Structure-based Analysis of High Pressure Adaptation of α-Actin
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m302328200
ISSN1083-351X
Autores Tópico(s)Physiological and biochemical adaptations
ResumoDeep-sea fishes occur to depths of several thousand meters, and at these abyssal depths encounter pressures that shallower living fishes cannot tolerate. Tolerance of abyssal pressures by deep-sea fish is likely to depend in part on adaptive modifications of proteins. However, the types of structural modifications to proteins that allow function at high pressure have not been discovered. To elucidate the mechanisms of protein adaptation to high pressure, we cloned the α-skeletal actin cDNAs from two abyssal Coryphaenoides species, C. armatus and C. yaquinae, and identified three amino acid substitutions, V54A or L67P, Q137K, and A155S, that distinguish these abyssal actins from orthologs of α-actin from non-abyssal Coryphaenoides. These substitutions, Q137K and A155S, prevent the dissociation reactions of ATP and Ca2+ from being influenced by high pressure. In particular, the lysine residue at position 137 results in a much smaller apparent volume change in the Ca2+ dissociation reaction. The V54A or L67P substitution reduces the volume change associated with actin polymerization and has a role in maintaining the DNase I activity of actin at high pressure. Together, these results indicate that a few amino acid substitutions in key functional positions can adaptively alter the pressure sensitivity of a protein. Deep-sea fishes occur to depths of several thousand meters, and at these abyssal depths encounter pressures that shallower living fishes cannot tolerate. Tolerance of abyssal pressures by deep-sea fish is likely to depend in part on adaptive modifications of proteins. However, the types of structural modifications to proteins that allow function at high pressure have not been discovered. To elucidate the mechanisms of protein adaptation to high pressure, we cloned the α-skeletal actin cDNAs from two abyssal Coryphaenoides species, C. armatus and C. yaquinae, and identified three amino acid substitutions, V54A or L67P, Q137K, and A155S, that distinguish these abyssal actins from orthologs of α-actin from non-abyssal Coryphaenoides. These substitutions, Q137K and A155S, prevent the dissociation reactions of ATP and Ca2+ from being influenced by high pressure. In particular, the lysine residue at position 137 results in a much smaller apparent volume change in the Ca2+ dissociation reaction. The V54A or L67P substitution reduces the volume change associated with actin polymerization and has a role in maintaining the DNase I activity of actin at high pressure. Together, these results indicate that a few amino acid substitutions in key functional positions can adaptively alter the pressure sensitivity of a protein. The deep-sea is typified by low temperature (1–4 °C), extremely high hydrostatic pressure, a lack of sunlight and a relatively low influx of utilizable organic material derived from primary production in surface waters. Among such environmental factors, hydrostatic pressure is thought to have the most influence on the vertical distribution of organisms and speciation in deep-sea (1France S.C. Mar. Biol. 1994; 118: 67-77Crossref Scopus (42) Google Scholar, 2France S.C. Kocher T.D. Mar. Biol. 1996; 126: 633-643Crossref Scopus (144) Google Scholar, 3Morita T. Mol. Phylogenet. Evol. 1999; 13: 447-454Crossref PubMed Scopus (46) Google Scholar). Hydrostatic pressure increases by approximately 0.1 megapascal (MPa) with every 10 m of depth in the ocean (4Saunders P.M. Fofonoff N.P. Deep-Sea. Res. 1976; 23: 109-111Google Scholar) and influences organism functions, especially those involving the formation of protein complexes, e.g. enzyme-substrate or protein-protein interaction (5Somero G.N. 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Zool. 2001; 289: 172-176Crossref PubMed Scopus (92) Google Scholar), and hypothetical models for protein adaptation to deep-sea pressure have been proposed (5Somero G.N. Am. Zool. 1990; 30: 123-135Crossref Scopus (19) Google Scholar); however, the primary structures of these unique proteins have not yet been determined. Marine fish belonging to the genus Coryphaenoides known as rattails or grenadiers have been studied extensively as an excellent model in which to elucidate adaptation to the deep sea (5Somero G.N. Am. Zool. 1990; 30: 123-135Crossref Scopus (19) Google Scholar, 6Gibbs A. Randall D.J. Farrell A.P. Deep-sea Fishes. Academic Press, New York1997: 239-278Google Scholar, 7Somero G.N. Annu. Rev. Physiol. 1992; 54: 557-577Crossref PubMed Scopus (278) Google Scholar, 8Siebenaller J.F. Hochachka P.W. Mommsen T.P. Biochemistry and Molecular Biology of Fish. Vol. 1. Elsevier Press, Amsterdam1991: 323-343Google Scholar, 9Siebenaller J.F. Murray T.F. Hochachka P.W. Mommsen T.P. Biochemistry and Molecular Biology of Fish. Vol. 5. Elsevier Press, Amsterdam1995: 147-174Google Scholar, 10Yancey P.H. Fyfe-Johnson A.L. Kelly R.H. Walker V.P. Aunon M.T. J. Exp. Zool. 2001; 289: 172-176Crossref PubMed Scopus (92) Google Scholar, 11Smith Jr., K.L. Nature. 1978; 274: 362-364Crossref Scopus (95) Google Scholar, 12Marshall N.B. Developments in the Deep-Sea Biology. Blandford Press, London1979Google Scholar), because of their widespread bathymetric distribution up to a depth of about 6000 m (13Iwamoto T. Stein D.L. Occas. Pap. Calif. Acad. Sci. 1974; 111: 1-79Google Scholar). An important parameter to study in the context of pressure is the change in volume that accompanies such events as protein-ligand interactions and protein-protein interactions, for the sign and magnitude of the reaction volume change determine the reaction's sensitivity to pressure. Swezey and Somero (14Swezey R.R. Somero G.N. Biochemistry. 1985; 24: 852-860Crossref PubMed Scopus (65) Google Scholar) have investigated the volume change (δV) that is associated with the polymerization of G-actin to F-actin α-skeletal actin from C. armatus (abyssal species) and C. acrolepis (non-abyssal species). The δV of actin from C. armatus was much smaller, which is advantageous for a deep-sea habitat, than that from C. acrolepis (14Swezey R.R. Somero G.N. Biochemistry. 1985; 24: 852-860Crossref PubMed Scopus (65) Google Scholar). Actin is the main component of the microfilament system in all eukaryotic cells and plays a central role in maintaining the cytoskeletal structure, cell motility, cell division, intracellular movements, and contractile processes (15Sheterline P. Clayton J. Sparrow J. Actin. 4th ed. Oxford University Press, New York1999Google Scholar, 16Pollard T.D. Blanchoin L. Mullins R.D. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 545-576Crossref PubMed Scopus (1182) Google Scholar). It is one of the most conserved proteins in eukaryotic cells, for example, α-skeletal actin proteins in carp and rat share 99.4% homology at the amino acid sequence level (17Watabe S. Hirayama Y. Imai J. Kikuchi K. Yamashita M. Fish. Sci. (Tokyo). 1995; 61: 998-1003Crossref Scopus (30) Google Scholar, 18Collins J.H. Elzinga M. J. Biol. Chem. 1975; 250: 5915-5920Abstract Full Text PDF PubMed Google Scholar). It is therefore surprising that differences in the δV of this highly conserved protein have been found between two species of Coryphaenoides that inhabit different niches. In this study, we have cloned and sequenced the α-skeletal actin cDNAs from two abyssal Coryphaenoides, C. armatus and C. yaquinae, of which C. yaquinae inhabits greater depths. These actins contain three unique amino acid substitutions compared with the previously sequenced α-skeletal actin from two non-abyssal Coryphaenoides, C. acrolepis and C. cinereus (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). Biochemical analyses of the α-actin molecules purified from the skeletal muscles of C. armatus, C. yaquinae, C. acrolepis, carp, and chicken show that these amino acid substitutions are responsible for the adaptation of α-actin to high pressures in abyssal species. Here we describe, for the first time, the mechanism of adaptation of deep-sea fishes to high pressures at the amino acid sequence level. Materials—C. acrolepis, C. armatus, and C. yaquinae were collected in large live-traps made from netting material by the R/V Soyo-maru of the National Research Institute of Fisheries Science. The sampling locations were 41–40.20′ N, 142–57.40′ E, 180 m for C. acrolepis (habitat depth, about 180–2000 m) and 44–00.70′ N, 145–22.20′ E, 3940 m for C. armatus (habitat depth, about 2700–5000 m), and 39–58.10′ N, 154–59.50′ E, 5600 m for C. yaquinae (habitat depth, about 4000–6400 m). Carp (Cyprinus carpio) and chicken samples were purchased from local stores. All samples were stored below –80 °C until use. Actin Protein—Actin was isolated from the skeletal muscle of each species according to Spudich and Watt (20Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) and purified by gel-filtration chromatography over Sephadex G-200 in G buffer (0.2 mm ATP, 0.2 mm CaCl2, 0.5 mm β-mercaptoethanol, Tris-HCl, pH 7.8). Actins were converted into the Mg2+-G form as described previously (21Chen X. Rubenstein P.A. J. Biol. Chem. 1995; 270: 11406-11414Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 22Pollard T.D. J. Cell Biol. 1986; 103: 2747-2754Crossref PubMed Scopus (601) Google Scholar). Mg2+-G-actin was used immediately after its conversion from Ca2+-G-actin. Actins were stored in G buffer at 4 °C after purification and used within 3 days. The concentration of G-actin was determined spectrophotometrically using an absorption coefficient of 0.63 ml/mg at 290 nm. Polymerization, Critical Concentration, and δV Assembly—Polymerization of Mg2+-actin was initiated by adding KCl and MgCl2 to final concentrations of 50 and 2 mm, respectively. Polymerization was monitored by light scattering, with excitation and emission wavelengths both set at 400 nm at 4 °C in a high pressure cell with a high pressure pump (PCI-400 cell and TP-500 pump Teramecs Co. Ltd., Kyoto, Japan). The change in light scattering was recorded as a function of time. The critical concentration was determined as described previously (23Chen X. Cook R.K. Rubenstein P.A. J. Cell Biol. 1993; 123: 1185-1195Crossref PubMed Scopus (88) Google Scholar, 24Tobacman L.S. Brenner S.L. Korn E.D. J. Biol. Chem. 1983; 258: 8806-8812Abstract Full Text PDF PubMed Google Scholar). The volume change (δV) in assembly of G-actin into F-actin was calculated by the method of Swezey and Somero (14Swezey R.R. Somero G.N. Biochemistry. 1985; 24: 852-860Crossref PubMed Scopus (65) Google Scholar). Isolation of α-Skeletal Actin cDNAs from C. armatus and C. yaquinae—C. armatus and C. yaquinae muscle cDNA libraries were constructed, and α-skeletal actin cDNAs from each library were cloned as described previously (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). Structures of actin were prepared using the program MOLSCRIPT (25Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) with data from the Protein Data Bank (accession number 1ATN). Phylogenetic Analysis—A molecular phylogenetic tree was constructed from the sequences of the actin coding regions. The DNADIST and NEIGHBOR programs in the PHYLIP version 3.5 program package (26Felsenstein J. Cladistics. 1986; 5: 164-166Google Scholar) were used for neighbor joining (27Saitou N. Nei M. Mol. Biol. Evol. 1987; 45: 406-425Google Scholar). Bootstrap analyses with 1000 replicates were performed to examine the confidence of nodes within the resultant topology. Molecular phylogenetic analysis showed that α-actin genes from the Coryphaenoides species were categorized into three types, actin 1, actin 2a, and actin 2b (see "Results"). The GenBank™ accession numbers of actin nucleotide sequences used in this study are give in parentheses: C. armatus 2a (AB086240), C. armatus 2b (AB086241), C. yaquinae 2a (AB086242), C. yaquinae 2b (AB086243), C. acrolepis 1 (AB021649), C. acrolepis 2a (AB021650), C. cinereus 1 (AB021651), C. cinereus 2a (AB021652); carp, C. carpio (D50025); medaka, Oryzias latipes (D87740); fugu 1, Fugu rubripes (U38850), fugu 2 (U38958); goldfish, Carassius auratus (D50029); tilapia, Oreochromis mossambicus (AB037866); zebrafish, Danio rerio (AF180887); chum salmon, Oncorhynchus keta (AB032464); Atlantic salmon, Salmo salar (AF304406); chicken, Gallus gallus (K02257); human, Homo sapiens (M20543); mouse, Mus musculus (M12234); rat, Rattus norvegicus (V01218); and bovine, Bos taurus (U02285). Medaka β-actin (D89627) was used as the outgroup gene. Quantification of Actin Isoforms—To identify the ratio of the α-actin isoforms, quantitative polymerase chain reaction with reverse transcription (RT-PCR) 1The abbreviations used are: RT, reverse transcription; δV, volume change; Quin 2, 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N, N, N′, N′-tetraacetic acid; V #, activation volume; ϵ-ATP, 1,N 6-etheno-ATP. and two-dimensional electrophoresis were performed. These analyses were repeatedly done four times using total RNAs, and actin proteins were isolated from four individuals in each species. The conditions for RT-PCR were as described previously (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar), except for the cycle number. The cycle number within a linear range of PCR amplification was determined to be 25 on the basis of the signal intensities of RT-PCR products with sequential cycles. The primer 5′-ATTGCTGACCGYATGCAGAA-3′ and NOT-1 d(T)18 primer amplified ∼480 bp and 680 bp, for actin 1 and actin 2b, respectively. RT-PCR products were subjected to 1.5% agarose gel electrophoresis. Two-dimensional electrophoresis was performed by Multiphor II electrophoresis unit with a pH range of 4.0–7.0 or 5.0–6.0 gel (24 cm) and 12.5% SDS-PAGE gel (Amersham Biosciences). The ratios of actin 2b to actin 2a, or actin 1 to actin 2a, were quantified using a computerized image analysis scanner STORM 860 (Amersham Bioscience). Quin 2 Assay—The dissociation rate constant of Ca2+ from actin was determined by assaying the fluorescence intensity increase of Quin 2 (8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N, N, N′, N′-tetraacetic acid) (28Kinosian H.J. Selden L.A. Estes J.E. Gershman L.C. J. Biol. Chem. 1993; 268: 8683-8691Abstract Full Text PDF PubMed Google Scholar). Ca2+-actin (5 μm) prepared by dialysis against G-buffer (free CaCl2) was added to 100 μm Quin 2 and 0.2 mm MgCl2, and the fluorescence was measured at respective excitation and emission wavelengths of 340 and 500 nm at 4 °C in a high pressure cell. For reaction rates, the relevant thermodynamic equation is, δln k/δP = –δV #/RT, where k is the rate constant for the reaction, P is the pressure, V # is activation volume, R is the gas constant, and T is the absolute temperature (29Morild E. Adv. Protein Chem. 1981; 34: 93-166Crossref PubMed Scopus (350) Google Scholar). The apparent binding rate constant and the apparent volume change (δV #) of the Ca2+ dissociation reaction were determined as described previously (29Morild E. Adv. Protein Chem. 1981; 34: 93-166Crossref PubMed Scopus (350) Google Scholar, 30Waechter F. Engel J. Eur. J. Biochem. 1975; 57: 453-459Crossref PubMed Scopus (55) Google Scholar). Nucleotide Exchange Assay—The apparent binding rate constant of ϵ-ATP (1,N 6-etheno-ATP) on actin was determined by displacing bound ATP with a large molar excess of ϵ-ATP such that the back reaction, ATP binding to actin, became negligible (28Kinosian H.J. Selden L.A. Estes J.E. Gershman L.C. J. Biol. Chem. 1993; 268: 8683-8691Abstract Full Text PDF PubMed Google Scholar). Actin (9 μm) prepared by dialysis against G-buffer (free ATP) was converted into Mg2+-G-actin and added to ϵ-ATP (0.1 mm), and the fluorescence was measured at respective excitation and emission wavelengths of 340 and 410 nm at 4 °C in a high pressure cell. The apparent binding constant and the apparent δV # in the ATP dissociation reaction were determined as described previously (29Morild E. Adv. Protein Chem. 1981; 34: 93-166Crossref PubMed Scopus (350) Google Scholar, 30Waechter F. Engel J. Eur. J. Biochem. 1975; 57: 453-459Crossref PubMed Scopus (55) Google Scholar). Intrinsic Tryptophan Fluorescence—The intrinsic tryptophan fluorescence spectrum of Ca2+-G-actin at 6.25 μm was recorded using a high pressure cell at respective excitation and emission wavelengths of 290 and 320–360 nm at 4 °C (31Lehrer S.S. Kerwar G. Biochemistry. 1972; 11: 1211-1217Crossref PubMed Scopus (226) Google Scholar). DNase I Inhibition Assay—The DNase I inhibition assay was performed as described previously (32Blikstad I. Markey F. Carlsson L. Persson T. Lindberg U. Cell. 1978; 15: 935-943Abstract Full Text PDF PubMed Scopus (453) Google Scholar). In brief, DNase I, either alone or combined with G-actin at various actin/DNase I ratios, was added to a control or salmon sperm DNA solution (100 mm Tris-HCl, pH 7.6, 4 mm MgSO4, 1.8 mm CaCl2), and the change in absorbance at 260 nm was recorded continuously at 20 °C in a high pressure cell. DNase I activity was calculated from the linear part of the plot of the increase in A 260 versus time. Actin Polymerization and Volume Change—We measured the polymerization kinetics of α-skeletal actin isolated from each species as a function of pressure at various actin concentrations by light scattering assay. Actins from all species except carp underwent polymerization at each pressure; carp actin polymerized only at pressures of 20 MPa or less. The half-time to steady state in polymerization and critical concentrations were determined from the polymerization kinetics (Fig. 1A). The half-times for all actins increased with high pressure, and at atmospheric pressure the polymerization half-time was shortest for carp. The half-times for chicken and non-abyssal actin species increased markedly above 20 MPa and, at 60 MPa were, respectively, about 5.6- and 7.3-fold higher than at atmospheric pressure. By contrast, the half-time of actin polymerization for the two abyssal species had increased only about 2.7-fold at 60 MPa (Fig. 1A). The critical concentrations of actin also increased with high pressure for each species (Fig. 1B). The critical concentrations of the two abyssal actin species were higher than those of other species at 20 MPa and lower pressures, and increased slightly from 0.1 to 60 MPa. The volume change (δV) associated with polymerization at each pressure was determined from the respective critical concentrations (Fig. 1C). The two abyssal actin species had a much smaller δV at each pressure than did the other species, in agreement with previous reports (14Swezey R.R. Somero G.N. Biochemistry. 1985; 24: 852-860Crossref PubMed Scopus (65) Google Scholar). The δV of actins from chicken and non-abyssal species decreased at high pressure, whereas those from the abyssal species showed little variation. These observations indicated that, for chicken and non-abyssal species, the coefficient of compressibility for F-actin was larger than that for G-actin and that the space produced by actin-actin interactions was reduced by high pressure. Clearly, there was no such reduction in space in actins from the abyssal species. Unexpectedly, the δV of carp actin, unlike that of other species, increased with high pressure, which indicated that for carp the coefficient of compressibility for G-actin was larger than that for F-actin; in other words, carp G-actin is softer than the G-actin of the other species. Thus, this would explain why carp actin was able to polymerize at pressures of only 20 MPa or less. cDNA Cloning and Deduced Amino Acid Sequences—Two α-skeletal actin isoforms were cloned from each of the two abyssal species and compared with the two non-abyssal actin species reported previously (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). Molecular phylogenic analysis showed that these four α-actin genes from the abyssal species were all categorized as actin 2. These categorizations were supported with the comparatively high bootstrap value (72%) (Fig. 2A). Therefore, the isoform with an identical amino acid sequence to that of actin 2 of the non-abyssal species was designated actin 2a, and the other was designated aemactin 2b and yaqactin 2b for C. armatus and C. yaquinae, respectively. Consequently, the non-abyssal actin 2 was re-designated actin 2a. The amino acid sequences of actin 1 and 2a differ by one amino acid residue at position 155, which is Ala-155 in actin 1 and Ser-155 in actin 2a (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). The sequence of actin 2b differs from that of actin 2a by two amino acids (either V54A or L67P and Q137K) (Table I). The x-ray crystallography structure of rabbit skeletal muscle actin shows that residues 54 and 67 are located in a β-sheet of subdomain 2 (residues 33–69), whereas residues 137 and 155 are located in the Ca2+- and ATP-binding sites, respectively (Fig. 3, A and B) (33Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1542) Google Scholar).Table IComparison of the deduced amino acid sequences of actin speciesActin typePosition235467137155165278299358Actin 1DEVLQAVALSActin 2a.....S....yaq actin 2b...PKS....aer actin 2b..A.KS....Carp.D.......TChickenED...SITMT Open table in a new tab Fig. 3Structure of actin with bound ATP and Ca2 + Blue sticks and a green ball represent ATP and Ca2+, respectively. A, ribbon drawing of actin (33Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1542) Google Scholar). The positions of substitutions found in this study are colored red. B, environment of ATP and Ca2+ in actin. Gln-137 and Ser-155 are indicated by ball-and-stick representation. The atoms carbon, oxygen, and nitrogen are colored gray, red, and purple, respectively. These images are created using MOLSCRIPT (25Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Quantification of Actin Isoforms—To identify the expression ratio of the α-actin isoforms, the expression of isoform mRNA was investigated using quantitative RT-PCR. Quantitative RT-PCR was carried out with one primer set, which amplified different length products from the two isoforms, about 480 bp for actin 1 and actin 2b, and about 680 bp for actin 2a, owing to the different lengths of their 3′ non-coding regions (Fig. 2B). Direct sequencing confirmed that the RT-PCR products were the expected actin isoforms. The results showed that there was differential expression of the two isoforms in each species. The expression ratio of actin 2b to actin 2a, or actin 1 to actin 2a, was 4.3 ± 0.18 for C. yaquinae, 4.1 ± 0.083 for C. armatus, and 0.67 ± 0.034 for C. acrolepis, respectively. We examined further the ratio of protein isoforms using two-dimensional electrophoresis. The abundance ratio of actin 2b to actin 2a was 4.8 ± 0.087 for C. yaquinae (Fig. 2C) and 4.5 ± 0.18 for C. armatus (data not shown), respectively. The protein isoforms from C. acrolepis, which have the same isoelectric point estimated, could not be separated by this electrophoresis method (data not shown). These results of the electrophoresis were not affected by dephosphorylation using Escherichia coli alkaline phosphatase (data not shown). These ratios reflect higher expression of the isoform that is more essential for the species habitat, as described below. Quin 2 and Nucleotide Exchange Assay—Sequence analysis showed that, although actin 2b from abyssal species has a lysine at position 137, the other isoform and all actins from the other species have a glutamine at this position, and all actin isoforms from abyssal species have a serine at residue 155. Actin binds ATP by sandwiching the ATP β- and γ-phosphates between two structurally equivalent β-hairpins (residues 11–18 and 154–161), which belong to homologous subdomains 1 and 3 (Fig. 3, A and B) (33Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1542) Google Scholar, 34Otterbein L.R. Graceffa P. Dominguez R. Science. 2001; 293: 708-711Crossref PubMed Scopus (413) Google Scholar). Actin also contains a tightly bound divalent cation (Ca2+ or Mg2+) in a deep hydrophilic pocket formed by the β- and γ-phosphates of the bound ATP and actin residues Asp-11, Gln-137, and Asp-154 (Fig. 3, A and B) (33Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1542) Google Scholar, 35Estes J.E. Selden L.A. Kinosian H.J. Gershman L.C. J. Muscle Res. Cell Motil. 1992; 13: 272-284Crossref PubMed Scopus (113) Google Scholar). Although actin has a higher affinity for Ca2+ than for Mg2+, the much higher cellular concentration of Mg2+ means that it will be the main occupant of the divalent cation site in vivo. We also investigated the effect of high pressures on the divalent cation and nucleotide binding of actin protein at various pressures (Fig. 4, A and B). Surprisingly, both dissociation rate constants of actin from abyssal species were much less affected by high pressures than those of the other actins, which increased rapidly at pressures greater than 20 MPa. The dissociation rate constant of Ca2+ in abyssal actins did not differ greatly from that of the other actins at atmospheric pressure, which means that the actins bind to Ca2+ with almost the same strength, whereas the differences in the dissociation rate constant of ATP suggest that Ser-155 actin binds ATP more tightly than Ala-155 actin, as pointed out previously (19Morita T. Fish. Sci. (Tokyo). 2000; 66: 1150-1157Crossref Scopus (17) Google Scholar). The apparent δV # value in the Ca2+ dissociation reaction at pressures greater than 20 MPa was estimated as –4.27 ± 1.86 cm3 mol–1 for abyssal species and –154.3 ± 0.583 cm3 mol–1 for the other species. The apparent δV # in the ATP dissociation reaction at more than 20 MPa was also determined to be –43.4 ± 0.47 cm3 mol–1 or more for abyssal actin, and as –80.4 ± 1.44, –90.0 ± 1.59, and –84.2 ± 0.995 cm3 mol–1 for chicken, carp, and non-abyssal actin, respectively. These results indicated that the smaller effect in both dissociation rate constants of pressure in abyssal actin results from Q137K and the differences in both constants among carp, chicken, and non-abyssal species from A155S. Effects of Pressure on Intrinsic Tryptophan Fluorescence Spectrum—Actin contains four tryptophan residues (at positions 74, 86, 340, and 356), which are all located in subdomain 1. The emission maximum of fluorescing tryptophan residues is 350 nm in a neutral water solution but shifts to shorter wavelengths in a hydrophobic environment, such as the interior of a folded protein (36Kuznetsova I.M. Yakusheva T.A. Turoverov K.K. FEBS Lett. 1999; 452: 205-210Crossref PubMed Scopus (40) Google Scholar). To investigate effect of pressures on the actin structure, we measured the intrinsic tryptophan fluorescence spectrum of Ca2+-G-actin at various pressures. Although the emission maximum did not shift, the fluorescence intensity of actins from chicken, carp, and non-abyssal species began to decrease at only 10 MPa (data not shown), and in particular the decrease for carp actin was larger than that for chicken and non-abyssal actin; by contrast, the fluorescence of the abyssal actins did not change even at 60 MPa (Fig. 5, A and B). Actin affinity for Ca2+ is greater than for Mg2+, but there were no differences in fluorescence intensity between the Ca2+- and Mg2+-G-actin forms for all actin species (data not shown). These results indicate that high pressure changes the environment of the tryptophan residues in actin; in other words, the structure of actin subdomain 1, which includes one of the β-hairpins that sandwiches the ATP β-and γ-phosphates and the Ca2+-binding sites. DNase I Inhibition Assay—Actin 2b of the two abyssal species contains either a V54A substitution or an L67P substitution (Table I). The x-ray crystallography structure shows that these residues are located in subdomain 2 (residues 33–69) (Fig. 3A). In actin-DNase I interactions, DNase I primarily contacts the DNase I-binding loop (residues 40–48) in subdomain 2 and interacts slightly with Thr-203 and Glu-207 in subdomain 4 (residues 181–269) (33Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1542) Google Scholar, 37Khaitlina S.Y. Moraczewska J. Strzelecka-Golaszewska H. Eur. J. Biochem. 1983; 218: 911-920Crossref Scopus (75) Google Scholar). To investigate whether these substitutions, V54A or
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