Light Microscopy Module Biophysics - 4 (LMM-B4)

Compare incorporation of protein aggregates into growing protein crystals on ISS (International Space Station) and on Earth. Measure growth rates in 1g (1 gravity) versus microgravity (micro-g) for different size aggregates of proteins. Compare the defect density and crystal quality via fluorescent-based atomic force microscopy and X-ray diffraction quality of crystals grown at different rates in a 1g environment

Interactions of the Neurotoxin Vipoxin in Solution Studied by Dynamic Light Scattering

AbstractThe neurotoxin vipoxin is the lethal component of the venom of Vipera ammodytes meridionalis. It is a heterodimer of a basic toxic His-48 phospholipase A2 (PLA2) and an acidic nontoxic Gln-48 PLA2. The shape of the neurotoxin and its separated components in solution as well as their interactions with calcium, the brain phospholipid phosphatidylcholine, and two inhibitors, elaidoylamide and vitamin E, were investigated by dynamic light scattering. Calcium binding is connected with a conformational change in vipoxin observed as a change of the hydrodynamic shape from oblate ellipsoid to a shape closer to a sphere. The Ca2+-bound form of vipoxin, which is catalytically active, is more compact and symmetric than the calcium-free heterodimer. Similar changes were observed as a result of the Ca2+-binding to the two separated subunits. In the presence of aggregated phosphatidylcholine, the neurotoxic complex dissociates to subunits. It is supposed that only the toxic component binds to the substrate, and the other subunit, which plays a chaperone function, remains in solution. The inhibition of vipoxin with the synthetic inhibitor elaidoylamide and the natural compound vitamin E changes the shape of the toxin from oblate to prolate ellipsoid. The inhibited toxin is more asymmetric in comparison to the native one. Similar, but not so pronounced, effects were observed after the inhibition of the monomeric and homodimeric forms of the toxic His-48 PLA2. Circular dichroism measurements in the presence of urea, methylurea, and ethylurea indicate a strong hydrophobic stabilization of the neurotoxin. Hydrophobic interactions stabilize not only the folded regions but also the regions of intersubunit contacts

CHL1 Is a Selective Organizer of the Presynaptic Machinery Chaperoning the SNARE Complex

Proteins constituting the presynaptic machinery of vesicle release undergo substantial conformational changes during the process of exocytosis. While changes in the conformation make proteins vulnerable to aggregation and degradation, little is known about synaptic chaperones which counteract these processes. We show that the cell adhesion molecule CHL1 directly interacts with and regulates the activity of the synaptic chaperones Hsc70, CSP and αSGT. CHL1, Hsc70, CSP and αSGT form predominantly CHL1/Hsc70/αSGT and CHL1/CSP complexes in synapses. Among the various complexes formed by CHL1, Hsc70, CSP and αSGT, SNAP25 and VAMP2 induce chaperone activity only in CHL1/Hsc70/αSGT and CHL1/CSP complexes, respectively, indicating a remarkable selectivity of a presynaptic chaperone activity for proteins of the exocytotic machinery. In mice with genetic ablation of CHL1, chaperone activity in synapses is reduced and the machinery for synaptic vesicle exocytosis and, in particular, the SNARE complex is unable to sustain prolonged synaptic activity. Thus, we reveal a novel role for a cell adhesion molecule in selective activation of the presynaptic chaperone machinery

Features of “All LNA” Duplexes Showing a New Type of Nucleic Acid Geometry

“Locked nucleic acids” (LNAs) belong to the backbone-modified nucleic acid family. The 2′-O,4′-C-methylene-β-D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of “all” LNA homoduplexes, however, exhibit significant differences in their overall geometry, in particular a decreased twist, roll and propeller twist. This results in a widening of the major groove, a decrease in helical winding, and an enlarged helical pitch. Therefore, the LNA duplex structure can no longer be described as a canonical A-type RNA geometry but can rather be brought into proximity to other backbone-modified nucleic acids, like glycol nucleic acids or peptide nucleic acids. LNA-modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery

Crystallization and preliminary X-ray diffraction studies of intact EF-Tu from Thermus aquaticus YT-1

AbstractMany attempts have been made to elucidate the three-dimensional structure from elongation factor Tu, but so far the only crystals suitable for X-ray crystallography contained a partially degraded protein. Here, we report the crystallization of a fully active, intact EF-Tu from Thermus aquaticus. The crystals belong to hexagonal space group P6322 and diffract up to 2.6 Å. The cell dimensions are a = b = 178 Å, c = 238 Å and 6 molecules are contained per asymmetric unit

Polysaccharide binding sites in hyaluronate lyase-crystal structures of native phage-encoded hyaluronate lyase and its complexes with ascorbic acid and lactose

Hyaluronate lyases are a class of endoglycosaminidase enzymes with a high level of complexity and heterogeneity. The main function of the Streptococcus pyogenes bacteriophage protein hyaluronate lyase, HylP2, is to degrade hyaluronan into unsaturated disaccharide units. HylP2 was cloned, over-expressed and purified to homogeneity. The recombinant HylP2 exists as a homotrimer with a molecular mass of approximately 110 kDa under physiological conditions. The HylP2 was crystallized and the crystals were soaked in two separate reservoir solutions containing ascorbic acid and lactose, respectively. The crystal structures of native HylP2 and its two complexes with ascorbic acid and lactose have been determined. HylP2 folds into four distinct domains with a central core consisting of 16 antiparallel β-strands forming an irregular triangular tube designated as triple-stranded β-helix. The structures of complexes show that three molecules each of ascorbic acid and lactose bind to protein at the sugar binding groove in the triple-stranded β-helix domain. Both ascorbic acid and lactose molecules occupy almost identical subsites in the long saccharide binding groove. Both ligands are involved in several hydrogen bonded interactions at each subsite. The binding characteristics and stereochemical properties indicate that Tyr264 may be involved in the catalytic activity of HylP2. The mutation of Tyr264 to Phe264 supports this observation

Production, purification and characterization of recombinant, full-length human claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-ß-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81

The crystal structure of an ‘All Locked’ nucleic acid duplex

‘Locked nucleic acids’ (LNAs) are known to introduce enhanced bio- and thermostability into natural nucleic acids rendering them powerful tools for diagnostic and therapeutic applications. We present the 1.9 Å X-ray structure of an ‘all LNA’ duplex containing exclusively modified β-d-2′-O-4′C-methylene ribofuranose nucleotides. The helix illustrates a new type of nucleic acid geometry that contributes to the understanding of the enhanced thermostability of LNA duplexes. A notable decrease of several local and overall helical parameters like twist, roll and propeller twist influence the structure of the LNA helix and result in a widening of the major groove, a decrease in helical winding and an enlarged helical pitch. A detailed structural comparison to the previously solved RNA crystal structure with the corresponding base pair sequence underlines the differences in conformation. The surrounding water network of the RNA and the LNA helix shows a similar hydration pattern

Resting-State Connectivity of the Left Frontal Cortex to the Default Mode and Dorsal Attention Network Supports Reserve in Mild Cognitive Impairment

Reserve refers to the phenomenon of relatively preserved cognition in disproportion to the extent of neuropathology, e.g., in Alzheimer’s disease. A putative functional neural substrate underlying reserve is global functional connectivity of the left lateral frontal cortex (LFC, Brodmann Area 6/44). Resting-state fMRI-assessed global LFC-connectivity is associated with protective factors (education) and better maintenance of memory in mild cognitive impairment (MCI). Since the LFC is a hub of the fronto-parietal control network that regulates the activity of other networks, the question arises whether LFC-connectivity to specific networks rather than the whole-brain may underlie reserve. We assessed resting-state fMRI in 24 MCI and 16 healthy controls (HC) and in an independent validation sample (23 MCI/32 HC). Seed-based LFC-connectivity to seven major resting-state networks (i.e., fronto-parietal, limbic, dorsal-attention, somatomotor, default-mode, ventral-attention, visual) was computed, reserve was quantified as residualized memory performance after accounting for age and hippocampal atrophy. In both samples of MCI, LFC-activity was anti-correlated with the default-mode network (DMN), but positively correlated with the dorsal-attention network (DAN). Greater education predicted stronger LFC-DMN-connectivity (anti-correlation) and LFC-DAN-connectivity. Stronger LFC-DMN and LFC-DAN-connectivity each predicted higher reserve, consistently in both MCI samples. No associations were detected for LFC-connectivity to other networks. These novel results extend our previous findings on global functional connectivity of the LFC, showing that LFC-connectivity specifically to the DAN and DMN, two core memory networks, enhances reserve in the memory domain in MCI

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin’s efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections