ABSTRACT
The microtubule cytoskeleton plays an important role in eukaryotic cells, e. g., in cell movement or morphogenesis. Microtubules, formed by assembly of tubulin dimers, are dynamic polymers changing randomly between periods of growing and shortening, a property known as dynamic instability. Another process characterizing the dynamic behaviour is the so-called treadmilling due to different binding constants of tubulin at both microtubule ends. In this study, we used tetramethylrhodamine (TMR)-labeled tubulin added to microtubule suspensions to determine the net exchange rate (NER) of tubulin dimers by fluorescence correlation spectroscopy (FCS) as a measure for microtubule dynamics. This approach, which seems to be suitable as a screening system to detect compounds influencing the NER of tubulin dimers into microtubules at steady-state, showed that taxol, nocodazole, colchicine, and vinblastine affect microtubule dynamics at concentrations as low as 10(-9)-10(-10) M.
Subject(s)
Microtubules/metabolism , Spectrometry, Fluorescence/methods , Tubulin/metabolism , Animals , Colchicine/metabolism , Colchicine/pharmacology , Dimerization , Fluorescent Dyes/chemistry , Microtubules/chemistry , Microtubules/drug effects , Nocodazole/metabolism , Nocodazole/pharmacology , Paclitaxel/metabolism , Paclitaxel/pharmacology , Rhodamines/chemistry , Tubulin/chemistry , Tubulin/drug effects , Vincristine/metabolism , Vincristine/pharmacologyABSTRACT
BACKGROUND: It is known that nonnative states of protein molecules can exist in living cells and can be involved in a number of physiological processes. It has also been established that the membrane surface can be responsible for the partial denaturation of proteins due to negative charges on it. The local decrease in the effective dielectric constant of water near the organic surface has been suggested to be an additional driving force for protein denaturation in the membrane field, but data to confirm this suggestion were lacking. RESULTS: Conformational transitions induced in beta-lactoglobulin by methanol, ethanol, isopropanol, dimethylformamide and dioxane were studied by near and far UV circular dichroism, steady-state tryptophan fluorescence and fluorescence decay of 8-anilinonaphthalene-1-sulfonate (8-ANS). The existence of at least two noncoinciding cooperative transitions has been established in all solvent systems studied. The first of these transitions describes the disruption of rigid tertiary structure in protein molecules, while the second reflects the formation of an expanded helical conformation typical of proteins in concentrated organic solvents. This means that the organic solvents provoke the formation of a denatured intermediate state with pronounced secondary structure and native-like compactness. We show that the positions of maxima in fI versus dielectric constant dependence virtually coincide for all five solvent systems studied. CONCLUSIONS: The decrease in the dielectric constant of the solvent induces in beta-lactoglobulin an equilibrium intermediate state. This state, being denatured, is relatively compact and has pronounced secondary structure and high affinity for the hydrophobic fluorescent probe 8-ANS, i.e. possesses all the properties of the molten globule intermediate state.