Biophysics and the Challenges of Emerging Threats
Springer, May 25, 2009 - Science - 179 pages
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.
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13C-edited/filtered Acad Sci U S A active Agmon amino acid analysis antibiotics atoms base flipping base pair Bashan binding Biochem Biochemistry Biophysics blinking catalysis cCTnC chain Chem complexes conformational contacts crystal structures CVNH docking domain dye stability dynamics entropy enzyme experimental experiments factor filtered fluorescence fluorophores folding rates free energy GODCAT HMQC-NOESY HSQC hydrophobic initial lifetimes interactions kinetics labeled protein large ribosomal subunit ligand loop mechanism methods methyltransferase Mol Biol Mol Cell molecular mRNA native Natl Acad Sci NMR spectroscopy NOESY NOESY-HSQC nucleobases nucleotides observed P-site tRNA peptide bond peptide bond formation peptidyl peptidyl transferase polypeptides Proc Natl Acad protein folding protons pulse r-proteins Ramakrishnan reaction coordinate receptor reducing agents region residues Rodnina rRNA Schluenzen Science scoring functions sequence signal single-molecule single-molecule fluorescence spectroscopy Stanford University Steitz Struct studies substrate target TCEP tRNA Troponin tunnel unlabeled Yonath