Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine

The nucleocapsid protein (NC) plays an important role in HIV-1, mainly through interactions with the genomic RNA and its DNA copies. Though the structures of several complexes of NC with oligonucleotides (ODNs) are known, detailed information on the ODN dynamics in the complexes is missing. To address this, we investigated the steady state and time-resolved fluorescence properties of 2-aminopurine (2Ap), a fluorescent adenine analog introduced at positions 2 and 5 of AACGCC and AATGCC sequences. In the absence of NC, 2Ap fluorescence was strongly quenched in the flexible ODNs, mainly through picosecond to nanosecond dynamic quenching by its neighboring bases. NC strongly restricted the ODN flexibility and 2Ap local mobility, impeding the collisions of 2Ap with its neighbors and thus, reducing its dynamic quenching. Phe16→Ala and Trp37→Leu mutations largely decreased the ability of NC to affect the local dynamics of 2Ap at positions 2 and 5, respectively, while a fingerless NC was totally ineffective. The restriction of 2Ap local mobility was thus associated with the NC hydrophobic platform at the top of the folded fingers. Since this platform supports the NC chaperone properties, the restriction of the local mobility of the bases is likely a mechanistic component of these properties

Interuniversity Online Courses as Possible Approach to Improve Teaching During Crisis: a Ukrainian Case Study

The war launched by Russia has created new challenges for universities, including massive student migration abroad and massive displacement of students within Ukraine from the frontline areas. Many students lost access to quality education or had their studies interrupted by the war. Recognizing these problems, universities are trying to find different solutions. One such approach may be to introduce inter-university online courses that will be recognized by partner universities. In this case study, we analyze the effectiveness of inter-university online courses as an approach to restoring education for students severely affected by the war and as a way to maintain the quality of education in small groups at universities. The online course "Integrated Life Science Course" was taken for analysis, which was taught both to biology students from different universities in Ukraine within the framework of the educational project supported by German Academic Exchange Service (DAAD). Using statistics on course registration and attendance, as well as interview methodology, we assessed students' motivation to participate in this online course, course satisfaction, and learning outcomes, and identified shortcomings and pitfalls to avoid in similar courses. The survey and the results of the final test show that the main motivator for students to register and study at the course was the desire to gain new knowledge for further professional growth. A scholarship was also an important argument to enroll in the course; however, it did not impact the motivation of students to study. The fraction of students that successfully passed the final exam (~70%) was equal in the cohorts that had and had not been awarded scholarships. Thus, the scholarship was not a motivator to complete the course and successfully pass the exam to receive the certificate. Therefore, in order for such courses to be productive and effective, it is necessary to emphasize student motivation during selecting procedure

Membrane insertion of—and membrane potential sensing by—semiconductor voltage nanosensors: Feasibility demonstration

We developed membrane voltage nanosensors that are based on inorganic semiconductor nanoparticles. We provide here a feasibility study for their utilization. We use a rationally designed peptide to functionalize the nanosensors, imparting them with the ability to self-insert into a lipid membrane with a desired orientation. Once inserted, these nanosensors could sense membrane potential via the quantum confined Stark effect, with a single-particle sensitivity. With further improvements, these nanosensors could potentially be used for simultaneous recording of action potentials from multiple neurons in a large field of view over a long duration and for recording electrical signals on the nanoscale, such as across one synapse

The small heat shock protein Hsp27 binds α-synuclein fibrils, preventing elongation and cytotoxicity

Proteostasis, or protein homeostasis, encompasses the maintenance of the conformational and functional integrity of the proteome and involves an integrated network of cellular pathways. Molecular chaperones, such as the small heat shock proteins (sHsps), are key elements of the proteostasis network that have crucial roles in inhibiting the aggregation of misfolded proteins. Failure of the proteostasis network can lead to the accumulation of misfolded proteins into intracellular and extracellular deposits. Deposits containing fibrillar forms of α-sy-nuclein (α-syn) are characteristic of neurodegenerative disorders including Parkinson\u27s disease and dementia with Lewy bodies. Here we show that the sHsp Hsp27 (HSPB1) binds to α-syn fibrils, inhibiting fibril growth by preventing elongation. Using total internal reflection fluorescence (TIRF)- based imaging methods, we show that Hsp27 binds along the surface of α-syn fibrils, decreasing their hydrophobicity. Binding of Hsp27 also inhibits cytotoxicity of α-syn fibrils. Our results demonstrate that the ability of sHsps, such as Hsp27, to bind fibrils represents an important mechanism through which they May mitigate cellular toxicity associated with aberrant protein aggregation. Fibril binding May represent a generic mechanism by which chaperone-active sHsps interact with aggregation-prone proteins, highlighting the potential to target sHsp activity to prevent or disrupt the onset and progression of α-syn aggregation associated with α-synucleinopathies

Lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation.

α-Synuclein (α-syn) is a 140-residue intrinsically disordered protein that is involved in neuronal and synaptic vesicle plasticity, but its aggregation to form amyloid fibrils is the hallmark of Parkinson's disease (PD). The interaction between α-syn and lipid surfaces is believed to be a key feature for mediation of its normal function, but under other circumstances it is able to modulate amyloid fibril formation. Using a combination of experimental and theoretical approaches, we identify the mechanism through which facile aggregation of α-syn is induced under conditions where it binds a lipid bilayer, and we show that the rate of primary nucleation can be enhanced by three orders of magnitude or more under such conditions. These results reveal the key role that membrane interactions can have in triggering conversion of α-syn from its soluble state to the aggregated state that is associated with neurodegeneration and to its associated disease states.This work was supported by the UK BBSRC and the Wellcome Trust (CMD, TPJK, MV), the Frances and Augustus Newman Foundation (TPJK), Magdalene College, Cambridge (AKB) , St John’s College, Cambridge (TCTM), the Cambridge Home and EU Scholarship Scheme (GM), Elan Pharmaceuticals (CMD, TPJK, MV, CG) and the Leverhulme Trust (AKB).This is the accepted manuscript. The final version is available from NPG at http://www.nature.com/nchembio/journal/v11/n3/abs/nchembio.1750.htm

Structural determinants of TAR RNA-DNA annealing in the absence and presence of HIV-1 nucleocapsid protein

Annealing of the TAR RNA hairpin to the cTAR DNA hairpin is required for the minus-strand transfer step of HIV-1 reverse transcription. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. To gain insight into the mechanism of NC-mediated TAR RNA–DNA annealing, we used structural probes (nucleases and potassium permanganate), gel retardation assays, fluorescence anisotropy and cTAR mutants under conditions allowing strand transfer. In the absence of NC, cTAR DNA-TAR RNA annealing depends on nucleation through the apical loops. We show that the annealing intermediate of the kissing pathway is a loop–loop kissing complex involving six base-pairs and that the apical stems are not destabilized by this loop–loop interaction. Our data support a dynamic structure of the cTAR hairpin in the absence of NC, involving equilibrium between both the closed conformation and the partially open ‘Y’ conformation. This study is the first to show that the apical and internal loops of cTAR are weak and strong binding sites for NC, respectively. NC slightly destabilizes the lower stem that is adjacent to the internal loop and shifts the equilibrium toward the ‘Y’ conformation exhibiting at least 12 unpaired nucleotides in its lower part

Sondes fluorescentes à émission duale pour la caractérisation d'interactions impliquant des protéines : application aux protéines rétrovirales

Dans ce travail, nous avons développé des sondes sensibles à l'environnement pour suivre les interactions de protéines entre elles, avec des acides nucléiques ou avec des membranes. Dans ce but, nous avons développés des fluorophores de la famille des 3-hydroxychromones (3HC) et des 3-hydroxyquinolones (3HQ). Ces sondes présentent à l'état excité une réaction de transfert de proton intramoléculaire (ESIPT) conduisant à deux bandes d'émission, permettant de caractériser les propriétés de cet environnement via le rapport d'intensités de ces deux bandes. La réaction ESIPT des 3HQs s'est révélée être irréversible et sensible à la polarité, à la basicité et la viscosité du milieu, contrairement aux 3HCs qui ne sont pas sensibles à cette basicité et donnent une réponse claire aux modifications de polarité et d'hydratation. Deux sondes ont été couplées au groupement amino-terminal de peptides préparés par synthèse en phase solide. En marquant la protéine de la nucléocapside (NC) de HIV-1 par la 2-furyl-3 hydroxychromone, une forte baisse, corrélée à la structure du complexe obtenu, du rapport d'intensité des deux bandes est observée lorsque NC se fixe à des oligonucléotides. Cette réponse peut être utilisée pour identifier le site de fixation préférentiel de NC. Cette sonde a également été appliquée avec succès pour suivre l'interaction d'un petit peptide avec son anticorps. Le marquage du domaine C-terminal de la protéine Vpr de HIV-1 avec la 4 -dimethylamino-3 hydroxyflavone sensible aux milieux apolaires a été utilisé pour déterminer la localisation du peptide lié à une biomembrane et préciser l'affinité du peptide en fonction de la phase et de la composition de la membrane.The task of the present work was to develop environment sensitive labels for monitoring protein interactions with nucleic acids, membranes and other proteins. Fluorophores of the 3 hydroxychromone (3HC) and 3 hydroxyquinolone (3HQ) family have been considered. They undergo an excited state intramolecular proton transfer (ESIPT) reaction leading to two emission bands thus allowing to characterize the environment properties through their intensities ratio. 3HQs were shown to undergo an irreversible ESIPT reaction and to be sensitive to the polarity, the H-bond basicity and the viscosity of the media. In contrast, the spectroscopic response of 3HCs is not sensitive to basicity and shows a clear response on polarity and hydration changes. Two different labels were proposed for sensing protein-DNA and protein-membrane interactions. Since DNA and membrane environment strongly differ by their polarity. The labels were coupled to the N terminal amino group of peptides prepared by solid phase synthesis. The probe based on 2-furyl-3 hydroxychromone was used for labeling HIV-1 nucleocapsid protein (NC) and shows a several-fold sequence-dependent decrease of the emission band ratio on NC-binding to oligonucleotides. This property was then used to identify the NC preferential binding site on oligonucleotides. The label was also successfully applied for sensing the interaction of a short peptide with an antibody. Labeling of the C-terminal domain of the HIV-1 Vpr protein with the 4 -dimethylamino-3 hydroxyflavone derivative was used to determine the location of the peptide in the membrane and the relationship between the affinity of the peptide and the membrane composition and phase

DESA1002 'Nine Quarter City' - <Kelly Millgate>

The Asakusa metro line is my response to our collective semester 2 project 'Nine Quarter city' - a generic city comprised of 9 actual cities to create distinct 'quarter's' all woven into an overall urban order. Being assigned none other than Tokyo - a major global city and technology hub - my design aims to reflect the unique atmosphere of Japan's capital, home to twelve million people. Bearing this in mind, I decided that it would be near impossible to recreate the complexity and bustle of the megacity. Thus early on, my concept was one of simplicity. In developing my design, it was key that the station be close to roads and walkways as well as central with respect to other sites. I decided on this particular location for these reasons, especially as it is situated on the major diagonal axis of our generic 'nine quarter city'. After deciding on the location of my site, the next step really was to understand my context. Using google earth as a guide, I noticed that the surrounding buildings were of similar height (a characteristic particularly of Asakusa) and confined to an order - making up the 'hard edge' of the streetscape. I tried to embody simplicity in numerous ways, essentially: - using a rectangle as the basis of my design - to emphasise straight, clean lines - double height ground floor - so the vastness and openness of the space is appreciated - inclusion of a Schwedler dome - a pure geometric form to which the eye can be drawn, while also symbolising the hub of the building. It that it is here the circulation takes place and movement can be seen notably through the criss-cross arrangement of escalators through the floors. - the use of a steel frame and double thickness glass. However, though I didn't want to compete vertically with the surrounding buildings, I felt at the same time there was a need for my building to be a part of what train stations typically symbolise - ie a recognisable meeting point for social interaction. I felt one way that I could make it stand out was not only by the inclusion of a glass space framed dome, but an interesting façade. I wanted the final facade to de different from the normal high tech Tokyo façade - in that it represents calm and simplicity in such an energetic city. I tried to achieve this by use of delicate, tessellating shapes and repeating the pattern on the glass façade. - not to block off the surrounding world, but merely screen the brutality of it