Single-molecule fluorescence spectroscopy

Single-molecule fluorescence spectroscopy is a powerful tool for the study of physical and biological processes through the use of fluorescent probes. By combining the femtoliter-sized observation volume of a confocal microscope with low concentrations of analytes, single fluorescent molecules can be observed as they freely diffuse in solution. From the many parameters of the fluorescence signal, a wealth of information is obtained about the structure, dynamics and interactions of the studied system. The objective of this thesis was the development, implementation and application of quantitative single-molecule fluorescence methods. To this end, a software framework for the analysis of solution-based single-molecule measurements of Förster resonance energy transfer (FRET) has been developed as part of the PAM software package. In addition, the new method of three-color photon distribution analysis (3C-PDA) is introduced in this thesis, enabling a quantitative analysis of single-molecule three-color FRET experiments. The developed analysis framework has been applied to elucidate coordinated conformational changes in the Hsp70 chaperone protein BiP, to study the conformational dynamics of a small fragment of the cellulosome, to investigate energy transfer pathways in complex artificial dye arrangements and to quantify the nanosecond dynamics of an intrinsically disordered peptide. For several studies, molecular dynamics (MD) simulations have also been used to support and cross-validate the experimental results. Here, the focus is to provide a comprehensive overview of the used methodologies, their theoretical background and their application to the various experimental systems

Single-molecule fluorescence spectroscopy

Single-molecule fluorescence spectroscopy is a powerful tool for the study of physical and biological processes through the use of fluorescent probes. By combining the femtoliter-sized observation volume of a confocal microscope with low concentrations of analytes, single fluorescent molecules can be observed as they freely diffuse in solution. From the many parameters of the fluorescence signal, a wealth of information is obtained about the structure, dynamics and interactions of the studied system. The objective of this thesis was the development, implementation and application of quantitative single-molecule fluorescence methods. To this end, a software framework for the analysis of solution-based single-molecule measurements of Förster resonance energy transfer (FRET) has been developed as part of the PAM software package. In addition, the new method of three-color photon distribution analysis (3C-PDA) is introduced in this thesis, enabling a quantitative analysis of single-molecule three-color FRET experiments. The developed analysis framework has been applied to elucidate coordinated conformational changes in the Hsp70 chaperone protein BiP, to study the conformational dynamics of a small fragment of the cellulosome, to investigate energy transfer pathways in complex artificial dye arrangements and to quantify the nanosecond dynamics of an intrinsically disordered peptide. For several studies, molecular dynamics (MD) simulations have also been used to support and cross-validate the experimental results. Here, the focus is to provide a comprehensive overview of the used methodologies, their theoretical background and their application to the various experimental systems

Cryptosporidium, Enterocytozoon, and Cyclospora Infections in Pediatric and Adult Patients with Diarrhea in Tanzania.

Cryptosporidiosis, microsporidiosis, and cyclosporiasis were studied in four groups of Tanzanian inpatients: adults with AIDS-associated diarrhea, children with chronic diarrhea (of whom 23 of 59 were positive [+] for human immunodeficiency virus [HIV]), children with acute diarrhea (of whom 15 of 55 were HIV+), and HIV control children without diarrhea. Cryptosporidium was identified in specimens from 6/86 adults, 5/59 children with chronic diarrhea (3/5, HIV+), 7/55 children with acute diarrhea (0/7, HIV+), and 0/20 control children. Among children with acute diarrhea, 7/7 with cryptosporidiosis were malnourished, compared with 10/48 without cryptosporidiosis (P < .01). Enterocytozoon was identified in specimens from 3/86 adults, 2/59 children with chronic diarrhea (1 HIV+), 0/55 children with acute diarrhea, and 4/20 control children. All four controls were underweight (P < .01). Cyclospora was identified in specimens from one adult and one child with acute diarrhea (HIV-). Thus, Cryptosporidium was the most frequent and Cyclospora the least frequent pathogen identified. Cryptosporidium and Enterocytozoon were associated with malnutrition. Asymptomatic fecal shedding of Enterocytozoon in otherwise healthy, HIV children has not been described previously

A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We test them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models

A new twist on PIFE: photoisomerisation-related fluorescence enhancement

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.Comment: No Comment