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 ¤1¤ Structure of G[iα1]áGppNHp, Autoinhibition in a G[α] Protein-Substrate
Complex[15]* ¤1¤

    1. [16]David E. Coleman[17]à and
    2. [18]Stephen R. Sprang[19]à[20]¤[21]¦

    1.


    From the ^¤Howard Hughes Medical Institute and the ^àDepartment of
    Biochemistry, The University of Texas Southwestern Medical Center,
    Dallas, Texas 75235-9050


   [22]Next Section

 ¤2¤ Abstract ¤2¤

   The structure of the G protein G[iα1] complexed with the
   nonhydrolyzable GTP analog guanosine-5′-(βγ-imino)triphosphate (GppNHp)
   has been determined at a resolution of 1.5 . In the active site of
   G[iα1]áGppNHp, a water molecule is hydrogen bonded to the side chain of
   Glu^43 and to an oxygen atom of the γ-phosphate group. The side chain
   of the essential catalytic residue Gln^204 assumes a conformation which
   is distinctly different from that observed in complexes with either
   guanosine 5′-O-3-thiotriphosphate or the transition state analog
   GDPáAlF[4] ^−. Hydrogen bonding and steric interactions position
   Gln^204 such that it interacts with a presumptive nucleophilic water
   molecule, but cannot interact with the pentacoordinate transition
   state. Gln^204 must be released from this auto-inhibited state to
   participate in catalysis. RGS proteins may accelerate the rate of GTP
   hydrolysis by G protein α subunits, in part, by inserting an amino acid
   side chain into the site occupied by Gln^204, thereby destabilizing the
   auto-inhibited state of Gα.

   The G[α] subunits of heterotrimeric G proteins are GTP hydrolyases
   which, upon receptor activation, bind to GTP and regulate effector
   molecules ([23]1, [24]2). G[α]-catalyzed hydrolysis of GTP to GDP
   releases the G[α]áGDP product complex from effector and allows its
   sequestration by inhibitory G[βγ]subunits. The rate of the hydrolytic
   reaction thus determines, in part, the length of time during which the
   effector is regulated by activated G proteins.

   Structures of the G[α] subunits G[tα](transducin), G[iα1], and G[sα]
   complexed with Mg^2+ and the nonhydrolyzable GTP analog GTPγS^1 have
   provided views of the active site in the ground state ([25]7-9). In
   particular, the positions of the presumptive nucleophilic water
   (W^nuc), Mg^2+, and the side chains of catalytically significant
   residues have been determined. Structures of G[iα1] and G[tα] complexed
   with GDPáAlF[4] ^−áW^nuc, a mimic of the pentavalent transition state
   for GTP hydrolysis, have also been determined ([26]8, [27]10).

   Mutagenesis experiments demonstrate that two residues of G[iα1],
   Arg^178 and Gln^204, are required for catalysis ([28]11, [29]12). In
   crystals of G[iα1]áGTPγS, which mimics the ground state EáS complex,
   the side chains of these residues are partly disordered and do not
   interact with the substrate analog. In contrast, in complexes of G[iα1]
   and its homolog G[tα] with GDPáAlF[4] ^−, both residues are well
   ordered and interact with AlF[4] ^− and its axial water ligand ([30]8,
   [31]10). It was proposed that Arg^178 and Gln^204stabilize the
   pentavalent transition state through an analogous set of interactions
   ([32]8, [33]10). It was also suggested that the low catalytic rate (k
   [cat] Å 2Ð4 min^−1) exhibited by G[iα1] ([34]14) and its homologs might
   be attributed to a high activation energy for the conformational
   rearrangement of Arg^178 and Gln^204 from the ground state to the
   transition state ([35]2). However, the nature of this rearrangement is
   not established, in part because the G[iα1]áGTPγS complex may not
   accurately mimic the true EáS complex. Specifically, the thiol
   substituent of the γ-phosphate in GTPγS may sterically perturb the
   catalytic site. The sulfur atom is both more bulky than the
   corresponding oxygen atom of GTP (van der Waals radii of 1.8 and 1.4 ,
   respectively) and has a longer bond length with the γ-phosphate atom
   (P-S, 1.86 ; P-O, 1.52 ). Hence, to obtain a more accurate view of
   the EáS ground state, we have determined a high resolution x-ray
   crystal structure of G[iα1] complexed with an alternative
   nonhydrolyzable GTP analog: GppNHp. In GppNHp all three terminal
   substituents of the γ-phosphate group are oxygen atoms.

   The active site of the G[iα1]áGppNHp complex differs from that with
   GTPγS in several respects, but most importantly in the conformation of
   the catalytic residue Gln^204. In this conformation, Gln^204 interacts
   with W^nuc, but neither contacts the nucleotide nor is positioned to
   interact with the pentavalent transition state. Thus, Gln^204 may
   participate directly in the G[iα1]áGTP ground state but must reorient
   to stabilize the transition state. RGS4, a member of the RGS family of
   G protein stimulatory factors ([36]15, [37]16), may accelerate
   hydrolysis of GTP by G[iα1], in part, by destabilizing the ground-state
   conformation of Gln^204.
   [38]Previous Section[39]Next Section

 ¤2¤ MATERIALS AND METHODS ¤2¤

   Non-myristoylated, recombinant rat G[iα1] was synthesized in E. coli
   and purified as described previously ([40]17). Crystals of G[iα1]
   complexed with GppNHp and Mg^2+ were grown and prepared for x-ray data
   collection as described except that GppNHp was substituted for GTPγS
   ([41]18). Crystals were transferred to a cryoprotection solution
   containing 15% (v/v) glycerol and frozen in liquid propane as described
   ([42]19). Two x-ray diffraction data sets were collected at 100 K, each
   from a single crystal. The first was measured at the Cornell
   synchrotron source (CHESS) A1 line (λ = 0.91 ) equipped with an ADSC
   Quantum 1 CCD detector. The crystal diffracted beyond 1.5 , but data
   were measured only to 1.7 . A second data set collected to 1.5  was
   collected at the CHESS F1 line (λ = 0.92 ) equipped with an ADSC
   Quantum 4 CCD detector. Data were processed using the DENZO/SCALEPACK
   programming package ([43]20). After simultaneous determination of the
   scale factors for both data sets using all observations, data between
   15Ð2.24  from the first data set and between 2.24Ð1.50  from the
   second were combined to obtain the final 15.00Ð1.50- data set
   (Table[44]I).
   View this table:
     * [45]In this window
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   Table I

   Data collection and final refinement statistics

   The structure of the G[iα1]áGppNHp complex was solved by molecular
   replacement, using the G[iα1]áGTPγS complex (PDB accession code
   [47]1gia) with the nucleotide, Mg^2+, and with waters removed as the
   starting model. SigmaA-weighted 2F[o] − F[c] andF[o] − F[c] difference
   maps ([48]21) indicated strong difference electron density for GppNHp,
   a previously unobserved active site water molecule, and new side chain
   positions for Glu^43 and Gln^204. Small changes in the side chain
   conformations of other residues were also observed. Simulated annealing
   omit maps ([49]22) were used to confirm that the observed differences
   were not caused by model bias. The protein model was manually rebuilt
   and ligands were added using the interactive graphics model building
   program O ([50]23). Positional and atomic temperature factor refinement
   was carried out on the rebuilt model using XPLOR ([51]24) followed by
   additional rounds of model building and refinement. In the final
   rounds, a bulk solvent correction ([52]25) was applied using XPLOR.
   Electron density about the active site is shown in Fig. [53]2. The
   free-R factor, computed using 5% of the data, was used to monitor the
   refinement ([54]26). The structure exhibits good stereochemistry, with
   94% of the residues in the most favored regions of the Ramachandran
   plot as analyzed by PROCHECK ([55]27). Comparisons and superposition of
   the model with other proteins was carried out using O. Data collection
   and refinement statistics are given in Table [56]I.
   [57]Figure 2
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   Figure 2

   The active site of G[iα1]in the (GTP analog) and (transition state
   analogáRGS4) bound complexes. Key features are labeled. A,
   G[iα1]áGppNHp; B, G[iα1]áGTPγS; C, RGS4áG[iα1]áGDPáAlF[4] ^−áH[2]O; and
   D, hypothetical RGS4áG[iα1]áGppNHp complex. The main chain segments of
   G[iα1] are coloredgreen (P-loop residues 38Ð48), blue (Switch I
   residues 178Ð184), and yellow (Switch II residues 200Ð208). The main
   chain segment of RGS4 in panels C and D is shown in red (residues
   126Ð131). The atoms and water molecules are colored as described in
   Fig. [61]1, except that the phosphorous atoms are in yellow, magnesium
   is blue, and the sulfur atom of GTPγS isgreen. In panel D, the region
   of the hypothetical model where Asn^128 of RGS4 and Gln^204 of G[iα1]
   collide is highlighted in cyan.

   The model of the RGS4áG[iα1]áGppNHp complex was made by superimposing
   the Cα atoms of residues 34Ð343 of G[iα1] from the G[iα1]áGppNHp
   complex onto the corresponding atoms of the RGS4áG[iα1]áGDPáAlF[4]
   ^−complex (PDB accession code [62]1agr). G[iα1]áGDPáAlF[4] ^− was then
   replaced by the superimposed G[iα1]áGppNHp complex. Analysis and in
   silico mutations were then performed on the resulting
   RGS4áG[iα1]áGppNHp model using O.
   [63]Previous Section[64]Next Section

 ¤2¤ RESULTS ¤2¤

   The structure of the G[iα1]áGppNHp complex (Fig.[65]2 A) differs from
   that of G[iα1]áGTPγS (Fig.[66]2 B) in two ways. First, there are small
   changes in the side-chain positions of several surface residues that
   are poorly ordered in crystals of G[iα1]áGTPγS. These differences are
   most likely because of damping of thermal motions in the frozen
   G[iα1]áGppNHp crystals. In contrast, 2.0  data from G[iα1]áGTPγS
   crystals were collected at 14 ¡C. Additionally, the G[iα1]áGppNHp data
   set was measured to 1.5 , permitting unambiguous assignment of side
   chain conformations.

   The second, and more interesting, group of changes relative to
   G[iα1]áGTPγS are observed in the active site and are most likely
   attributable to GppNHp (Fig. [67]2 A). For the most part, the active
   site is similar to that observed in the G[iα1]áGTPγS complex (Fig.
   [68]2 B). All of the interactions between the guanine nucleotide,
   Mg^2+, and the protein observed in the latter complex are also present
   in G[iα1]áGppNHp. The presumptive water nucleophile (W^nuc) is also
   clearly observed (Fig.[69]1). A new feature of the active site is a
   highly ordered (B = 21.9^2) water molecule (W^600) bound to the O1G
   oxygen of the γ-phosphate (Fig. [70]1and Fig. [71]2 A). Modeling
   indicates that this water molecule cannot bind to the G[iα1]áGTPγS
   complex because it would be sterically excluded by the γ-thiophosphate
   sulfur atom of GTPγS. The conformation of Glu^43 is altered, and its
   carboxylate moiety forms a hydrogen bond to W^600 and consequently
   cannot form the hydrogen bond to Arg^242 that is present in
   G[iα1]áGTPγS (Fig. [72]2, A and B). In G[iα1]áGppNHp, Glu^43 and
   Arg^178 form a doubly hydrogen-bonded ion pair (Fig.[73]2 A), in
   contrast to the less intimate ion pair interaction observed in the
   G[iα1]áGTPγS complex (Fig.[74]2 B). This enhanced salt-bridge may
   strengthen binding of GTP in the ground state. Remarkably, the same
   interaction occurs in crystals of G[iα1]áGDP complexed with G protein
   βγ subunits ([75]28). The conformations of Arg^178 in G[iα1]áGppNHp and
   G[iα1]áGTPγS differ slightly, but in neither case does Arg^178 interact
   with the GTP substrate analog. In contrast, in the G[tα]áGTPγS complex,
   the corresponding residue, Arg^174, interacts directly with the oxygen
   bridging the β- and γ-phosphate atoms and the γ-thiophosphate sulfur
   atom. The bridging NH group of GppNHp would not be capable of this
   interaction however.
   [76]Figure 1
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   Figure 1

   Electron density about the active site of the G[iα1]á GppNHp complex.
   The 1.5- 2F[o] − F[c] electron density map (blue) was calculated using
   SigmaA-weighted phases derived from the model, contoured at 1.5 ς. The
   model is shown as aball-and-stick representation. Red, oxygen;yellow,
   carbon; blue, nitrogen; green, phosphorous; silver, magnesium. The
   figure was generated using the program BOBSCRIPT ([80]51) and rendered
   with RASTER3D ([81]52), and POVRAY.

   The orientation of Gln^204 in the GppNHp complex is particularly
   interesting. In the G[iα1]áGTPγS complex the side chain of this
   residue, which assumes the favoredgauche + χ1 conformation, is poorly
   ordered and directed out of the active site, and does not appear to
   interact with any other residues (Fig. [82]2 B). Similar conformations
   for the corresponding catalytic glutamine (Gln^cat) are observed in the
   GTPγS-G[tα], -G[sα], and -G[sα]áadenylyl cyclase complexes ([83]7,
   [84]9, [85]29). In contrast, Gln^204 exhibits strong density for
   thegauche − χ1 conformation in the G[iα1]áGppNHp complex and makes
   several contacts with other moieties (Figs. [86]1 and [87]2 A).
   Notably, while Gln^204 does not interact directly with the substrate
   analog, its side chain amino group forms a hydrogen bond to W^nuc. The
   orientation of the amide group is defined by the functionality of the
   three other moieties to which W^nuc is hydrogen bonded: the O1G oxygen
   of the γ-phosphate group, which is proposed to act as a catalytic base
   ([88]30, [89]31), the main chain carbonyl oxygen of Thr^181, and the
   main chain NH group of Gln^204 (Fig. [90]2 A). Gln^204 is also anchored
   by a hydrogen bond between its carboxamide oxygen atom and the hydroxyl
   moiety of Ser^206.

   The thiophosphate moiety of GTPγS does not prevent Gln^204from adopting
   the conformation observed in the GppNHp complex; however, it does block
   entry of W^600 to the active site. W^600, in turn prevents Gln^204 from
   assuming the conformation that is partly populated in the GTPγS
   complex. In the GppNHp complex, the hydrogen bond between the phosphate
   O1G oxygen and W^nuc is shorter than the corresponding interaction in
   the GTPγS complex (2.82 versus 3.27 ). The position of W^nuc closer to
   the γ-phosphate permits a more favorable interaction with the side
   chain of Gln^204. The well ordered conformation of Gln^204 in the
   GppNHp complex may be attributed to hydrogen bonds formed with W^nuc
   and Ser^206 but also to the conformational restriction imposed by W^600
   as well as the cryogenic data measurement conditions. In other G
   protein-GppNHp or -GppCp complexes, Gln^cat also interacts with W^nuc
   ([91]32, [92]33). However, in these cases Gln^cat assumes the gauche +
   χ1 conformation.
   [93]Previous Section[94]Next Section

 ¤2¤ DISCUSSION ¤2¤

   The most intriguing feature of the G[iα1]áGppNHp complex is the
   conformation of the side chain of Gln^204 and its implications for the
   mechanism of both intrinsic and RGS-stimulated GTP hydrolysis. As
   established by mutagenesis studies ([95]11), Gln^204, and the
   corresponding residues in other G proteins ([96]12, [97]34, [98]35), is
   absolutely required for enzymatic activity. Studies of
   substrate-assisted catalysis using GTPase-deficient G[sα]have
   demonstrated that a hydrogen donor group near the γ-phosphate can
   substitute for Gln^cat ([99]36). However, the function of Gln^cat
   during GTP hydrolysis by G proteins has been debated. Hydrolysis of GTP
   in G proteins is believed to occur by a direct, in-line attack on the
   γ-phosphate atom by a nucleophilic water ([100]37-39). Crystal
   structures of G[iα1] and other G proteins have identified, near the
   γ-phosphate group, a well ordered water molecule (W^nuc) that is
   positioned to carry out a nucleophilic attack ([101]7-9, [102]32,
   [103]40, [104]41). Although Gln^204 is near W^nuc in G[iα1]áGTPγS, it
   does not directly contact it. Further, W^nuc is observed in
   Gln^204 → Leu G[iα1]áGTPγS, and Gln^61 → Leu RasáGppNHp (Gln^61 is
   Gln^cat in Ras) ([105]8, [106]42), indicating that Gln^catis not
   required for binding of W^nuc in the ground state. It has been pointed
   out that the basicity of glutamine is low, and it is therefore unlikely
   that Gln^cat acts as the general base that deprotonates W^nuc
   ([107]43). Rather, an oxygen of the presumably dianionic γ-phosphate is
   proposed to serve this function in Ras ([108]30, [109]31). Hence,
   Gln^cat may only polarize W^nuc in the ground state.

   X-ray crystallographic studies have indicated that Gln^catstabilizes
   the transition state ([110]2), as originally proposed by Priveet al.
   ([111]43). Structures of G[iα1] and G[tα] complexed with the transition
   state analog GDPáAlF[4] ^−, reveal Gln^catpositioned within the active
   site and directly interacting with a fluorine substituent and W^nuc of
   the GDPáAlF[4] ^−áW^nuc complex. Fig.[112]2 C shows these interactions
   in the RGS4áG[iα1]áGDPáAlF[4] ^−complex ([113]44). It was proposed that
   the amino group of Gln^cat stabilizes negative character on the
   equatorial oxygen of the transition state and its carbamoyl oxygen
   stabilizes the attacking nucleophilic water. A similar configuration is
   observed in the Ras-GAPáRasáGDPáAlF[4] ^− and the
   Rho-GAPáCdc42HsáGDPáAlF4^− complexes ([114]45-47). In addition,
   mutations that perturb the transition state conformation of Gln^cat
   abolish GTPase activity ([115]42, [116]48). These observations indicate
   that Gln^cat stabilizes the transition state for GTP hydrolysis.

   The GppNHp complex provides novel insights into both the mechanism of
   GTP hydrolysis as well as to the role of both Arg^cat and Gln^cat in
   the ground state EáS complex. GppNHp, but not GTPγS, permits a water
   molecule, W^600, to occupy a position in which it could act as the
   ultimate proton acceptor from W^nuc. Water molecules in similar, but
   not identical, positions are present in the GppNHp- or GppCp-bound
   complexes of Ras and Rac1 ([117]32, [118]40). A proton could be relayed
   from W^nuc to W^600 via O1G of the GTP γ-phosphate. This substituent
   does not otherwise participate in hydrogen bonds with the protein and
   corresponds to the thiol of GTPγS. The basicity of W^600may be enhanced
   by hydrogen bond formation with Glu^43, which is well conserved in Gα
   proteins with the exception of G[zα] where it is replaced with an
   asparagine residue. Glu^43 also forms a hydrogen-bonded ion pair with
   Arg^178. In this conformation, Arg^178 is restrained from interacting
   with the γ-phosphate of GTP. Transfer of a proton from W^nuc to W^600
   would tend to weaken this ion pair, releasing Arg^178 to stabilize the
   incipient pentacoordinate phosphoryl transition state. W^600 also
   blocks the side chain of Gln^204 from interacting with the
   pentacoordinate phosphate. Thus, until it diffuses from the active
   site, W^600 impedes the reorganization of the catalytic site that is
   required for transition state stabilization.

   Gln^204 is anchored in a noncatalytic conformation by hydrogen bonds to
   both W^nuc and Ser^206(Ser^206 is substituted by an Asp in G[αs],). In
   the ground state, Gln^204 could orient and perhaps activate W^nuc;
   however, to stabilize the transition state as represented by G protein
   GDPáAlF[4] ^−complexes, Gln^204 must sever its hydrogen bond with
   Ser^206 and W^nuc and rotate Å120¡ about χ[1] and Å90¡ about χ[2] and
   χ[3] (to gauche+ and gauche−, respectively) such that its carbamoyl
   group donates a hydrogen bond to the equatorial oxygen of the
   pentacoordinate γ-phosphoryl group and accepts a hydrogen bond from
   W^nuc. Such would incur a substantial penalty in catalytic efficiency
   and perhaps account, at least in part, for the low catalytic rate of
   GTP hydrolysis in G[α] and perhaps in other G proteins.

   We propose that the ground state G[iα1]áGTP complex is Òauto-inhibitedÓ
   with Gln^cat locked into an unproductive conformation. Active site
   residues in the EF-TuáGppNHp complex also assumes anti-catalytic
   positions; in this case His^cat, the residue corresponding to Gln^cat,
   cannot interact with the substrates because of steric interference by
   other active site residues ([119]41). In G[iα1], catalysis could occur
   only if the bonds that hold Gln^cat in this position are broken, and
   the side chain freed to interact with the pentacoordinate transition
   state. This model predicts that changes that disrupt the ground state
   conformation of Gln^204, while not otherwise compromising the active
   site, would increasek [cat].

   RGS proteins, which accelerate the rate of GTP hydrolysis by G[iα1] by
   50Ð100-fold, may act in part by destabilizing the ground state
   conformation of Gln^cat, as well as stabilizing its productive
   conformation in the transition state ([120]44). The crystal structure
   of the RGS4áG[iα1]áGDPáAlF[4] ^−complex demonstrates that RGS4
   stabilizes the active site of G[iα1] in the conformation corresponding
   to that of the transition state complex (Fig. [121]2 C) ([122]44). No
   residues from RGS4 are inserted into the active site except Asn^128,
   which could enhance catalysis by aiding in binding, orienting, and
   polarizing W^nuc in the pre-transition state complex ([123]44).

   However, superposition of G[iα1]áGppNHp and G[iα1]áGDPáAlF[4] ^− from
   the RGS4áG[iα1]áGDPáAlF[4] ^−complex reveals that the carbamoyl groups
   of Gln^204 and Asn^128 occupy nearly the same positions, although the
   side chains approach from opposite directions (Fig. [124]2 D). Further,
   both residues are positioned such that they can bind the nucleophilic
   water and Ser^206. We suggest that Asn^128 of RGS4 displaces the side
   chain of Gln^204 from its Òanti-catalyticÓ position in the ground
   state, freeing it to participate in stabilization of the transition
   state.

   Mutational analysis of RGS proteins supports this hypothesis. Mutation
   of Asn^131 in hRGSr (analogous to Asn^128 of RGS4) to either serine or
   glutamine resulted in a relatively small decrease in the k [cat] of
   G[tα] ([125]49). In addition, hRGSr in which Asn^131 was mutated to
   leucine or alanine also retains substantial stimulatory activity, and
   the loss of activity that was observed could be attributed to weakened
   binding of these mutants to G[tα]. Similar mutagenic studies have been
   performed with RGS4 ([126]50). Mutants of Asn^128analogous to those of
   hRGSr Asn^131 were modeled in the structure of the ÒRGS4áG[iα1]áGppNHpÓ
   complex. In all cases these residues were in steric conflict with
   Gln^204. These findings indicate that the bulk and binding of the
   residue at position 128 is important to the stimulatory activity of
   RGS4 although it is unlikely that it has a direct catalytic role in
   stimulation of GTPase activity ([127]49).

   The evidence presented is consistent with a self-inhibited or
   anti-catalytic model of the ground state of G[α] proteins, and a role
   for RGS proteins in stimulating GTPase activity by releasing G[α]
   subunits from this ground state while stabilizing the transition state.
   [128]Previous Section[129]Next Section

 ¤2¤ Footnotes ¤2¤

     * [130]↵* The costs of publication of this article were defrayed in
       part by the payment of page charges. The article must therefore be
       hereby marked ÒadvertisementÓ in accordance with 18 U.S.C. Section
       1734 solely to indicate this fact.
       The atomic coordinates and structure factors (code1cip) has been
       deposited in the Protein Data Bank, Brookhaven National Laboratory,
       Upton, NY.
     * [131]↵¦ To whom correspondence should be addressed: Howard Hughes
       Medical Institute, The University of Texas Southwestern Medical
       Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9050.
       Tel.: 214-648-5008; Fax: 214-648-6336; E-mail:
       Sprang{at}howie.swmed.edu.
     * Abbreviations:

        GTPγS
                guanosine 5′-O-3-thiotriphosphate

        RGS
                regulator of G protein signaling

        GppNHp
                guanosine-5′-(βγ-imino)triphosphate

        GppCHp
                guanosine-5′-(βγ-methylene)triphosphate

        W^nuc
                nucleophilic water

        Gln^cat and Arg^cat
                conserved catalytic residues in Gα subunits

     *
          + Received March 3, 1999.
     * The American Society for Biochemistry and Molecular Biology, Inc.

   [132]Previous Section

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