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Dissecting interdomain communication within cAPK regulatory subunit type IIß using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS)

¹ Department of Chemistry and Biochemistry,
² Howard Hughes Medical Institute, and
³ Department of Medicine, University of California, San Diego, La Jolla, California 92093, USA
⁴ Sierra Analytics, LLC, Modesto, California 95355, USA

Reprint requests to: Virgil L. Woods Jr., Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; e-mail: vwoods{at}ucsd.edu; fax: (858) 534-2180.

(RECEIVED April 27, 2003; FINAL REVISION May 29, 2003; ACCEPTED May 30, 2003)

⁵ Present address: ExSAR Corporation, 11 Deer Park Drive, Suite 103, Monmouth Junction, NJ 08852, USA.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03166903.

	Abstract

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material Competing interests References

 
cAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively). Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RIIß subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain.

Keywords: cAPK; PKA; regulatory subunit; amide hydrogen exchange; mass spectrometry

Abbreviations: cAB-A, cAMP-binding domain A • cAB-B, cAMP-binding domain B • cAMP, adenosine 3',5'-cyclic monophosphate • cAPK, cAMP-dependent protein kinase • cGMP, guanosine 3',5'-cyclic monophosphate • C-subunit, catalytic subunit • DD, dimerization/docking domain • DTT, dithiothreitol • EDTA, ethylenediaminetetraacetic acid • EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid • GuHCl, guanidine hydrochloride • MES, 2-(N-morpholino) ethanesulfonic acid • MOPS, 3-(N-morpholino) propanesulfonic acid • NaCl, sodium chloride • PBC, phosphate binding cassette • R-subunit, regulatory subunit • TFA, trifluoroacetic acid • DXMS, enhanced hydrogen/deuterium exchange mass spectrometry

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material Competing interests References

 
cAMP-dependent protein kinase (cAPK), a kinase involved in the regulation of many processes, is down-regulated by binding to its regulatory (R) subunit to form an inactive heterotetrameric complex. This complex consists of two catalytic (C) subunits bound to an R-subunit dimer. The enzyme is activated when four cAMP molecules bind allosterically to the R-subunits, causing dissociation of the holoenzyme and the release of active C-subunits (Hofmann et al. 1975; Rosen and Erlichman 1975). Four R-subunit isoforms have been identified (RI, RIß, RII, and RIIß) and differ by autophosphorylation (Rosen and Erlichman 1975), disulfide bonding (Zick and Taylor 1982; Bubis et al. 1987), molecular weight (Zoller et al. 1979), cellular localization (Rubin 1979), tissue distribution (Jahnsen et al. 1986; Clegg et al. 1988; Cummings et al. 1996), antigenicities (Fleischer et al. 1976; Hofmann et al. 1977), structural differences (Su et al. 1995; Diller et al. 2001; Newlon et al. 2001; Banky et al. 2003; R. Lipsitz and P. Jennings, unpubl.), and unique physiological roles (Brandon et al. 1994; Cummings et al. 1996; Schreyer et al. 2001; Amieux et al. 2002). The molecular architecture of the isoforms is, nevertheless, identical. Each contains an N-terminal dimerization/docking domain, a pseudosubstrate inhibitory sequence-containing linker region, and two tandem, C-terminal cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively), that bind one cAMP per domain (Fig. 1).

View larger version (43K): [in this window] [in a new window]   Figure 1. Overview of the R-subunits and the RIIß phosphate binding cassettes (PBC). (A) (Top) Schematic diagram of R-subunit domain organization. Dimerization/docking domain is shown in brown; cAMP-binding domains A and B are shown in tan and in gray, respectively; and the pseudosubstrate inhibitor sequence is shown in yellow. (Bottom) Ribbon diagram of the cAMP-binding domains with the PBC-conserved Arg residues highlighted in orange. (B, C) Close-up diagram highlighting the network of interactions originating from Arg230 and Arg359 (B and C, respectively). Acidic and basic side-chain atoms are shown in red and blue, respectively. Hydrogen bonds are designated with dashed lines, and water molecules are shown in aqua. Residues labeled as, e.g., G220:N, indicate that the H-bond is directed to the amide group of G220.

The Arg-to-Lys mutation allows us to understand the structural importance of this residue. Not only is this Arg critical for the binding of cAMP but also it can play an important role in organizing each domain and in orchestrating the allosteric properties of this protein as it shuttles between two conformational states: one in which it is bound to cAMP and the other in which it is bound to C (Herberg et al. 1996; Steinberg et al. 1996; Canaves et al. 2000; K.M. Zawadzki and S.S. Taylor, unpubl.). To better understand how the mutation of the conserved Arg affects the dynamic properties of the R-subunit, we have used amide hydrogen/deuterium exchange measured by mass spectrometry. A powerful approach is that of Zhang and Smith (1993), in which amide hydrogen exchange is followed by pepsin proteolysis, HPLC separation, and electrospray mass spectrometry. This or analogous methods have been increasingly used in the past decade to study protein structure (Zhang and Smith 1993; Resing et al. 1999), protein dynamics (Neubert et al. 1997; Engen and Smith 2001; Hoofnagle et al. 2001), protein–ligand interactions (Engen et al. 1999; Andersen et al. 2001; Anand et al. 2002), and protein–protein interactions (Mandell et al. 1998, 2001; Ehring 1999; Anand et al. 2002). We previously described (Hamuro et al. 2002a,b, 2003) the development and use of an improved version of this approach that is referred to as enhanced deuterium exchange-mass spectrometry (DXMS; Woods 1997 Woods 2001a,b; Woods and Hamuro 2001; Hamuro et al. 2002b). A similar approach using MALDI mass spectrometry has been used for mapping conformational changes in the RI isoform (Anand et al. 2002).

RIIß is a unique isoform because it is the predominant R isoform in the brain and adipose tissue of a variety of mammals, with limited expression elsewhere, and is implicated to play a role in metabolism, obesity, and diabetes (Stein et al. 1987; Cummings et al. 1996). DXMS analysis of the Arg-to-Lys mutations in the cAB-A and cAB-B domains of RIIß (R230K and R359K, respectively) provides insight into the effects of the mutation on RIIß dynamics. Specifically, to what extent do the PBC-conserved Arg residues organize the structure of the cAMP-binding domains? How do the mutations alter the local and global dynamics? How does mutation in one domain affect amide hydrogen exchange in the nonmutated domain? Identifying sites of altered amide hydrogen exchange will provide insight into which regions of the cAMP-binding domains relay interdomain communication signals. The dynamic properties of the mutant proteins can then be compared with the wild-type RIIß in the presence and absence of cAMP to separate the effects of removing cAMP from the effects of the Arg mutation.

The results reveal that mutation of Arg230 in the cAB-A domain increases amide hydrogen exchange throughout the cAB-A domain ß barrel. Mutation of Arg359 in the cAB-B domain affects amide hydrogen exchange not only throughout the cAB-B domain ß barrel and but also in the cAB-A domain C-helix and DD domain. These results indicate that communication flows from the cAB-B domain into the cAB-A and the DD domains, and this pathway of communication can account for the highly allosteric properties of this protein.

	Results

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RIIß mutants proteolytic fragmentation
Digestion and HPLC separation conditions that produced RIIß fragments of optimal size and distribution for exchange analyses for the wild-type protein were previously established (Hamuro et al. 2003). These conditions were applied to the two mutant RIIß proteins studied here. The conditions generated >100 identified and analyzed peptides for the R230K mutant and >60 analyzable peptides for the R359K mutant. Thirty-eight peptides, still covering 97% of the entire sequence, were selected for the analysis of R230K mutant and 26 peptides, covering 75% of the sequence were selected for R359K (Fig. 2). The digestion pattern of wild-type RIIß and that of R230K were very similar, evidenced by the coverage map that shows many common pepsin-generated fragments (Fig. 2, black lines and gray lines). The digestion pattern of the N-terminal 300 residues of R359K is similar to that of R230K and wild-type RIIß, but fragmentation of the R359K cAB-B domain is distinct. With the cleavage conditions that were optimized for wild-type RIIß, very few pepsin fragments were observed in the C-terminal of the R359K protein. The reason for this poor coverage is not clear, but evidently, the mutation alters the conformation of the protein such that its susceptibility toward pepsin digestion has changed. Circular dichroism and C-subunit binding assays, however, indicate that the R359K protein is still functional and folded in a manner similar to the wild-type protein (K.M. Zawadzki and S.S. Taylor, unpubl.).

View larger version (14K): [in this window] [in a new window]   Figure 2. Pepsin digestion map of R230K and R359K RIIß. Black lines indicate peptides analyzed in all wild type, R230K, and R359K. Gray lines indicate peptides analyzed in wild type and R230K. Dashed lines indicate peptides analyzed in R230K and R359K. White lines indicate peptides analyzed in wild-type protein only.

The change in amide exchange of select RIIß peptides upon mutation of the conserved Arg residues was determined (Table 1; Fig. 3). Peptides that provided the most extensive coverage of the protein without overlapping were chosen for amide hydrogen exchange analysis. Residues within the cAMP-binding domains with amide exchange that was altered by introduction of an Arg mutation are mapped onto the crystallographic structure in Figure 4. The R230K and R359K proteins are altered not only by the introduction of the Arg-to-Lys mutation but also by the removal of cAMP from the mutated cAMP-binding pocket. The nonmutated binding pocket retains its cAMP molecule (Herberg et al. 1996; K.M. Zawadzki and S.S. Taylor, unpubl.). To distinguish altered amide hydrogen exchange due to the mutation from altered exchange due to cAMP removal, each Arg mutant protein was compared with both apo and cAMP-bound wild-type RIIß. For example, in the R230K protein, the cAB-A domain should be compared with the cAB-A domain in the cAMP-free wild-type protein (both are lacking cAMP), and the cAB-B domain should be compared with the cAB-B domain from the cAMP-bound wild-type protein (both are cAMP saturated).

View larger version (27K): [in this window] [in a new window]   Figure 3. Deuteration levels of RIIß. Each horizontal grouping of bars represents, from top to bottom, cAMP-bound wild type, R230K, R359K, and cAMP-free wild type. Each data set contains six time points: 10-s, 30-s, 100-s, 300-s, 1000-s, and 3000-s on-exchange.

View larger version (31K): [in this window] [in a new window]   Figure 4. Difference in deuteration between Arg mutant and wild-type proteins mapped onto ribbon diagram of RIIß residues 130–412. Residues showing increased deuteration upon mutation are in red, decreased deuteration upon mutation are in blue, and little change in deuteration are in gray. Arg230 (A) and Arg359 (B) are shown in green.

View larger version (32K): [in this window] [in a new window]   Figure 5. Number of deuterons incorporated as a function of time for various peptides in wild-type cAMP-free (open circles), wild-type cAMP-bound (solid circles), R230K (triangles), and R359K (squares) RIIß. (A) Amide hydrogen exchange of dimerization/docking domain helix I residues 12–15 and 15–19. (B) Residues within cAMP-binding domain A with amide exchange that is altered upon Arg mutation. Ribbon diagram of the cA domain highlighting (red) residues 171–188 (ß1,ß2), 191–197 (ß3,ß4), 203–219 (ß5), 228–233 (PBC), 236–242 (ß7,ß8), 245–246 (ß8), 247–250 (B), 253–268 (C), and 271–281 (C). Arg230 is shown in green, and the conserved Asp residues, which interact with Arg230, are shown in white. The plot for residues 236–242 is representative of the plots for 191–197, 203–219, 245–246, 247–250, and 228–233 is representative of 171–188. (C) Residues within cAMP-binding domain B whose amide exchange is altered upon Arg mutation. Ribbon diagram of the cAB-B domain highlighting residues 287–300 (A,ß1). Arg359 is shown in green; the conserved Asp residues, which interact with Arg359, are shown in white; and Arg381 is shown in purple.

Although the R359K mutant protein gave virtually identical hydrogen/deuterium exchange pattern as wild-type proteins for residues 1–250, indicating the mutation at Arg359 did not affect the majority of cAB-A domain, the R359K mutation did affect two peptides within the cAB-A domain C-helix. Residues 253–268 and 271–281 display an increase in exchange for the R359K mutant compared with cAMP-free and cAMP-bound wild type and the R230K protein (Fig. 5B), indicating a coupling between Arg359 and the cAB-A domain C-helix. No cAMP-binding effect was observed in the cAB-A domain C-helices of the wild-type proteins, indicating that this increase in exchange is due exclusively to the Arg359 mutation.

cAMP-binding domain B
Only three peptides were observed in the cAB-B domain of R359K mutant, and all three peptides are highly deuterated (Fig. 3; Supplemental Material). Residues 287–300 (A, ß1) in the R359K protein display the largest increase in exchange compared with cAMP-free and cAMP-bound wild type and the R230K protein (Fig. 5C; Table 1). A significant increase in exchange is also observed in residues 305–311 when comparing the deuteration level of 305–312 in wild type and that of 305–311 in R359K (Fig. 3; Supplemental Material). The exchange patterns of the wild-type proteins in these regions were identical, indicating that the increased exchange for R359K RIIß is due exclusively to the Arg359 mutation. None of the peptides generated from the cAB-B domain of R230K mutant showed significantly different exchange patterns from those of wild-type proteins, indicating that the structure and dynamics of this domain was not affected by the mutation at Arg230.

	Discussion

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material Competing interests References

 
Amide hydrogen exchange analysis of RIIß proteins containing the conserved arginine mutations allows us to examine the effects that a single point mutation has on both local and global dynamics. Not only can we gain insight into the extent to which these critical residues contribute to binding of cAMP, but we can also further understand the structural organization of each domain and the mechanism by which the two domains allosterically orchestrate the binding of either cAMP or the C-subunit.

Phosphate-binding cassettes
The amide hydrogen exchange data has provided novel and unanticipated information regarding the conserved PBCs. Despite the strong and multifaceted interactions between cAMP and the PBC that were revealed in the crystal structures (Fig. 1), the PBC amide hydrogens of both RI and RIIß remain relatively protected from solvent exchange even when cAMP is removed (Anand et al. 2002; Hamuro et al. 2003). As seen in the R230K protein, however, the mutation of the conserved Arg residue is capable of dramatically increasing the amide hydrogen exchange of the PBC well beyond the subtle increased observed from the removal of cAMP (Fig. 5B, residues 228–234). Arg230 thus plays a role not only in binding cAMP but also in coordinating the organization of the cAB-A domain PBC. The cAB-B domain PBC peptide was not identified in the R359K mutant protein, so we are unable to draw a conclusion as to the effect of the cAB-B domain mutation on its PBC.

Localized and global effects of the arginine mutations
Arg230 mutation
Neither the dynamics of the cAB-B domain nor of the surface that is predicted to be the C-subunit binding site was significantly affected by the mutation of Arg230. However, it is possible that there are unobserved effects in the region, as the majority of the linker region (residues 79–128) is fully deuterated even at the shortest exchange time, meaning that all the exchange events in this region occur before the time window used here.

Mutation of Arg230 in the cAB-A domain has a major effect on the exchange of backbone amides throughout the ß barrel subdomain of domain A. The entire ß barrel seems to "loosen up" and become more dynamic, as evidenced by the increased amide hydrogen exchange in all the peptides observed in the region 171–250, including ß3–6, the PBC, and ß7–8 (Figs. 2–4). These results are consistent with the observed decrease in stability of the cAB-A domain upon mutation of Arg230 (K.M. Zawadzki and S.S. Taylor, unpubl.).

In the crystal structure (Diller et al. 2001), Arg230 makes a critical contact with the exocyclic oxygen of cAMP, and these contacts are well documented. A number of interactions between Arg230 and the rest of the domain, primarily within the loop preceding ß-sheet 3, also exist and are summarized in Figures 1 and 6A. This network of interactions radiates outward from the cAMP-binding site and likely provides a mechanism for mediating the effects that the Arg230 mutation has on the dynamics of the cAB-A domain. The removal of cAMP does not interfere with the ability of Arg230 to integrate domain A and the overall dynamics of the ß barrel. Instead, the effects of removing cAMP are more subtle and appear to allosterically regulate its ability to toggle between the two conformational states that the domain assumes: the dissociated cAMP-bound conformation and the holoenzyme conformation in which the C-subunit is bound.

View larger version (46K): [in this window] [in a new window]   Figure 6. (A) Diagram highlighting the hydrogen bond interactions of Asp187. In addition to the Asp187-Arg230 (orange)-cAMP (yellow) bridge, Asp187 also interacts with the backbone of ß-sheet 3. Acidic residues are shown in red, and basic residues are shown in blue. Water molecules are shown in aqua. Hydrogen bond distances are given in angstroms. (B) RIa and RIIß surface diagrams highlighting the contacts between the cAB-A domain C-helix (green ribbon) and the cAB-B domain (green). The cAB-B domain B-helix (red) stacks against the cAB-A domain PBC.

Previous DXMS experiments on wild-type RIIß indicated that cAMP binding influences amide hydrogen exchange within the DD domain helix I (Hamuro et al. 2003). Results from the R359K mutant protein analysis provided more definitive support for this hypothesis by indicating that the decrease in amide hydrogen exchange occurs only when the cAB-B domain is vacant. This is an intriguing result because helix I of the dimerization/docking domain provides the docking site for A-kinase anchoring proteins (AKAPs; Li and Rubin 1995; R. Lipsitz and P. Jennings, unpubl.), which function to localize the R-subunits to various cellular organelles. Interestingly, evidence of crosstalk between the nucleotide binding domains and the DD domain has previously been observed in cGMP-dependent protein kinase (PKG), which is homologous to cAPK (Chu et al. 1997). Crosstalk between the nucleotide binding domains and the DD domain, then, could be a characteristic of several kinases.

A major effect of the Arg359 mutation is to significantly enhance the dynamical properties of the cAB-A domain C-helix, which is predicted to be the primary docking site for the C-subunit in the RIIß holoenzyme (Hamuro et al. 2003). Even though Arg381 of the cAB-B domain links directly to the cAMP-binding pocket of the cAB-A domain (Fig. 5C), the dynamic properties of the cAB-A domain are not globally affected by mutation of Arg359. Communication between the cAB-B domain binding site and the cAB-A domain C-helix had been hypothesized to play an important role in the cAPK activation mechanism. The sequential binding of cAMP to domain B and then domain A is believed to be mediated by the cAB-A domain C-helix (Ogreid and Døskeland 1981a, b; Herberg et al. 1996), and this study provides the first evidence of this communication at the molecular level. However, this study does not support an ordered, obligatory cAMP-binding pathway in RIIß.

The coupling between the cAB-B domain mutation and amide hydrogen exchange in the cAB-A domain C-helix also confirms predictions based on the crystal structures of RI and RIIß; that is, mainly the C-helix of the cAB-A domain is structurally linked to the cAB-B domain (Fig. 6B). The interactions are primarily hydrophobic (Su et al. 1995; Diller et al. 2001), and it was predicted that the position of the C-helix segment would be determined by the positioning of the cAB-B domain. The domain interface in RI is quite different from RIIß, but the orientation of the C-helix relative to the B domain is conserved in both isoforms at the C terminus.

Understanding how cAMP binding mediates a biological response
The cAMP-binding domains found in the R-subunits are highly conserved signaling modules that have been preserved from bacteria to man. This study has provided us with a better understanding of the conformational steps that allow cAMP binding to promote cAPK activation. Consequently, we want to also understand how the binding of cAMP propagates signals across R-subunit molecules that are then converted into a biological response. By using the cAB-A domain of RI and RIIß as models, we can describe the domains as containing two functional shells (Fig. 7A).

View larger version (39K): [in this window] [in a new window]   Figure 7. (A) Amino acid side-chain and backbone interactions connecting shells I and II in the R-subunit. Residues of shell I (PBC, gold), shell II (ß2–ß3 turn, blue; cAB-B domain A, cranberry; and cAB-A domain B and C, silver) are displayed. Two numbers are listed for each amino acid; the first corresponds to RI numbering, and the second corresponds to RIIß numbering. Interactions conserved between RI and RIIß are shown with black dotted lines. Interactions unique to RI are shown with solid blue lines, and interactions unique to RIIß are shown with solid green lines. The residues that provide the primary isoform difference between the functional shells (Arg241/262, Lys240/261, and Asp267/288) are designated with a box. (B, C) Ribbon diagrams of RI and RIIß demonstrating the isoform-specific connection between the cAB-A domain PBC and C-helix.

Although the interactions between the conserved Arg residues in domain A and the ß2–ß3 turn are conserved in RI and RIIß, the contacts between shell I (PBC) and the C-helix of shell II show some striking differences between the RI and RIIß isoforms. These differences presumably contribute to isoform-specific activation mechanisms (K.M. Zawadzki and S.S. Taylor, unpubl.). A direct connection between the PBC of domain A and the C-helix can be found in the Glu200-Arg241 interaction of RI (Fig. 7B). Arg241 is critical for cooperative cAMP binding and holoenzyme activation (Symcox et al. 1994). In contrast, in RIIß, Asp288 of the cAB-B domain A-helix bridges Arg262 (C-helix) and Tyr226 (PBC), therefore mediating the PBC and C-helix interaction of RIIß (Fig. 7C). Asp288, then, is a functional member of shell II within RIIß. The homologous Asp in RI (Asp267) forms a hydrogen bond with Arg241 but does not contact shell I; therefore, it is not a functional member of shell II.

As we begin to look at the genomic diversity of this critical cAMP-binding motif (Canaves and Taylor 2002), it is clear that access to the cAMP-binding site will be regulated by the adjacent domain or by another protein. The diversity that we see in two related isoforms, RI and RIIß, will be expanded many fold as we look at the diverse ways in which this motif has been used throughout evolution to mediate biological responses.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material Competing interests References

 
Expression and purification
Transformed Escherichia coli BL21 (DE3) cells (Novagen) were induced with 0.4 mM dioxane-free isopropyl-ß-D-thiogalactopyranoside (IPTG) at 25°C. Protein expression was allowed to continue for 8 h. The wild-type protein was purified as described previously (Zawadzki et al. 2003) by using cAMP-agarose resin. Nucleotide-bound protein was isolated by eluting at room temperature with buffer containing 25 mM cAMP, whereas nucleotide-free protein was isolated by eluting with 25 mM cGMP (J. Jones, pers. comm.).

The two mutants were purified by co-lysis with a poly-His tagged C-subunit (Cox et al. 1994) as described elsewhere (K.M. Zawadzki and S.S. Taylor, unpubl.). The pellets from cells expressing poly-His C-subunit (4 L) were co-lysed with cells expressing the mutant R-subunit (6 L) in lysis buffer (20 mM MOPS, 100 mM NaCl, 5 mM 2-mercaptoethanol, 0.01% TritonX-100 at pH 8.0). The mass of C-subunit pellet was twice the mass of the R-subunit pellet. After lysis, the supernatant was incubated with nickel agarose for 1 h at 4°C, the resin was washed with buffer, and the R-subunit was eluted with buffer containing 10 mM cAMP. The protein eluates were then immediately dialyzed overnight at 4°C in buffer A (20 mM MOPS, 200 mM NaCl, 2 mM EDTA, 2 mM EGTA, 10 mM DTT at pH 7.0). Typical yields were 0.5 to 1 mg/L of protein cell culture.

DXMS analysis
General operational procedure
A 20 µL hydrogen-exchanged protein solution (5 µM) was quenched by shifting to pH 2.2 to 2.5, 0°C with 30 µL of 3.2 M GuHCl in 0.8% formic acid as previously described (Hamuro et al. 2003). At 0°C, the quenched solution was immediately pushed over a small solid-phase pepsin column with 0.05% TFA (200 µL/min) for 2 min with contemporaneous collection of proteolytic products by a C18 column as previously described (Hamuro et al. 2002a, 2003). Subsequently, the C18 column was eluted with a linear gradient of 10% to 50% B over 30 min (solvent A was 0.05% TFA in water, and solvent B was 80% acetonitrile, 20% water, 0.01% TFA). Mass spectrometric analyses were carried out with a Finnigan LCQ mass spectrometer with capillary temperature at 200°C. The sequences of pepsin-generated peptides were quickly determined by acquisition and analysis of data-dependent MS/MS data sets obtained from quenched undeuterated protein, and the isotopic envelope centroids of peptides from deuterated samples were determined from raw LC-MS data as previously described, using specialized DXMS data reduction software developed in collaboration with Sierra Analytics, LLC (Hamuro et al. 2002a, 2003).

Deuterium exchange experiments
Deuterated samples were prepared at 22°C by diluting 2 µL of RIIß (cAMP-bound wild-type, cAMP-free wild-type, R230K, and R359K) stock solution with 18 µL of deuterated buffer (20 mM MOPS, 50 mM NaCl, 1 mM DTT at pD 7.4), followed by "on-exchange" incubation for varying times (10, 30, 100, 300, 1000, 3000 s) prior to quenching in 30 µL of 0.8% formic acid and 3.2 M GuHCl at 0°C. These functionally deuterated samples were then subjected to DXMS processing as above, along with control samples of nondeuterated and fully deuterated RIIß (incubated in 0.5% formic acid in 90% D₂O for 24 h at 22°C). Corrections for back-exchange were made as previously described (Hamuro et al. 2002a, 2003). Typical deuteron recovery of fully deuterated sample was 82%.

	Electronic supplemental material

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material Competing interests References

 
The file RIIbArgDXMS.doc contains the plots of number of deuterons as a function of time for all peptides analyzed in this study.

	Competing interests

TOP Abstract Introduction Results Discussion Materials and methods Electronic supplemental material Competing interests References

 
Authors declare competing interests; see Web version for details.

	Acknowledgments

 
We thank Simon Brown for assistance in protein purification and Elzbieta Radzio-Andzelm for assistance in figure preparation. These studies were funded by University of California BioStar Technology transfer grants S97–90 and S99–44 (V.L.W.), and University of California Life Sciences Informatics (LSI) Technology transfer grant L98–30 (V.L.W.). ExSAR, Inc. is the matching partner for these grants. This work was also supported by NIH grant GM34921 (to S.S.T.). K.M.Z. was supported by a Department of Education Graduate Assistance in Areas of National Need (GAANN) Fellowship and by NIH Training Grant GM08326–10.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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