PubMed · 14461225
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
E LAAS. 1961. [Fluorescence macroscopy].. https://pubmed.ncbi.nlm.nih.gov/14461225/
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfolded-state ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through Förster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of approximately equal to 20 micros at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of approximately equal to 2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants.
The fluorescence excitation-emission matrices of dissolved organic matter (DOM) are investigated between 10 and 45 degrees C for river and waste waters and organic matter standards. With increased temperature, fluorescence intensity is quenched. It is demonstrated that for a range of river and wastewater samples, that tryptophan-like fluorescence exhibits a greater range of quenching (between 20+/-4% and 35+/-5%) than fulvic-like fluorescence (19+/-4 to 26+/-3%) over this temperature range. Humic substance standards exhibit similar fulvic-like (23+/-4%) fluorescence thermal quenching properties to river water samples (23+/-3%); however none of the samples exhibit quenching of tryptophan-like fluorescence to the same extent as the tryptophan standards (approximately 50%). Thermal fluorescence quenching is related to the exposure of the fluorophores to the heat source; our findings suggest that the tryptophan-like groups within DOM is more exposed in untreated wastewaters than in treated wastewaters riverine DOM. Thermal fluorescence properties have the potential to be used to source DOM, to provide additional chemical structural information, to temperature correct laser-induced remotely sensed DOM fluorescence, and to characterise DOM through the wastewater treatment process.
The formation of sterol and palmitoyl sphingomyelin enriched ordered domains in a fluid bilayer was examined using domain selective fluorescent reporter molecules (cholestatrienol and trans-parinaric acid containing lipids) together with a quencher molecule in the fluid phase. The aim of the study was to explore how stable the ordered domains were and how different, biologically interesting, membrane intercalators could affect domain stability and sterol distribution between domains. We show that sterols easily can be displaced from ordered domains by a variety of saturated, single- and double-chain membrane intercalators with a small polar group as a common denominator. Of the two-chain intercalators examined, both palmitoyl ceramide and palmitoyl dihydroceramide were effective in displacing sterols from ordered domains. Of the single-chain intercalators, hexadecanol and hexadecyl amide displaced the sterol from sterol/sphingomyelin domains, whereas palmitic acid, sphingosine and sphinganine failed to do so. All molecules examined stabilized the sphingomyelin-rich domains, as reported by trans-parinaric-sphingomyelin and by scanning calorimetry. Parallels between the displacement of sterol from ordered domains in our model membrane system and the ability of the above mentioned molecules to alter the chemical activity and distribution of sterols in biological membranes are discussed.