Search results for "Fluorescence Correlation Spectroscopy"
showing 10 items of 29 documents
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…
Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles
2005
Although sharing a T = 1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence cor…
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…
Assembly of fluorescent chimeric virus-like particles of canine parvovirus in insect cells
2004
Canine parvovirus (CPV) is a small non-enveloped ssDNA virus composed of the viral proteins VP1, VP2, and VP3 with a T=1 icosahedral symmetry. VP2 is nested in VP1 and the two proteins are produced by differential splicing of a primary transcript of the right ORF of the viral genome. The VP2 protein can be further proteolytically cleaved to form VP3. Previous studies have shown that VP1 and VP3 are unnecessary for capsid formation and consequently, that VP2 alone is sufficient for assembly. We have hypothesized that insertion of the enhanced green fluorescent protein (EGFP) at the N-terminus of VP2 could be carried out without altering assembly. To investigate the possibility to develop flu…
Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy.
2003
Abstract Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89±0.74)10 8 cm2s 1 and an apparent hydrodynamic radius of 83.35±21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.5710 7 cm2s 1), showing that the fusion proteins were anchored in the viral envelope…
Diffusion of single molecular and macromolecular probes during the free radical bulk polymerization of MMA : towards a better understanding of the Tr…
2016
Free radical bulk polymerizations exhibit complex kinetics due to the viscosity increase during the polymerization process. Especially the termination rate constant can be strongly influenced by the mobility of polymer chains in the polymerization mixture. As a consequence an autoacceleration period, the so-called Trommsdorff effect, can be observed often. In order to investigate this behaviour on a nanoscopic scale, we directly visualized the mobility of molecules and macromolecules in polymerizing MMA solutions using a combination of highly sensitive fluorescence correlation spectroscopy and widefield fluorescence microscopy. For this purpose, rather monodisperse PMMA chains were synthesi…
HPMA-Based Nanoparticles for Fast, Bioorthogonal iEDDA Ligation
2019
Contains fulltext : 216143.pdf (Publisher’s version ) (Open Access) Fast and bioorthogonally reacting nanoparticles are attractive tools for biomedical applications such as tumor pretargeting. In this study, we designed an amphiphilic block copolymer system based on HPMA using different strategies to introduce the highly reactive click units 1,2,4,5-tetrazines (Tz) either at the chain end (Tz-CTA) or statistical into the hydrophobic block. This reactive group undergoes a rapid, bioorthogonal inverse electron-demand Diels-Alder reaction (iEDDA) with trans-cyclooctenes (TCO). Subsequently, this polymer platform was used for the preparation of different Tz-covered nanoparticles, such as micell…
Aggregation behavior of amphiphilic p(HPMA)-co-p(LMA) copolymers studied by FCS and EPR spectroscopy.
2012
A combined study of fluorescence correlation spectroscopy and electron paramagnetic resonance spectroscopy gave a unique picture of p(HPMA)-co-p(LMA) copolymers in aqueous solutions, ranging from the size of micelles and aggregates to the composition of the interior of these self-assembled systems. P(HPMA)-co-p(LMA) copolymers have shown high potential as brain drug delivery systems, and a detailed study of their physicochemical properties can help to elucidate their mechanism of action. Applying two complementary techniques, we found that the self-assembly behavior as well as the strength of hydrophobic attraction of the amphiphilic copolymers can be tuned by the hydrophobic LMA content or…
Toward oxygen binding curves of single respiratory proteins
2004
Oxygen binding curves of single molecules promise to discriminate between different models describing cooperativity because load distributions are accessible. Individual tarantula hemocyanins could be detected by fluorescence correlation spectroscopy using intrinsic tryptophan fluorescence as sensor of bound oxygen. However, imaging of immobilized proteins was not possible due to fast photo-bleaching. It is shown that tetra-methyl-carboxy-rhodamine (TAMRA), commonly used as a fluorescence label in single-molecule spectroscopy, can also be applied to monitor bound oxygen. The dye's fluorescence is quenched due to Förster energy transfer to the oxygenated active sites of hemocyanin.
Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling.
2010
This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNA(Phe). LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I…