Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

Abstract

Conformational and dynamic properties of actin filaments polymerized from ATP-or ADP-actin monomers were compared by using fluorescence spectroscopic methods. The fluorescence intensity of IAEDANS attached to the Cys 374 residue of actin was smaller in filaments from ADP-actin than in filaments from ATPactin monomers, which reflected a nucleotide-induced conformational difference in subdomain 1 of the monomer. Radial coordinate calculations revealed that this conformational difference did not modify the distance of Cys 374 from the longitudinal filament axis. Temperaturedependent fluorescence resonance energy transfer measurements between donor and acceptor molecules on Cys 374 of neighboring actin protomers revealed that the inter-monomer flexibility of filaments assembled from ADP-actin monomers were substantially greater than the one of filaments from ATP-actin monomers. Flexibility was reduced by phalloidin in both types of filaments.Actin is one of the most abundant proteins in biological systems and responsible for a number of cell functions in vivo (1, 2). Two principal forms of actin exist in living cells, the monomeric and the filamentous. Actins biological function depends on the actual dynamic and conformational properties of the protein and on the dynamic equilibrium between the two principal forms (1, 2).The polymerization of actin monomers with bound ATP is accompanied by the parallel hydrolysis of the nucleotide. However, the biological relevance of ATP hydrolysis by actin is still not well understood. Interestingly, ADP-monomeric actin also forms filaments (3), therefore, the presence of ATP and its hydrolysis are not essential for filament assembly. Previously, the hydrolysis of actin-bound ATP was assumed to play a key role in the steady-state treadmilling of actin filaments (4). Alternatively, Carlier (5, 6) suggested that ATP hydrolysis facilitated the rapid de-polymerization of actin.Recently, Janmey and colleagues (7) provided evidence that actin filaments polymerized from ATP-actin monomers were significantly stiffer than the ones obtained from ADP-monomers. Considering that both types of filaments consist mainly of ADP-actin protomers, this observation was explained by the assumption that the ATP-actin monomers were conformationally trapped following the hydrolysis of ATP, and the energy released during the hydrolysis was stored as elastic energy (7). The authors proposed that this elastic energy could play an important role when the actin filament interacted with actinbinding proteins. Although this exciting observation was later supported by other laboratories, a number of experimental results were contradictory.The direct effect of ATP on actin filaments was confirmed and extended to phalloidin-labeled actin by fluorescence microscopy experiments (8). Three-dimensional reconstructions from electron micrographs revealed similar effects of the nucleotides on the flexibility of actin filament (9, 10). The results of phosphorescence spectroscopic experiments apparently also supported the c...

Substrate-induced domain movement in a bifunctional protein, DcpA, regulates cyclic di-GMP turnover: Functional implications of a highly conserved motif

“…Taking these into consideration, the energy transfer efficiency between IAEDANS-IAF in the labeled protein was found to be 54 ± 3%. The "R 0 " value at 50% transfer efficiency was 42.5 Å (κ 2 = 0.48) and 45.0 Å (κ 2 = 0.67), and it is within the 1% of error of the value reported in the literature (45.5 Å) (28,29). This can happen due to various reasons like solvent and nature of protein etc.…”

New Aspects of the Spontaneous Polymerization of Actin in the Presence of Salts

“…It does not exclude more subtle but functionally important conformational differences, such as the grater flexibility of intersubunit contacts of the C-terminus in F-actin assembled from monomers of ADP-actin, as suggested by spectroscopic studies with probes placed on Cys374 of actin. 63,64 The difference in UD formation, observed here, is presumably also related to the state of the C-terminus, as the Cys374 is the primary site of PDM binding to actin. 15 These and other data for the nucleotide-dependent, 65-70 divalent-cation-dependent, 65,70-76 and ionic-strengthdependent changes in the conformations of G-actin and F-actin 26,42,44,71,72,77 suggest a role for intrastrand contacts between the C-terminus and the D-loop of subdomain 2 of actin 32,[34][35][36][37] in the reorganization of the filament structure during polymerization.…”

Nucleotide dependent differences between the α-skeletal and α-cardiac actin isoforms

“…It is well known that α-skeletal actin filaments can generate a more stable and rigid conformation when they are prepared from ATP-actin monomers compared to the filaments prepared from ADP-actin monomers [9][10][11][12]. There was previously no report about such nucleotide dependence in the case of the α-cardiac actin isoform.…”

“…The characterisation of the ADPactin revealed a change in the subdomain structure and the dynamics of the protein as well [10,11]. Increased inter-monomer flexibility and decreased stiffness were observed when the actin filaments were polymerised from ADP-actin monomers [9,12].…”

“…The characterisation of the ADPactin revealed a change in the subdomain structure and the dynamics of the protein as well [10,11]. Increased inter-monomer flexibility and decreased stiffness were observed when the actin filaments were polymerised from ADP-actin monomers [9,12].The work of some actin-binding proteins (e.g. thymosin β4, ARP 2/3 and cofilin) in the cardiac muscle tissue is strongly influenced by the quality of the actin-bound nucleotide [13][14][15].…”