Expression of a Nonpolymerizable Actin Mutant in Sf9 Cells

Joel, Peteranne B.; Fagnant, Patricia M.; Trybus, Kathleen M.

doi:10.1021/bi048899a

Cited by 53 publications

(59 citation statements)

References 28 publications

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“…Studying the effect of these mutations in vitro has been slowed by the lack of a high yield expression system for human actins. Our laboratory has previously expressed a nonpolymerizable cytoplasmic actin mutant from Drosophila in the baculovirus/Sf9 cell expression system (14). Here we developed a method for purification of overexpressed, untagged human ␣-cardiac actin that provides functional recombinant actin in good yield and with only trace contaminants.…”

Section: Discussionmentioning

confidence: 99%

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Functional Consequences of a Mutation in an Expressed Human α-Cardiac Actin at a Site Implicated in Familial Hypertrophic Cardiomyopathy

Bookwalter

Trybus

2006

Journal of Biological Chemistry

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Point mutations in human ␣-cardiac actin cause familial hypertrophic cardiomyopathy. Functional characterization of these actin mutants has been limited by the lack of a high level expression system for human cardiac actin. Here, wild-type (WT) human ␣-cardiac actin and a mutant E99K actin have been expressed and purified from the baculovirus/insect cell expression system. Glu-99 in subdomain 1 of actin is thought to interact with a positively charged cluster located in the lower 50-kDa domain of the myosin motor domain. Actin-activated ATPase measurements using the expressed actins and ␤-cardiac myosin showed that the mutation increased the K m for actin 4-fold (4.7 ؎ 0.7 M for WT versus 19.1 ؎ 3.0 M for the mutant), whereas the V max values were similar. The mutation slightly decreased the affinity of actin for S1 in the absence of nucleotide, which can partly be accounted for by a slower rate of association. The in vitro motility for the E99K mutant was consistently lower than WT over a range of ionic strengths, which is likely related to the lower average force supported by the mutant actin. The thermal stability of the E99K was comparable to that of WTactin, implying no folding defects. The lower density of negative charge in subdomain 1 of actin therefore weakens the actomyosin interaction sufficiently to decrease the force and motion generating capacity of E99K actin, thus providing the primary insult that ultimately leads to the disease phenotype.

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Section: Discussionmentioning

confidence: 99%

“…Our laboratory has succeeded in expressing actin in high yields in the baculovirus/Sf9 cell system (14). Here we cloned and expressed human ␣-cardiac actin (WT) and the charge reversal mutant E99K implicated in HCM, in the baculovirus expression system.…”

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confidence: 99%

Functional Consequences of a Mutation in an Expressed Human α-Cardiac Actin at a Site Implicated in Familial Hypertrophic Cardiomyopathy

Bookwalter

Trybus

2006

Journal of Biological Chemistry

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“…Infection of Sf9 cells with recombinant baculovirus encoding the AP-actin construct, lysis of the cells, and dialysis of the cell lysate have been described (8). The dialysate was fractionated on a Q-Sepharose column (1.5 ϫ 30 cm for a 4 billion cell culture preparation) using a 500-ml gradient from 0.1 to 0.4 M KCl (10 mM imidazole, pH 7.5, 0.1 mM CaCl 2 , 0.5 mM DTT, 0.5 mM Na 2 ATP, 1 g/ml leupeptin).…”

Section: Methodsmentioning

confidence: 99%

“…The dialysate was fractionated on a Q-Sepharose column (1.5 ϫ 30 cm for a 4 billion cell culture preparation) using a 500-ml gradient from 0.1 to 0.4 M KCl (10 mM imidazole, pH 7.5, 0.1 mM CaCl 2 , 0.5 mM DTT, 0.5 mM Na 2 ATP, 1 g/ml leupeptin). Pooled fractions from the Q Sepharose were concentrated with an Amicon Ultacentrifugal filter device (Millipore Corp.) and applied to a Sephacryl S300 column as described (8). Some preparations were run on a second Q-Sepharose column following the Sephacryl S300 column.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structures of Expressed Non-polymerizable Monomeric Actin in the ADP and ATP States

Rould¹,

Wan²,

Joel³

et al. 2006

J. Biol. Chem.

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166

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ATP hydrolysis drives actin filament dynamics. The effect known as treadmilling arises from the preferential addition of ATP-actin monomers to the plus or barbed end of actin and the preferential dissociation of ADP-actin from the minus or pointed end. Hydrolysis of ATP occurs after the monomer is incorporated into the filament. In addition, actin-binding proteins often have significantly different affinities for the ADPbound versus ATP-bound forms of actin. Both of these lines of evidence strongly suggest that there must be significant structural changes in actin induced by ATP hydrolysis. Indirect solution techniques such as proteolytic digestion rates and fluorescence studies (reviewed in Ref. 1) are in agreement with conformational differences between the ADP and ATP states, but direct structural evidence has been lacking until recently.Based on crystal structures of a tetramethylrhodaminelabeled monomeric actin (TMR-actin) 2 with ADP (2) or AMP-PNP (3) at the active site, Dominguez and co-workers proposed the provocative idea that ATP hydrolysis initiates a series of changes originating at the active site, that ultimately cause a loop-to-helix transition in the DNase binding loop in subdomain 2 of actin ("D-loop," residues in subdomain 2 of actin which comprise part of the DNase I binding site). They suggested that this was the long sought after change in structure between ADP and ATP actin. The cleft between subdomains 2 and 4 of actin remained closed in both nucleotide states. Despite this observation, an opposing point of view (4) held that the more important conformational change is an opening of the cleft upon ATP hydrolysis, a change that would be compatible with that observed for other nucleotide hydrolyzing proteins. Docking of actin crystal structures into negatively stained images of F-actin was also consistent with the idea that ADPactin had a more open cleft than the triphosphate state (5). If the latter view is correct, one would have to suppose that the modification of Cys 374 by TMR stabilized the closed conformation and inactivated the nucleotide sensing mechanism by virtue of the binding of rhodamine between subdomains 1 and 3, a potential hinge region (6) of the molecule. It has been suggested (4) that the short helix observed in the crystal structure of the ADP state of TMR-actin could be formed as a result of contacts with neighboring molecules in the crystal. We provide further evidence to support this idea, and suggest a pathway through which nucleotide-dependent changes may propagate through fortuitous crystal packing interactions from one monomer to the D-loop region of another in the TMR-actin crystals.Here we crystallized and expressed a cytoplasmic actin that was rendered incapable of polymerization by virtue of two surface mutations in subdomain 4 (A204E/P243K). This strategy negates concerns raised about the TMR modification of actin.De novo crystallization of AP-actin with either ATP or ADP at the active site reveals obligatory nucleotide-dependent confor-* This work was suppor...

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“…. Actin residues 204 and 247 are expected to be part of the subunit-subunit interface in F-actin, as mutation of these residues results in loss of polymerization 35 . The overall structural conservation between actin and ParM does not extend to this region, consistent with different filamentous interfaces.…”

Section: Supplementary Materialsmentioning

confidence: 99%

The structure of bacterial ParM filaments

Orlova

Garner

Galkin

et al. 2007

Nat Struct Mol Biol

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Bacterial ParM is a homolog of eukaryotic actin and is involved in moving plasmids so that they segregate properly during cell division. Using cryo-EM and three-dimensional reconstruction, we show that ParM filaments have a different structure from F-actin, with very different subunitsubunit interfaces. These interfaces result in the helical handedness of the ParM filament being opposite to that of F-actin. Like F-actin, ParM filaments have a variable twist, and we show that this involves domain-domain rotations within the ParM subunit. The present results yield new insights into polymorphisms within F-actin, as well as the evolution of polymer families.ParM is a bacterial actin homolog involved in plasmid segregation 1,2 . The structures of both ParM and MreB 3 , another bacterial actin homolog, as well as FtsZ 4 , a tubulin homolog, show that the eukaryotic cytoskeleton is similar to a prokaryotic cytoskeleton that has only recently been studied. Although the higher-order structure of FtsZ is still unknown, it has been assumed that both ParM and MreB assemble into filaments with an arrangement of subunits very similar to that found in F-actin. However, a crystal structure of monomeric ParM has unexpectedly shown that its main differences from actin are in regions expected to be involved in the filamentous subunit-subunit interface 2 . A previous study 2 has used EM of negatively stained ParM filaments to generate a low-resolution three-dimensional reconstruction, which suggests that the ParM filament is very similar to F-actin.We have used negative staining, cryo-EM and quick-freeze/deep-etch EM to examine filaments formed by the ParM protein from Escherichia coli. The question that we intended to answer was how similar to F-actin the ParM filaments appear at higher resolution. We show that the ParM filaments are substantially different from F-actin, with a very different subunit-subunit interface. Because of the very different interface, the helical handedness of the ParM filaments is opposite to that of F-actin. RESULTS Variable twist in ParM filamentsEM shows that ParM forms long filaments when incubated with the nonhydrolyzable ATP analog AMP-PNP (Fig. 1). We analyzed both negatively stained (Fig. 1a) and frozenhydrated ParM filaments (Fig. 1b), but took advantage of the iterative helical real-space reconstruction (IHRSR) method 5 to surmount the problems posed by flexible and disordered filaments. In this method, rather than individual filaments being treated as ideal helices with a uniform structure, short segments are analyzed, classified and reconstructed separately. One of the most obvious forms of disorder revealed by such a method is the presence of variable twist in the ParM filaments, even greater than what has been shown for F-actin 6-8 . The variability in average twist angles found in unstained frozen-hydrated ParM segments (28,886 segments, each segment ~480 Å long) is shown in Figure 2a. This variability arises from different segments within the same filaments, rather than different ...

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Expression of a Nonpolymerizable Actin Mutant in Sf9 Cells

Cited by 53 publications

References 28 publications

Functional Consequences of a Mutation in an Expressed Human α-Cardiac Actin at a Site Implicated in Familial Hypertrophic Cardiomyopathy

Functional Consequences of a Mutation in an Expressed Human α-Cardiac Actin at a Site Implicated in Familial Hypertrophic Cardiomyopathy

Crystal Structures of Expressed Non-polymerizable Monomeric Actin in the ADP and ATP States

The structure of bacterial ParM filaments

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