51 research outputs found
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
Org Biomol Chem
A new fluorescent label N-[4'-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-beta-alanine () was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form. The label was coupled to the N-terminus of penetratin, a cell penetrating peptide, in order to study its interactions with lipid membranes and internalization inside the cells. As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase. After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed, in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides bound to cell membrane structures were observed. These observations confirmed the ability of penetratin to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of peptides with living cells and their internalization mechanisms
Sensing peptide–oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein
We present a new methodology for site-specific sensing of peptide–oligonucleotide (ODN) interactions using a solvatochromic fluorescent label based on 3-hydroxychromone (3HC). This label was covalently attached to the N-terminus of a peptide corresponding to the zinc finger domain of the HIV-1 nucleocapsid protein (NC). On interaction with target ODNs, the labeled peptide shows strong changes in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from the bulk water by the ODN bases. Remarkably, this two-color response depends on the ODN sequence and correlates with the 3D structure of the corresponding complexes, suggesting that the 3HC label monitors the peptide–ODN interactions site-specifically. By measuring the two-color ratio, we were also able to determine the peptide–ODN-binding parameters and distinguish multiple binding sites in ODNs, which is rather difficult using other fluorescence methods. Moreover, this method was found to be more sensitive than the commonly used steady-state fluorescence anisotropy, especially in the case of small ODNs. The described methodology could become a new universal tool for investigating peptide–ODN interactions
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
Repeats in the α-Synuclein Sequence Determine its Conformation on Membranes and Influence Aggregation Properties
A Four-Amino Acid Linker between Repeats in the α‑Synuclein Sequence Is Important for Fibril Formation
α-Synuclein
is a 140-amino acid protein that can switch conformation
among intrinsically disordered in solution, helical on a membrane,
and β-sheet in amyloid fibrils. Using the fluorescence of single-tryptophan
mutants, we determined the immersion of different regions of the protein
into lipid membranes. Our results suggest the presence of a flexible
break close to residues 52–55 between two helical domains.
The four-amino acid linker is not necessary for membrane binding but
is important for fibril formation. A deletion mutant lacking this
linker aggregates extremely slowly and slightly inhibits wild-type
aggregation, possibly by blocking the growing ends of fibrils
The mode of α-synuclein binding to membranes depends on lipid composition and lipid to protein ratio.
AbstractInteractions of the presynaptic protein α-synuclein with membranes are involved in its physiological action as well as in the pathological misfolding and aggregation related to Parkinsons’s disease. We studied the conformation and orientation of α-synuclein bound to model vesicular membranes using multiparametric response polarity-sensitive fluorescent probes together with CD and EPR measurements. At low lipid to α-synuclein ratio the protein binds membranes through its N-terminal domain. When lipids are in excess, the α-helical content and the role of the C-terminus in binding increase. Highly rigid membranes also induce a greater α-helical content and a lower polarity of the protein microenvironment
Interactions of α-Synuclein with lipids and artificial membranes monitored by ESIPT probes.
Non-uniform self-assembly:On the anisotropic architecture of α-synuclein supra-fibrillar aggregates
Although the function of biopolymer hydrogels in nature depends on structural anisotropy at mesoscopic length scales, the self-assembly of such anisotropic structures in vitro is challenging. Here we show that fibrils of the protein α-synuclein spontaneously self-assemble into structurally anisotropic hydrogel particles. While the fibrils in the interior of these supra-fibrillar aggregates (SFAs) are randomly oriented, the fibrils in the periphery prefer to cross neighboring fibrils at high angles. This difference in organization coincides with a significant difference in polarity of the environment in the central and peripheral parts of the SFA. We rationalize the structural anisotropy of SFAs in the light of the observation that αS fibrils bind a substantial amount of counterions. We propose that, with the progress of protein polymerization into fibrils, this binding of counterions changes the ionic environment which triggers a change in fibril organization resulting in anisotropy in the architecture of hydrogel particles
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