Intermonomer flexibility of Ca‐ and Mg‐actin filaments at different pH values

Abstract

The¯uorescence resonance energy transfer parameter, f, is de®ned as the e ciency of the energy transfer normalized by the quantum yield of the donor in the presence of acceptor. It is possible to characterize the¯exibility of the protein matrix between the appropriate¯uorescent probes by monitoring the temperature dependence of f. The intermonomer¯exi-bility of the Ca-actin and Mg-actin ®laments was characterized by using this method at pH values of 6.5 and 7.4. The protomers were labeled on Cys374 with donor [N-(((iodoacetyl)amino)ethyl)-5-naphthylamine-1-sulfonate; IAEDANS] or acceptor [5-(iodoacetamido)¯uorescein; IAF] molecules. The temperature pro®le of f suggested that the intermonomer¯exibility of actin ®laments was larger at pH 7.4 than pH 6.5 in the case of Mg-F-actin while this di erence was absent in the case of Ca-F-actin. More rigid intermonomer connection was identi®ed at both pH values between the protomers of Mg-F-actin compared to the Ca-F-actin. The results were further supported by time dependent¯uorescence measurements made on IAEDANS and IAF labeled Mg-and Ca-actin ®laments at pH 6.5 and 7.4. Our spectroscopic results may suggest that the altered function of muscle following the change of pH within the muscle cells under physiological or pathological conditions might be a ected by the modi®ed dynamic properties of the magnesium saturated actin ®laments. The change of the intracellular pH does not have an e ect on the intermonomer exibility of the Ca-actin ®laments.Keywords: muscle; cations; protein dynamics;¯uorescence resonance energy transfer;¯uorescence anisotropy decay.The dynamic properties of the actin ®lament have been widely investigated since it was discovered and named by Straub in 1942 [1]. He proposed that the actin ®lament was an active and essential part of the muscle function. A large number of experiments con®rm that the actin ®laments can change their dynamic and/or conformational properties due to the effect of the altered physico-chemical properties of the surroundings or to the effects of other proteins involved in the force generation [2±7]. The actin ®laments present in different dynamic states [6,8±12] can have an effect on the function of the acto±myosin complex through the altered¯exibility of the protein matrix [7,12], which can also have signi®cant functional effects at the cellular level [6]. The better understanding of the relationship between the¯exibility and the function of actin ®laments seems to be important for describing the molecular details of the muscle contraction under physiological and pathological conditions. The pH can change within the muscle cells under physiological and pathological conditions [13±21]. In skeletal muscle cells the pH can decrease physiologically during intense exertion. Renaud and colleagues found that the intracellular pH of the frog sartorius muscles could decrease from 7.1 to 6.5 after a fatiguing stimulation period [16]. The drop in the cytoplasmic pH to 6.12 was also observed in human muscle investigated in vivo during and...

Synthesis and hydrolysis of pppGpp in mycobacteria: A ligand mediated conformational switch in Rel

“…All values were obtained after single exponential fit. The measured τ and τ r for IAEDANS and IAF are within limits and similar values have been reported earlier [52]. The orientation factor, κ 2 was assumed to be 0.476 as the rotational correlation time (τ r ) for both donor and acceptor is more than their lifetime (τ) and therefore, the probe is not an isotropic rotor during the lifetime of the excited state [38][39][40].…”

Conformational dynamics of actin: Effectors and implications for biological function

“…It was also proved by fluorescence spectroscopy and EPR measurements that the C-terminal part of the monomer became more rigid when the bound calcium is replaced by magnesium [Nyitrai et al, 1997]. The effects of cations are highly influenced by the environmental pH [Hild et al, 2002] that can intracellularly change under physiological (e.g., fatigue) and pathological (e.g., ischemia) conditions [Mohabir et al, 1991; Thompson et al, 1992]. The interprotomer flexibility of Mg 2+ -F-actin was found to be lower than that of Ca 2+ -F-actin in the range between pH 6.5 and pH 7.4 [Hild et al, 2002], while there was no such pH induced difference in the case of Ca 2+ -F-actin.…”

“…The effects of cations are highly influenced by the environmental pH [Hild et al, 2002] that can intracellularly change under physiological (e.g., fatigue) and pathological (e.g., ischemia) conditions [Mohabir et al, 1991; Thompson et al, 1992]. The interprotomer flexibility of Mg 2+ -F-actin was found to be lower than that of Ca 2+ -F-actin in the range between pH 6.5 and pH 7.4 [Hild et al, 2002], while there was no such pH induced difference in the case of Ca 2+ -F-actin. The interprotomer connections were more rigid at both pH 6.5 and 7.4 in Mg 2+ -F-actin than in Ca 2+ -F-actin [Hild et al, 2002].…”

“…The interprotomer flexibility of Mg 2+ -F-actin was found to be lower than that of Ca 2+ -F-actin in the range between pH 6.5 and pH 7.4 [Hild et al, 2002], while there was no such pH induced difference in the case of Ca 2+ -F-actin. The interprotomer connections were more rigid at both pH 6.5 and 7.4 in Mg 2+ -F-actin than in Ca 2+ -F-actin [Hild et al, 2002]. …”

The effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin filaments

“…Actin was labeled with fluorescent probes (IAEDANS (on Cys 374 ), IAF (Cys 374 ) or FC (Gln 41 )) as described previously [8][9][10]. The labeling ratios (the ratio of the probe concentration to the actin concentration) for IAEDANS, IAF and FC were 0.9, 0.6 and 0.6, respectively.…”