SNPs). 2. Knowledge of the stability of DNA duplexes is also of great importance for the construction of DNA-based nanostructures and devices. Conformational changes occuring in artificially generated DNA structures can be used to produce motion on the nanometer scale. Usually, DNA devices are driven by the manual addition of fuel molecules or by the periodic variation of buffer conditions. One prominent example of such a conformational change is the formation of the so-called i-motif, which is a folded four-stranded DNA structure characterized by noncanonical hemiprotonated cytosine-cytosine base-pairs. In order to achieve controlled autonomous motion, the oscillating pH-value of a chemical oscillator has been employed to drive the i-motif periodically through its conformational states. The experiments were conducted with the DNA switch in solution and attached to a solid substrate and constitute the first example of DNA-based devices driven autonomously by a chemical non-equilibrium reaction. 3. Finally, a DNA-crosslinked and switchable polyacrylamide hydrogel is introduced, which is used to trap and release fluorescent colloidal quantum dots in response to externally applied programmable DNA signal strands. Trapping and release of the nanoparticles is demonstrated by studying their diffusion properties using single molecule fluorescence microscopy, single particle tracking and fluorescence correlation spectroscopy. Due to the biocompatibility of the polymerized acrylamide and the crosslinking DNA strands, such gels could find application in the context of controlled drug delivery, where the autonomous release of a drug-carrying nanoparticle could be triggered by naturally occurring, potentially disease-related DNA or RNA strands.

The first human monoclonal islet cell antibodies of the IgG class (MICA 1-6) obtained from an individual with Type 1 (insulin-dependent) diabetes mellitus were cytoplasmic islet cell antibodies selected by the indirect immunofluorescence test on pancreas sections. Surprisingly, they all recognized the 64 kDa autoantigen glutamate decarboxylase. In this study we investigated which typical features of cytoplasmic islet cell antibodies are represented by these monoclonals. We show by double immunofluorescence testing that MICA 1-6 stain pancreatic beta cells which is in agreement with the beta-cell specific expression of glutamate decarboxylase. In contrast an islet-reactive IgM monoclonal antibody obtained from a pre-diabetic individual stained all islet cells but lacked the tissue specificity of MICA 1-6 and must therefore be considered as a polyreactive IgM-antibody. We further demonstrate that MICA 1-6 revealed typical features of epitope sensitivity to biochemical treatment of the target tissue which has been demonstrated for islet cell antibodies, and which has been used to argue for a lipid rather than a protein nature of target antigens. Our results provide direct evidence that the epitopes recognized by the MICA are destroyed by methanol/chloroform treatment but reveal a high stability to Pronase digestion compared to proinsulin epitopes. Conformational protein epitopes in glutamate decarboxylase therefore show a sensitivity to biochemical treatment of sections such as ganglioside epitopes. MICA 1-6 share typical features of islet cell and 64 kDa antibodies and reveal that glutamate decarboxylase-reactive islet cell antibodies represent a subgroup of islet cell antibodies present in islet cell antibody-positive sera.

Fri, 1 Jan 1988 12:00:00 +0100 http://epub.ub.uni-muenchen.de/2236/ http://epub.ub.uni-muenchen.de/2236/1/128.pdf Köhler, W.; Friedrich, J.; Scheer, Hugo Köhler, W.; Friedrich, J. und Scheer, Hugo (1988): Conformational barriers in low-temperature proteins and glasses. In: Physical Reviews, Vol. A37, Nr. 2: pp. 660-662. Biologie

Sat, 1 Jan 1977 12:00:00 +0100 http://epub.ub.uni-muenchen.de/2574/ http://epub.ub.uni-muenchen.de/2574/1/2574.pdf Scheer, Hugo; Kufer, Werner Scheer, Hugo und Kufer, Werner (1977): Conformational studies of phycocyanin from Spirulina platensis. In: Zeitschrift für Naturforschung C, Vol. 32C, Nr. 1-2: pp. 513-518. Biologie