Fluorescence quenching protein folding

Similarly, l-anilinonaphthalene-8-sulfonic acid (ANS) only emits in a hydrophobic environment, being almost completely quenched in aqueous solution. ANS and some other dyes, including 6-(p-toluidinyl)naphthalene-2-sulfo-nate, pyrene, l,6-diphenyl-l,3,5-hexatriene, fluorescein, and rhodamine derivatives attached to long acyl chains or to fatty acids that localize in the cellular membranes were used as probes for hydrophobic sites in proteins, protein folding, imaging of membranes of the cell, and solvent polarity. Pyrene-labeled fatty acids were used to detect the fusion of two membranes. When present in a membrane at sufficiently high concentrations, pyrene excimers (excited-state dimers) are formed that emit at 470 nm. Upon fusion with other membranes, probe concentration decreases, and excimer fluorescence is replaced by monomer fluorescence at 400 nm. This process can be monitored by ratiometric detection of pyrene labels. 

Zhuang,XW, Ha, T, Kim, HD, Centner, T, Labeit, S, and Chu,S, Fluorescence quenching A tool for single-molecule protein- folding study. Proceedings oftheNational Academy of Sciences of the United States of America 97 (2000) 14241-14244. 

The fluorescence of purified histones has been studied by several different groups, 90 95) with the most detailed studies being on calf thymus histone HI. Histone HI, which binds to the outside of core particles, contains one tyrosine and no tryptophan. This protein exhibits a substantial increase in fluorescence intensity in going from a denatured to a folded state.<90) Collisional quenching studies indicate that the tyrosine of the folded HI is in a buried environ-ment.(91) Libertini and Small(94) have identified three emissions from this residue when in the unfolded state with peaks near 300, 340, and 400 nm. The 340-nm peak was ascribed to tyrosinate (vide infra), and several possibilities were considered for the 400-nm component, including room temperature phosphorescence, emission of a charge transfer complex, or dityrosine. Dityrosine has the appropriate spectral characteristics, but would require... 

Incorporation of two different fluorescent amino acids into single proteins can expand the scope of fluorescence analysis from the simple quenching analysis as described above to a detailed study on conformational changes associated with folding processes. Fluorescence resonance energy transfer... 

Figure 2.26 Stern-Volmer plots lor quenching of fluorescence in (a) tryptophan in solution (b) tryptophan residues in a globular protein, by quencher molecule, Q. The smaller slope (/

---