Biomedical subjects

E Reisler

Publications and source records attributed to E Reisler.

At least 19 recordsLinked to original sources

PubMed1997

Fluorescence probing of yeast actin subdomain 3/4 hydrophobic loop 262-274. Actin-actin and actin-myosin interactions in actin filaments.

Residues 262-274 form a loop between subdomains 3 and 4 of actin. This loop may play an important role in actin filament formation and stabilization. To assess directly the behavior of this loop, we mutated Ser265 of yeast actin to cysteine (S265C) and created another mutant (S265C/C374A) by changing Cys374 of S265C actin to alanine. These changes allowed us to attach a pyrene maleimide stoichiometrically to either Cys374 or Cys265. These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal stability of labeled or unlabeled G-actin. The presence of the loop cysteine, either labeled or unlabeled, did not affect the actin-activated S1 ATPase activity or the in vitro motility of the actin. Both mutant actins, either labeled or unlabeled, nucleated filament formation considerably faster than wild-type (WT) actin, although the critical concentration was not affected. Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C374A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was approximately 50% less than the sum of the fluorescence of the individual fluorophores. Quenching was also observed in copolymers of labeled WT and S265C/C374A actins. An excimer peak was present in the emission spectrum of labeled S265C F-actin and in the labeled S265C/C374A-WT actin copolymers. These results show that in the filaments, the C-terminal pyrene of a substantial fraction of monomers directly interacts with the loop pyrene of neighboring monomers, bringing the two cysteine sulfurs to within 18 A of one another. Finally, when bound to labeled S265C/C374A F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe. Both proteins had no effect on excimer fluorescence. These results help establish the orientation of monomers in F-actin and show that the binding of S1 to actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.

Actins↗

PubMed1997

Activation of regulated actin by SH1-modified myosin subfragment 1.

The reactive SH1 (Cys-707) group of the myosin subfragment 1 (S1) has been used frequently as an attachment site for fluorescent and spin probes in solution and muscle fiber experiments. In this study we examined (i) the motor function of SH1 spin-labeled heavy meromyosin (HMM) in the in vitro motility assays and (ii) the effect of SH1-modified S1 on the motility of regulated actin, i.e., actin complexed with tropomyosin and troponin. N-ethylmaleimide (NEM), N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-iodacetamide (IASL), N-[[(iodoacetyl)amino]ethyl]1-sulfo-5-naphthylamine (IAEDANS), and iodoacetamide (IAA) were used to selectively modify the SH1 group on S1; the SH1 group on HMM was labeled with IASL. In the in vitro motility assays, 10-20% of unregulated actin filaments moved at a speed of approximately 1 microm/s over a surface coated with 90-95% modified IASL-HMM. Actin sliding was not observed with 95-98% modified IASL-HMM. The sliding of regulated actin over unmodified HMM was activated by the addition of S1 modified with any of the SH1 reagents to the in vitro motility assay solutions; both the speeds and the percentage of the moving filaments increased at pCa 5, 7, and 8. To shed light on the activation of regulated actin sliding by SH1-modifed S1, acto-S1 ATPase and the binding to actin were determined for IASL-S1. While the binding affinities to actin were similar for IASL-S1 and unmodified S1 in the presence and absence of ADP and ATP, the Km and Vmax values were approximately 10-fold lower for the modified protein. It is concluded that the activation of regulated actin by SH1-modifed S1 facilitates the interaction of unmodified HMM heads with actin and thus can increase the sliding speeds and the percentage of regulated actin filaments that move in the in vitro motility assays.

Actins↗

PubMed1997

Effect of complexes of ADP and phosphate analogs on the conformation of the Cys707-Cys697 region of myosin subfragment 1.

Recent crystallographic studies have suggested structural differences between the complexes of S1.Mg.ADP with the phosphate analogs aluminium fluoride (AlF4-), vanadate (VO(4)3-) and beryllium fluoride (BeFx) [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960-8972; Smith, R. & Rayment, I. (1996) Biochemistry 35, 5404-5417]. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1 thiol) and Cys707-Cys697 (SH1-SH2) cross-linking, were used to compare the S1.ADP.BeFx, S1.ADP. AlF4- and S1.ADP-VO(4)3- complexes with specific states of the myosin-ATPase pathway. Modification of Cys707 with the fluorescent monofunctional reagents 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and N-iodoacetyl-N'-(5-sulfo-1-naphtyl)ethylenediamine has shown that the reactivity of the SH1 group depends on the nucleotide bound to S1. The observed rates of Cys707 modification at 20 degrees C lead to the conclusion that S1.ADP.BeFx is similar to S1*.ATP, while S1.ADP.AlF4- and S1.ADP.VO(4)3- are more similar to S1**.ADP.Pi. The conformations of the analog states were also compared by monitoring the dissociation of the fluorescent nucleotide analog 1-N6-ethenoadenosine diphosphate (ADP[C2H2]) from the active site of Cys707-modified (by N-ethylmaleimide) and Cys707-Cys697-cross-linked (by N,N'-p-phenylene dimaleimide) S1.ADP[C2H2].AlF4- and S1.ADP[C2H2]. BeFx. Our results suggest that the conformations of the S1.ADP.AlF4-, S1.ADP.VO(4)3- and S1.ADP.BeFx complexes in the Cys707-Cys697 region are distinct from each other, with the former two at least partially resembling the S1**.ADP.Pi state, while the latter is similar to the prehydrolyzed S1*.ATP state.

Adenosine Diphosphate↗

PubMed1997

Nucleotide and actin binding properties of the isolated motor domain from Dictyostelium discoideum myosin.

Nucleotide and actin binding properties of the truncated myosin head (S1dC) from Dictyostelium myosin II were studied in solution using rabbit skeletal myosin subfragment 1 as a reference material. S1dC and subfragment 1 had similar affinities for ADP analogues, epsilon ADP and TNP-ADP. The complexes of epsilon ADP and BeFx or AIF4- were less stable with S1dC than with subfragment 1. Stern-Volmer constants for acrylamide quenching of S1dC complexes with epsilon ADP, epsilon ADP.AIF4- and epsilon ADP.BeFx were 2.6, 2.9 and 2.2 M-1, respectively. The corresponding values for subfragment 1 were 2.6, 1.5 and 1.1 M-1. The environment of the nucleotide binding site was probed by using a hydrophobic fluorescent probe, PPBA. PPBA was a competitive inhibitor of S1dC Ca(2+)-ATPase (Ki = 1.6 microM). The binding of nucleotides to subfragment 1 enhanced PPBA fluorescence and caused blue shifts in the wavelength of its maximum emission in the order: ATP approximately ADP.AIF4- approximately ADP.BeFx > ATP gamma S > ADP > PPi. In the case of S1dC, the effects of different nucleotides were smaller and indistinguishable from each other. S1dC bound actin tighter than S1 (Kd = 7 nM and 60 nM, respectively). The actin activated MgATPase activity of S1dC varied between preparations, and the Vmax and K(m) values ranged between 3 and 7 s-1 and 60 and 190 microM, respectively. S1dC showed lower structural stability than S1 as revealed by their thermal inactivations at 35 degrees C. These results show that the nucleotide and actin binding of S1dC and subfragment 1 are similar but there are some differences in nucleotide and phosphate analogue-induced changes and the communication between the nucleotide and actin binding sites in these proteins.

Acrylamide↗

PubMed1996

Mutational analysis of the role of the N terminus of actin in actomyosin interactions. Comparison with other mutant actins and implications for the cross-bridge cycle.

Yeast actin mutants with acidic residues at the N terminus either neutralized (DNEQ) or deleted (delta-DSE) were used to assess the role of N-terminal acidic residues in the interactions of actin with myosin in the contractile cycle. Cosedimentation experiments revealed an approximately 3-fold decrease in the binding constant for DNEQ and delta-DSE actins to myosin subfragment-1 (S1) relative to that of wild type actin both in the presence of MgATP and in the absence of nucleotides (strong binding). DNEQ and delta-DSE actins protected S1 from tryptic digestion as well as the wild type and rabbit actins. The activation of S1 ATPase by DNEQ and delta-DSE actins (up to 50 microM) was very low but increased greatly after cross-linking these mutant actins to S1 by dimethyl suberimidate. Thus, the increased dissociation of mutant actins from S1 in the presence of ATP is the main cause for the low acto-S1 ATPase activities. At low-ionic strength conditions and in the presence of methylcellulose, the DNEQ and delta-DSE actins moved in the in vitro motility assays at a mean velocity similar to that of wild type actin (3.0 microns/s). Yet, the sliding velocity of the N-terminal and D24A/D25A and E99A/E100A mutant actins decreased relative to that of the wild type at all levels of external load introduced into the assay and at low densities of heavy meromyosin (HMM) on the cover slip. This indicates a lower relative force generation with the mutant actins. In contrast, the force generated under the same conditions with the 4Ac mutant actin (with four acidic charges at the N terminus) was higher than with wild type actin. At higher-ionic strength conditions (I = 150 mM), the sliding of the DNEQ and delta-DSE as well as that of the D24A/D25A and E99A/E100A actins ceased even in the presence of methylcellulose, while I341A actin (deficient in strong binding to myosin) still moved. These results indicate the importance of electrostatic actomyosin interactions under physiological salt conditions and show functionally distinct roles for the different myosin binding sites on actin.

Actins↗

PubMed1996

Polymerization and in vitro motility properties of yeast actin: a comparison with rabbit skeletal alpha-actin.

Actin purified from the yeast (Saccharomyces cerevisae) was polymerized faster than rabbit skeletal alpha-actin by MgCl2. The two actins polymerized at similar rates in the presence of CaCl2. Yeast actin, up to 25 microM, was not polymerized by KCl (100-300 mM); the monovalent salt also inhibited the MgCl2-induced polymerization of actin. The local structure of the subdomain-2 region in yeast actin filaments was probed by subtilisin and trypsin digestions. Loop 38-52 appeared more flexible and accessible to subtilisin in yeast than in rabbit actin. In contrast, tryptic digestions at Lys-61 and -68 occurred at the same rate for yeast and alpha-actin filaments. Modification of yeast actin by a sulfhydryl reagent CPM [7-(diethylamino)-3-(4'-maleimidophenyl)-4-methylcoumain] was specific to the Cys-374 residue; no labeling of a yeast actin mutant containing an alanine substitution for cysteine 374 was observed. The rates of Cys-374 labeling by CPM were similar for yeast and muscle actin, suggesting a similar environment for the C terminus in both polymers. In the in vitro motility assays, yeast actin required higher concentrations of heavy meromyosin (HMM) for its sliding than did the rabbit actin. At saturating concentrations of HMM, the sliding velocities of both actins were the same (3.0 microns/s). Relative forces generated by HMM with yeast and muscle actin were assessed by monitoring their in vitro motility in the presence of NEM-HMM load. The sliding of yeast actin was stopped at a level of external load (molar ratio NEM-HMM/HMM = 0.25) lower than that of muscle actin (NEM-HMM/HMM = 0.43), suggesting lower force production with yeast actin. These results are discussed in terms of the myosin cross-bridge cycle and actomyosin interactions.

Actins↗

PubMed1996

Complexes of myosin subfragment-1 with adenosine diphosphate and phosphate analogs: probes of active site and protein conformation.

Previous work has revealed phosphate-dependent differences in the complexes formed from myosin subfragment-1 with adenosine diphosphate (S1.ADP) and aluminum fluoride (AlF4-) or beryllium fluoride (BeFx) [Phan and Reisler, Biophys. J., 66 (1994) A78], with the former resembling more the S1**.ADP.Pi state while the latter resembles more the S1.ATP state. In this work, the conformations of the S1.epsilon ADP.AlF4- and S1.epsilon ADP.BeFx, complexes were examined by nucleotide chase and collisional quenching experiments. epsilon ADP release from S1.epsilon ADP.AlF4- was slower than that from S1.epsilon ADP.BeFx. However, acrylamide titrations of S1.epsilon ADP.AlF4- and S1.epsilon ADP.BeFx showed little difference in nucleotide protection from quenching between the two complexes. This contrasts with the earlier observation on phosphate analog-dependent changes in the reactivity of the SH1 group on S1. To confirm phosphate-related perturbation of the SH1-SH2 sequence, emission spectra of fluorescein (IAF)-labeled SH1 and IANBD-labeled SH2 were recorded for S1 complexes with nucleotides and phosphate analogs. Considerable differences were found between the BeFx and AlF4- complexes with S1.MgADP for both SH1- and SH2-labeled proteins. These results are consistent with a recent crystallographic study of S1 complexes with ADP and phosphate analogs [Fisher et al., Biophys. J., 68 (1995) 19S] and the idea that the opening of the nucleotide cleft on S1 does not change much during ATP hydrolysis [Franks-Skiba et al., Biochemistry, 33 (1994) 12720], while significant changes in the SH1-SH2 region accompany phosphate cleavage.

Acrylamides↗

PubMed1996

Mutational analysis of the role of hydrophobic residues in the 338-348 helix on actin in actomyosin interactions.

Yeast actin mutants with alanines replacing I341 and I345 were studied to assess the role of hydrophobic residues in the alpha-helix 338-348 in interactions with myosin. In structural models of the actomyosin complex, this helix on actin was assigned a prominent role in the strong binding of myosin to actin. Substitution of I341 with alanine reduced the strong binding of actin to myosin subfragment-1 (S1) 9-fold compared to wild-type actin. In addition, the Vmax of the actin-activated S1 ATPase was reduced 4-fold with no change in the Km. In contrast, substitution of I345 with alanine had no significant effect on either the strong binding to S1 or the actin activation of S1 ATPase. The I341A actin filaments were found to slide in the in vitro motility assays at a lower mean velocity (1.6 +/- 0.4 microns/s) than wild-type actin filaments (2.6 +/- 0.3 microns/s). Only 65% of the mutant actin filaments moved in such assays in comparison to 95% of the wild-type filaments. However, addition of 2.0 mM MgADP to the motility assay buffer induced movement of all the I341A filaments at a velocity (1.6 +/- 0.1 microns/s) similar to that of wild-type actin (1.7 +/- 0.1 microns/s). The decrease in motility of the I341A actin filaments in the absence of ADP was attributed to a negative load slowing the mutant filaments and the smaller force produced by the heavy meromyosin and I341A actin system. The latter conclusion was confirmed by showing that a greater percentage of NEM-modified heavy meromyosin (external load) was required for arresting the motion of wild-type actin in the in vitro motility assay than that needed for stopping the I341A filaments.

Actin Cytoskeleton↗

PubMed1996

The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head.

A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin.

Actins↗

PubMed1996

Intermolecular coupling between loop 38-52 and the C-terminus in actin filaments.

The recently reported structural connectivity in F-actin between the DNase I binding loop on actin (residues 38-52) and the C-terminus region was investigated by fluorescence and proteolytic digestion methods. The binding of copper to Cys-374 on F- but not G-actin quenched the fluorescence of dansyl ethylenediamine (DED) attached to Gin-41 by more than 50%. The blocking of copper binding to DED-actin by N-ethylmaleimide labeling of Cys-374 on actin abolished the fluorescence quenching. The quenching of DED-actin fluorescence was restored in copolymers (1:9) of N-ethylmaleimide-DED-actin with unlabeled actin. The quenching of DED-actin fluorescence by copper was also abolished in copolymers (1:4) of DED-actin and N-ethylmaleimide-actin. These results show intermolecular coupling between loop 38-52 and the C-terminus in F-actin. Consistent with this, the rate of subtilisin cleavage of actin at loop 38-52 was increased by the bound copper by more than 10-fold in F-actin but not in G-actin. Neither acto-myosin subfragment-1 (S1) ATPase activity nor the tryptic digestion of G-actin and F-actin at the Lys-61 and Lys-69 sites were affected by the bound copper. These observations suggest that copper binding to Cys-374 does not induce extensive changes in actin structure and that the perturbation of loop 38-52 environment results from changes in the intermolecular contacts in F-actin.

Actins↗

PubMed1996

Cross-bridge binding to actin and force generation in skinned fibers of the rabbit psoas muscle in the presence of antibody fragments against the N-terminus of actin.

To assess the significance of the NH2-terminus of actin for cross-bridge action in muscle, skinned fibers of rabbit psoas muscle were equilibrated with Fab fragments of antibodies directed against the first seven N-terminal residues of actin. With the antibody fragment, active force is more inhibited than relaxed fiber stiffness, or stiffness in rigor or in the presence of magnesium pyrophosphate. Inhibition of stiffness in rigor or with magnesium pyrophosphate does not necessarily indicate involvement of the NH2-terminus of actin in strong cross bridge binding to actin but may simply result from the large size of the Fab. At high Fab concentrations, active force is essentially abolished, whereas stiffness is still detectible under all conditions. Thus, complete inhibition of active force apparently is not due to interference with cross-bridge binding to actin but may result from the Fab-mimicking inhibition of the thin filament by Troponin-1 binding to the NH2-terminus of actin at low Ca2+. However, although Troponin-1 is released from the NH2-terminus at high Ca2+, the Fab is not, thus disallowing force generation upon increase in Ca2+. These data are consistent with involvement of the NH2-terminus of actin in both weak cross-bridge binding to actin and Ca2+ regulation of the thin filament.

Actins↗

PubMed1996

Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41.

Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin.

Actins↗

PubMed1995

A novel 27/16 kDa form of subtilisin cleaved actin: structural and functional consequences of cleavage between Ser234 and Ser235.

A new 27/16 kDa form of cleaved actin was prepared by subtilisin cleavage between Ser234 and Ser235 of F(MgADP)-actin complexed with BeFx. The cleavage had little effect on actin-actin interactions as probed in polymerization measurements and by electron microscopy. In circular dichroism melting experiments the thermostability of F-actin was reduced by about 10 degrees C by this cleavage. The in vitro motility and Vmax, but not Km, of actomyosin ATPase were decreased by about 20% upon 27/16 kDa cleavage of F-actin. The binding of tropomyosin to actin was unchanged by this modification.

Actins↗

PubMed1995

Role of charged amino acid pairs in subdomain-1 of actin in interactions with myosin.

Yeast actin mutants with alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D81, and E83/K84 were studied to assess their role in interactions with myosin. In a previous report Dictyostelium actin filaments with residues D24/D25 or E99/E100 replaced with histidines showed complete or partial loss of filament sliding in the in vitro motility assay [Johara, M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2127-2131]. In the motility experiments reported here, actin filaments with alanines substituted at D24/D25 or E99/E100 moved in the presence of 0.7% methylcellulose at velocities similar to those of wild-type yeast actin. Without methylcellulose, mutant filaments dissociated from the assay surface upon addition of ATP with little or no sliding detected. In contrast to this, filaments with alanines substituted at D80/D81 or E83/K84 were motile in the presence and absence of methylcellulose. Direct binding measurements involving cosedimentation of D24A/D25A and E99A/E100A actins with myosin subfragment-1 (S-1) in the presence of ATP revealed 3- and 2-fold decreases in their binding constants, respectively, compared to wild-type actin. In the absence of ATP all yeast actins had a similar affinity for S-1. A large decrease in the activation of S-1 ATPase was observed for both D24A/D25A and E99A/E100A actins. The D80A/D81A and E83A/K84A actin filaments showed normal S-1 binding and activation of ATPase activity. These results demonstrate the involvement of the D24/D25 and E99/E100 charged residues in the weak binding of myosin to actin and reveal that D80/D81 and E83/K84 residues in the 79-92 helix do not modulate actomyosin interactions.

Actins↗

PubMed1995

Flexation of caldesmon: effect of conformation on the properties of caldesmon.

The contribution of the extended and bent forms of caldesmon to its function was investigated by examining chemically modified forms of this protein. The bent 'hairpin' form of caldesmon was enhanced between pH 6.0 and 8.0 and at low ionic strengths, as reported by an increase in excimer fluorescence of pyrene-labelled caldesmon under these conditions. The presence of nucleotides also produced significant conformational changes in caldesmon, as detected by fluorescence measurements and protease digestions. Titrations of pyrene caldesmon with actin, heavy meromyosin, and calmodulin resulted in a decrease in excimer fluorescence. The function of the bent form of caldesmon was investigated by using intramolecular 1-ethyl-3-(3-dimethylamino propyl) carbodiimide-crosslinked caldesmon. The inhibition of acto-S-1 ATPase activity by crosslinked caldesmon was less efficient compared with that by pyrene modified and control caldesmons. Caldesmon's ability to switch from an activator to an inhibitor of actin-activated ATPase of myosin was also affected by the folding. Cosedimentation experiments revealed normal binding of crosslinked caldesmon to smooth muscle myosin. These results indicate the importance of caldesmon's transition from extended to folded forms and suggest possible functional roles for these different forms of caldesmon.

Actins↗

PubMed1995

Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41.

Gln-41 on G-actin was specifically labeled with a fluorescent probe, dansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2+ with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2+ quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCl2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.

Actins↗

PubMed1995

Actin's view of actomyosin interface.

Actomyosin interactions were examined by using yeast actin mutants with alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D81, and E83/K84. In the in vitro motility experiments, actin filaments of D24A/D25A or E99A/E100A mutants moved in the presence of 0.7% methylcellulose at the velocities of wild-type actin. Without methylcellulose, these mutant filaments, but not the D80/D81 or E83/K84 filaments, dissociated from the assay surface upon addition of ATP. Measurements of myosin subfragment-1 (S1) binding to D24A/D25A- and E99A/E100A-polymerized actins in the presence of ATP revealed a three- and twofold decrease in their binding constant, respectively, compared with wild-type actin. In contrast to this, all monomeric actins had the same binding affinity for S1. The rates and extents of polymerization of D24A/D25A and E99A/E100A actins by S1 were reduced in comparison to wild-type actin. The local structure of subdomain-2 on actin, as probed by subtilisin cleavage, was not altered for either mutant. A twofold decrease in nucleotide exchange was detected for the D24A/D25A mutant actin. These results demonstrate the involvement of the D24/D25 and E99/E100 residues in the weak binding of myosin to actin and reveal that residues D80/D81 and E83/K84 do not modulate actomyosin interactions.

Actins↗

PubMed1994

Sequence 18-29 on actin: antibody and spectroscopic probing of conformational changes.

Experimental evidence for the involvement of the 18-29 site within actin subdomain-1 in the actomyosin weak binding interface includes the inhibition of actomyosin ATPase activity by specific peptide antibodies [Adams, S., & Reisler, E. (1993) Biochemistry 32, 5051-5056] and by the Dictyostelium actin mutant D24H/D25H [Johara, M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2127-2131]. In this work, the effect of the 18-29 peptide antibodies on the polymerization and conformation of actin has been characterized. Binding of antibody to the 18-29 site strongly inhibited the MgCl2-induced polymerization of G-actin, had a much weaker impact on the CaCl2 polymerization of actin, and showed very little effect on the NaCl polymerization of G-actin. These observations were linked to the binding of the 18-29 antibody to the different forms of actin. In sedimentation assays, the (18-29) IgG bound more strongly to Mg-F- and Mg-G-actins than to Ca-F- and Ca-G-actins, respectively. The binding of IgG to F-actin decreased sharply with an increase in ionic strength. Antibody binding to the 18-29 site induced conformational changes within the nucleotide cleft, both slowing the rate of nucleotide exchange and increasing the fluorescence intensity of actin-bound epsilon ATP. The increased fluorescence of a dansyl probe attached to Gln-41 and a pyrene probe attached to Cys-374 demonstrated that antibody binding also caused local perturbations in the DNase I loop of subdomain-2 and at the C-terminus of actin. These results are discussed in terms of actin plasticity and its implications for actomyosin interactions.

Actins↗