Search results for "Fluorescence Correlation Spectroscopy"
showing 9 items of 29 documents
Synthesis, Characterization and Preliminary Biological Evaluation of P(HPMA)-b-P(LLA) Copolymers: A New Type of Functional Biocompatible Block Copoly…
2010
We describe a synthetic pathway to functional P(HPMA)-b-P(LLA) block copolymers. The synthesis relies on a combination of ring-opening polymerization of L-lactide, conversion into a chain transfer agent (CTA) for the RAFT polymerization of pentafluorophenyl methacrylate. A series of block copolymers was prepared that exhibited molecular weights $\overline M _{\rm n}$ ranging from 7 600 to 34 300 g · mol(-1) , with moderate PDI between 1.3 and 1.45. These reactive precursor polymers have been transformed into biocompatible P(HPMA)-b-P(LLA) copolymers and their fluorescently labeled derivatives by facile replacement of the pentafluorophenyl groups. The fluorescence label attached to this new …
Fluorescence Correlation Spectroscopy in Dilute Polymer Solutions: Effects of Molar Mass Dispersity and the Type of Fluorescent Labeling
2015
Fluorescence correlation spectroscopy (FCS) has become an important tool in polymer science. Among various other applications the method is often applied to measure the hydrodynamic radius and the degree of fluorescent labeling of polymers in dilute solutions. Here we show that such measurements can be strongly affected by the molar mass dispersity of the studied polymers and the way of labeling. As model systems we used polystyrene and poly(methyl methacrylate) synthesized by atom transfer radical polymerization or free-radical polymerization. Thus, the polymers were either end-labeled bearing one fluorophore per chain or side-labeled with a number of fluorophores per chain proportional to…
Disassembly of structurally modified viral nanoparticles: characterization by fluorescence correlation spectroscopy.
2005
Abstract Analysis of the breakdown products of engineered viral particles can give useful information on the particle structure. We used various methods to breakdown both a recombinant enveloped virus and virus-like particles (VLPs) from two non-enveloped viruses and analysed the resulting subunits by fluorescence correlation spectroscopy (FCS). Analysis of the enveloped baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), displaying the green fluorescent protein (GFP) fused to its envelope protein gp64 was performed in the presence and absence of 5 mM SDS and 25 mM DTT. Without treatment, the viral particle showed a diffusion time of 3.3 ms. In the presence of SDS…
FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.
2021
International audience; Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever- increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among d…
Modeling of Particle Number Fluctuations in Entire Cells
2012
In a recent study we developed a method to model protein diffusion in cells [1], where special attention was given to generating from image data of the measured cell a realistic digital model cell in which protein dynamics were simulated. The method was shown to be well suited for modeling non-equilibrium situations that arise, e.g., in photobleaching experiments, and to be capable of producing more detailed information about protein motion than traditional modeling.Another experimental way to assess protein dynamics is to study fluctuations in the local protein number, as it is done, e.g., in fluorescence correlation spectroscopy (FCS), or in similar measurements that apply single-plane il…
In Vitro–In Vivo Fluctuation Spectroscopies
2010
Fluorescence correlation spectroscopy (FCS) was first developed for biophysical studies in analogy with photon scattering correlation spectroscopy. Although it is mainly devoted to the study of freely diffusing particles, FCS is actually able to discern between different kinds of motions, such as diffusion, anomalous diffusion, or drift motions. The frontier application of FCS nowadays is in medical studies both within cells and on the cell membranes, and in the investigation of single molecules in solid matrices. In this field, FCS originated also image correlation spectroscopy methods. The whole field can be unified under the name of fluorescence fluctuation spectroscopy (FFS). We present…
Conformational Dynamics of the Dengue Virus Protease Revealed by Fluorescence Correlation and Single-Molecule FRET Studies.
2021
The dengue virus protease (DENV-PR) represents an attractive target for counteracting DENV infections. It is generally assumed that DENV-PR can exist in an open and a closed conformation and that active site directed ligands stabilize the closed state. While crystal structures of both the open and the closed conformation were successfully resolved, information about the prevalence of these conformations in solution remains elusive. Herein, we address the question of whether there is an equilibrium between different conformations in solution which can be influenced by addition of a competitive inhibitor. To this end, DENV-PR was statistically labeled by two dye molecules constituting a FRET …
Fluorescence Decay Time of Single Semiconductor Nanocrystals
2002
We present fluorescence decay measurements of single ZnS covered CdSe nanocrystals. It is shown that the fluorescence decay time is fluctuating during the investigation leading to a multiexponential decay even for a single nanocrystal. In combination with measurements of the fluorescence blinking behavior we find that a high fluorescence intensity is correlated with a long fluorescence decay time. This is consistent with a model of fluctuating nonradiative decay channels leading to variable dynamic quenching processes of the excited state.
Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes
2021
AbstractRhodesain is the lysosomal cathepsin L-like cysteine protease ofT. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression ofT. brucei rhodesiensepro-rhodesain inE. coliand determined its crystal structure. The trypanosomal pr…