Biophysics and the Challenges of Emerging Threats

Single-molecule techniques eliminate ensemble averaging, thus revealing transient or rare species in heterogeneous systems [1-3]. These approaches have been employed to probe myriad biological phenomena, including protein and RNA folding [4-6], enzyme kinetics [7, 8], and even protein biosynthesis [1, 9, 10]. In particular, immobilization-based fluorescence te- niques such as total internal reflection fluorescence microscopy (TIRF-M) have recently allowed for the observation of multiple events on the millis- onds to seconds timescale [11-13]. Single-molecule fluorescence methods are challenged by the instability of single fluorophores. The organic fluorophores commonly employed in single-molecule studies of biological systems display fast photobleaching, intensity fluctuations on the millisecond timescale (blinking), or both. These phenomena limit observation time and complicate the interpretation of fl- rescence fluctuations [14, 15]. Molecular oxygen (O) modulates dye stability. Triplet O efficiently 2 2 quenches dye triplet states responsible for blinking. This results in the for- tion of singlet oxygen [16-18]. Singlet O reacts efficiently with organic dyes, 2 amino acids, and nucleobases [19, 20]. Oxidized dyes are no longer fluor- cent; oxidative damage impairs the folding and function of biomolecules. In the presence of saturating dissolved O , blinking of fluorescent dyes is sup- 2 pressed, but oxidative damage to dyes and biomolecules is rapid. Enzymatic O -scavenging systems are commonly employed to ameliorate dye instability. 2 Small molecules are often employed to suppress blinking at low O levels.

Proceedings of the NATO Advanced Study Institute on Biophysics and the Challenges of Emerging ThreatsErice, Sicily, Italy19-30 June 2007

Presents state of the art information on NMR spectroscopy, and its place in the broader field of biophysics

A Simple Model for Protein Folding.- Complementarity of Hydrophobic/Hydrophilic Properties In Protein-Ligand Complexes: A New Tool to Improve Docking Results.- Structures of Cvnh Family Lectins.- Biophysical Approaches To Study Dna Base Flipping.- The Diversity of Nuclear Magnetic Resonance Spectroscopy.- Improved Dye Stability in Single-Molecule Fluorescence Experiments.- The Evaluation of Isotope Editing and Filtering for Protein-Ligand Interaction Elucidation by Nmr.- Ribosome: an Ancient Cellular Nano-Machine for Genetic Code Translation.