The Structure and Dynamics of Partially Folded Actin

Abstract

Steady-state and time-resolved intrinsic fluorescence, fluorescence quenching by acrylamide, and surface testing by hydrophobic label ANS were used to study the structure of inactivated alpha-actin. The results are discussed together with that of earlier experiments on sedimentation, anisotropy of fluorescence, and CD spectrum in the near- and far-UV regions. A dramatic increase in ANS binding to inactivated actin in comparison with native and unfolded protein indicates that the inactivated actin has solvent-exposed hydrophobic clusters on the surface. It results in specific association of actin macromolecules (sedimentation constants for native and inactivated actin are 3 and 20 S, respectively) and, consequently, in irreversibility of native-inactivated actin transition. It was found that, though the fluorescence spectrum of inactivated actin is red-shifted, the efficiency of the acrylamide collision quenching is even lower than that of the intact protein. It suggests that tryptophan residues of inactivated actin are located in the inner region of protein formed by polar groups, which are highly packed. It correlates with the pronounced near-UV CD spectrum of inactivated actin. The experimentally found tryptophan fluorescence lifetimes allowed evaluation rotational correlation times on the basis of Perrin plots. It is found that oscillations of tryptophan residues in inactivated actin are restricted in comparison with native one. The inactivated actin properties were invariant with experimental conditions (ionic strength, the presence of reducing agents), the way of inactivation (Ca2+ and/or ATP removal, heating, 3-5 M urea or 1.5 M GdmCl treatment), and protein concentration (within the limits 0.005-1.0 mg/mL). The same state of actin appears on the refolding from the completely unfolded state. Thermodynamic stability, pronounced secondary structure, and the existing hydrophobic clusters, tested by ANS fluorescence and reversibility of transition inactivated-unfolded forms, allowed us to suggest that inactivated actin can be intermediate in the folding-unfolding pathway.

Actin as a potential target for decavanadate

“…In this state, the tryptophan residues are located in a more polar and mobile environments. It was described that the form and position of the fluorescence spectrum of inactivated actin are independent of the solution ionic strength, the presence of reducing agents, the excessive quantity of ATP and Ca 2+ , the protein concentration and the way of the inactivation [28].…”

“…It was observed that the parameter A has a value of 2.6 and 1.4 in the absence or in the presence of 1300 μM decavanadate, respectively. According to Turoverov [28], inactivated actin has the parameter A = 1.30. Therefore, it is suggested that V 10 promotes actin inactivation in the concentration range used (0-1300 μM decavanadate).…”

Partially Folded Conformations in the Folding Pathway of Bovine Carbonic Anhydrase II: A Fluorescence Spectroscopic Analysis

“…[30] Fluorescence was excited at the long-wave absorption edge where the contribution of tyrosine residues is negligible. Fluorescence intensity was recorded at 320 and 365 nm.…”

Specificity of the proteasome population secreted from cells into the culture medium

“…The endoribonuclease activity was measured as described in [2]. The proteasome peptidase activity was measured from the release of (7-amino)-4-methylcumarin using a custom-made fluorimeter [15].Proteasomes excreted into the c-medium and cellular proteasomes were characterized by comparison of their proteolytic activities. Three peptidase activities CELL BIOLOGY…”

“…The endoribonuclease activity was measured as described in [2]. The proteasome peptidase activity was measured from the release of (7-amino)-4-methylcumarin using a custom-made fluorimeter [15].…”