Dynamics of the Active Loop of Snake Toxins As Probed by Time-Resolved Polarized Tryptophan Fluorescence

Abstract

The local environment and dynamics of the single tryptophan residue in the respective active loops of cardiotoxin and alpha-neurotoxin from Naja nigricollis and of erabutoxin b from Laticauda semifasciata have been studied by steady-state and time-resolved polarized fluorescence and analyzed with distributions of decay times. Trp11 in loop I of cardiotoxin exhibits a very broad and complex distribution of fluorescence lifetimes at 20 degrees C. Despite its relatively external location in the toxin, the residue appears to be partly shielded from water and shows restricted but significant conformational fluctuations on the picosecond and nanosecond time scales. The thermal stability of cardiotoxin allowed a study of its static and dynamic fluorescence properties over a large range of temperatures. Interconversions in the intermediate nanosecond range lead to a thorough reorganization of the cardiotoxin fluorescence lifetime distribution with temperature. On the contrary, the fluorescence kinetics of Trp29 in loop II of the two neurotoxins is dominated by about 80% of a major decay time, which suggests that a nearly unique local conformation of the toxin is maintained over all time scales above the sub-nanosecond range. The fluorescence anisotropy decays show that the residue also has extremely limited rotational freedom down to the picosecond time scale. These findings are in good agreement with structural and dynamic information previously reported on the different toxins from NMR and X-ray crystallographic studies. The different dynamic properties around the tryptophan residue of the cardiotoxin and neurotoxin active loops can be analyzed within the frame of their different respective mechanisms of toxicity.

The isolated H4‐H5 cytoplasmic loop of Na,K‐ATPase overexpressed in Escherichia coli retains its ability to bind ATP

“…Considering the excitation and emission wavelengths used, the other shorter components may include or reflect some possible contamination by tryptophan fluorescence from the protein. However, the 24 ns lifetime is well outside the range of possible fluorescence lifetimes of tryptophan, which has a reported radiative lifetime of 21 ns [15], setting a theoretical upper limit, while it never exhibits experimental fluorescence lifetimes significantly in excess of 10 ns. Therefore, this long lifetime is to be specifically ascribed to the fluorescence of the ATP analogue bound to the H 4 ‐H 5 protein.…”

“…Time sampling was 49 ps/channel and 2048 channels were used. All details of data analysis by the maximum entropy method (FAME, MEDC Ltd, UK) are given elsewhere [15]. The final lifetime distribution a(τ) is split into as many species as there are peaks separated by two well defined minima, over which the lifetime τ i and relative integrated pre‐exponential amplitude c i of each species i are computed.…”

Steady State and Time-Resolved Fluorescence Study of Residual Structures in an Unfolded Form of Yeast Phosphoglycerate Kinase

“…The correction factor, , for the differing sensitivities to polarization was determined as previously described (38). For each sample, approximately 10-20 millions counts were stored in the total fluorescence decay [I VV (t) + 2 I VH (t)] with each polarized curve being collected over 2048 channels at 23 ps/channel.…”

“…According to this description, the analysis was then performed as described in detail previously (38), except that 120 iterations were systematically performed.…”

Fluorescence quenching of tryptophan by trivalent lanthanide ions in aqueous media

“…From the photochemical standpoint it contributes to the photoreactions of proteins. Moreover, it has been widely used as a fluorescent probe for conformational change studies of enzymes and proteins (Eftink and Ghiron, 1976;Calhoun et al, 1983;Raymond, 1986;Blandin et al, 1994). A number of works have concerned the study of the interactions in the excited singlet and triplet states between tryptophan or indole derivatives and various molecules and ions, and related luminescence quenching phenomena (Fox et al, 1993;Kim et al, 1994;Eftink et al, 1994;Aaron et al, 1984;Anantharaman et al, 1983;Tine et al, 1983;1986;1989 , Tb 3 and Yb 3 ) in aqueous (pH 5.8) and buffer (HEPES pH 7.5) solutions at 298 K. The goal of the study was to investigate the usefulness and efficiency of trivalent Ln 3 ions as probes for studying the fluorescence quenching of biologically important molecules such as tryptophan, in aqueous media.…”