Rotational freedom of tryptophan residues in proteins and peptides

Abstract

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.

Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium.

“…Therefore, the polarization mainly refers to the internal segmental mobility of Trp. Various examples of this phenomenon have already been shown for other proteins (Lakowicz et al, 1983;Lakowicz, 1983). Therefore, the variation in flexibility shown by propylamine transferase is probably indicative of local Trp dynamics and not necessarily of the entire protein motion.…”

“…Broad transitions characterized by non-coincident midpoints can be observed. The polarization decreased to 0.10 at 80 C. A depolarizing transfer between Tyr and Trp (Lakowicz, 1983) must be ruled out because the excitation was at 295 nm. Spectroscopic measurements above 75 "C become difficult and care must be taken to detect and minimize artifacts.…”

“…'The polarization value at 25°C is very close to its maximum value, i. e. Po, the limiting polarization (Lakowicz, 1983) for this protein. In fact, polarization in a very viscous medium such as glycerol at low temperature (-10°C), where the protein rotational motion is severely restricted, exhibits very little effect (Chen et al, 1969;Lakowicz, 1983). The low polarization value measured at room temperature may be explained by considering that the irregular shape and/or the relatively high molecular mass of the protein should generate such a slow overall molecular tumbling that a protein rotational correlation time greater than the excited-state lifetime of fluorophores (Trp) can be suspected.…”

Effect of unfolding on the tryptophanyl fluorescence lifetime distribution in apomyoglobin

“…The modulation frequency was variable from 4 to 250 MHz. A solution of pterphenyl (from Kodak) in cycloexane was placed in the reference cell to correct for "color error" (Lakowicz et al, 1981). A lifetime of 1.000 ns was assigned to the reference solution.…”

Sex and age alter plasma membranes of cultured fibroblasts

“…All fluorescence lifetimes were simultaneously measured relative to a reference solution of dimethyl-p-bis[2-(5-phenyloxazolyl)]benzene (i. e. dimethyl POPOP) in absolute ethanol [42]. Multiple lifetimes were resolved by the method of Weber [43] and the PDP-8E computer.…”