Rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. A steady-state and time-resolved anisotropy study

Abstract

The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2 and its zymogen (prophospholipase A2) have been studied by polarized fluorescence using steady-state and time-resolved single-photon counting techniques. The motion of Trp-3 in phospholipase A2 consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about +/- 20 degrees accompanied by slower isotropic rotation of the entire protein. The rotational correlation times for overall particle rotational diffusion are consistent with conventional hydrodynamic theory. When phospholipase A2 binds to micelles of n-hexadecylphosphocholine, the amplitude of the fast ring rotation decreases. The whole particle rotational correlation time of the enzyme/micelle complex is smaller than the minimum value calculated from hydrodynamic theory. A similar result is obtained for the micelle itself by using the lipophilic probe transparinaric acid. These low values for the particle correlation times can be understood by postulating that an isotropic motion of the fluorophore in the small detergent particles contributes to the angular reorientation of the fluorophore. The internal reorientational motion of the tryptophan in the zymogen, prophospholipase A2, is of larger amplitude than that observed for the enzyme; specifically, the proenzyme exhibits a motion with a significant amplitude on the nanosecond time scale. This additional freedom of motion is attributed to segmental mobility of the N-terminal residues of prophospholipase A2. This demonstrates that this region of the protein is flexible in the zymogen but not in the processed enzyme. The implications of these findings for the mechanism of surface activation of phospholipase A2 are discussed by analogy with a trypsinogen-trypsin activation model.

Molecular interaction and functional regulation of connexin50 gap junctions by calmodulin

“…We first examined the dansyl fluorescence anisotropy changes to confirm the formation of complex in the low-micromolar range. Fluorescence anisotropy is dependent on the rotational correlation time of fluorophore in the sample, and is often used to reflect the hydrodynamic properties of biomolecules [45]. The increase in anisotropy often arises from slower tumbling of the fluorophore and thus reports the formation of a larger complex.…”

Native state dynamics affects the folding transition of porcine pancreatic phospholipase A2

“…Numerous studies within the context of interfacial enzyme kinetics and activation have shown such native state heterogeneity for ppPLA 2 and the prominent role of its N-terminus therein [64][65][66][67]. The involvement of Trp3 in interfacial activation was confirmed by fluorescence measurements [68,69]. Likewise, the four N-terminal residues of ppPLA 2 were found to be unstructured in the solution structure, but were "immobilized" by micelle binding [19,21].…”

Studies of tryptophan fluorescence using bacteriophage T4 lysozyme

“…(Reviews of the literature concerning this topic are given in [1,2,8,10,13,15,17].) The decay of the fluorescence of proteins can usually be represented as a sum of two or three exponential components.…”

“…There is one qualitative observation that seems to be relevant to this issue and this suggests a connection between quenching and kinetic complexity. We have found [1,8,17] that in quite a few cases (not restricted to T4 lysozyme), the fluorescence decay complexity is greater for those cases where the quantum yield is low. This suggests that there is a connection between collisional quenching and fluorescence decay complexity.…”