Search PubMed⌕ Search

Biomedical subjects

Victor Krasnikov

Publications and source records attributed to Victor Krasnikov.

5 recordsLinked to original sources
PubMed2006

Dual-color fluorescence-burst analysis to probe protein efflux through the mechanosensitive channel MscL.

The mechanosensitive channel protein of large conductance, MscL, from Escherichia coli has been implicated in protein efflux, but the passage of proteins through the channel has never been demonstrated. We used dual-color fluorescence-burst analysis to evaluate the efflux of fluorescent labeled compounds through MscL. The method correlates the fluctuations in intensity of fluorescent labeled membranes and encapsulated (macro)molecules (labeled with second fluorophore) for each liposome diffusing through the observation volume. The analysis provides quantitative information on the concentration of macromolecules inside the liposomes and the fraction of functional channel proteins. For MscL, reconstituted in large unilamellar vesicles, we show that insulin, bovine pancreas trypsin inhibitor, and other compounds smaller than 6.5 kDa can pass through MscL, whereas larger macromolecules cannot.

Animals↗
PubMed2006

On the decrease in lateral mobility of phospholipids by sugars.

Upon cold and drought stress, sucrose and trehalose protect membrane structures from fusion and leakage. Similarly, these sugars protect membrane proteins from inactivation during dehydration. We studied the interactions between sugars and phospholipid membranes in giant unilamellar vesicles with the fluorescent lipid analog 3,3'-dioctadecyloxacarbocyanine perchlorate incorporated. Using fluorescence correlation spectroscopy, it was found that sucrose decreased the lateral mobility of phospholipids in the fully rehydrated, liquid crystalline membrane more than other sugars did, including trehalose. To describe the nature of the difference in the interaction of phospholipids with sucrose and trehalose, atomistic molecular dynamics studies were performed. Simulations up to 100 ns showed that sucrose interacted with more phospholipid headgroups simultaneously than trehalose, resulting in a larger decrease of the lateral mobility. Using coarse-grained molecular dynamics, we show that this increase in interactions can lead to a relatively large decrease in lateral phospholipid mobility.

Carbocyanines↗
PubMed2006

The oligomeric state and stability of the mannitol transporter, EnzymeII(mtl), from Escherichia coli: a fluorescence correlation spectroscopy study.

Numerous membrane proteins function as oligomers both at the structural and functional levels. The mannitol transporter from Escherichia coli, EnzymeII(mtl), is a member of the phosphoenolpyruvate-dependent phosphotransferase system. During the transport cycle, mannitol is phosphorylated and released into the cytoplasm as mannitol-1-phosphate. Several studies have shown that EII(mtl) functions as an oligomeric species. However, the oligomerization number and stability of the oligomeric complex during different steps of the catalytic cycle, e.g., substrate binding and/or phosphorylation of the carrier, is still under discussion. In this paper, we have addressed the oligomeric state and stability of EII(mtl) using fluorescence correlation spectroscopy. A functional double-cysteine mutant was site-specifically labeled with either Alexa Fluor 488 or Alexa Fluor 633. The subunit exchange of these two batches of proteins was followed in time during different steps of the catalytic cycle. The most important conclusions are that (1) in a detergent-solubilized state, EII(mtl) is functional as a very stable dimer; (2) the stability of the complex can be manipulated by changing the intermicellar attractive forces between PEG-based detergent micelles; (3) substrate binding destabilizes the complex whereas phosphorylation increases the stability; and (4) substrate binding to the phosphorylated species partly antagonizes the stabilizing effect.

Binding Sites↗
PubMed2004

Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles.

GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain (raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) (complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.

ATP-Binding Cassette Transporters↗