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• W1615954049 abstract "Transient state kinetic studies indicate that substrate phosphorylation in protein kinase A is partially rate-limited by conformational changes, some of which may be associated with nucleotide binding (Shaffer, J., and Adams, J. A. (1999)Biochemistry 38, 12072–12079). To assess whether specific structural changes are associated with the binding of nucleotides, hydrogen-deuterium exchange experiments were performed on the enzyme in the absence and presence of ADP. Four regions of the protein are protected from exchange in the presence of ADP. Two regions encompass the catalytic and glycine-rich loops and are integral parts of the active site. Conversely, protection of probes in the C terminus is consistent with nucleotide-induced domain closure. One protected probe encompasses a portion of helix C, a secondary structural element that does not make any direct contacts with the nucleotide but has been reported to undergo segmental motion upon the activation of some protein kinases. The combined data suggest that binding of the nucleotide has distal structural effects that may include stabilizing the closed state of the enzyme and altering the position of a critical helix outside the active site. The latter represents the first evidence that the nucleotide alone can induce changes in helix C in solution. Transient state kinetic studies indicate that substrate phosphorylation in protein kinase A is partially rate-limited by conformational changes, some of which may be associated with nucleotide binding (Shaffer, J., and Adams, J. A. (1999)Biochemistry 38, 12072–12079). To assess whether specific structural changes are associated with the binding of nucleotides, hydrogen-deuterium exchange experiments were performed on the enzyme in the absence and presence of ADP. Four regions of the protein are protected from exchange in the presence of ADP. Two regions encompass the catalytic and glycine-rich loops and are integral parts of the active site. Conversely, protection of probes in the C terminus is consistent with nucleotide-induced domain closure. One protected probe encompasses a portion of helix C, a secondary structural element that does not make any direct contacts with the nucleotide but has been reported to undergo segmental motion upon the activation of some protein kinases. The combined data suggest that binding of the nucleotide has distal structural effects that may include stabilizing the closed state of the enzyme and altering the position of a critical helix outside the active site. The latter represents the first evidence that the nucleotide alone can induce changes in helix C in solution. Protein kinases are the essential enzymes that direct protein phosphorylation in the cell. The results of this posttranslational modification on protein structure and function can have extraordinary effects ranging from changes in carbohydrate and neurotransmitter metabolism to organelle trafficking and cell division. Given the general role that protein phosphorylation plays in these and many other signal transduction pathways, understanding how these enzymes process substrates has become key to understanding cell function. The insights derived from biophysical studies will support the intense consideration that this enzyme family is now being given as chemotherapeutic targets (1Cohen P. Curr. Opin. Chem. Biol. 1999; 3: 459-465Crossref PubMed Scopus (177) Google Scholar). As essential components for normal cell function, protein kinases are tightly regulated through a broad host of processes including phosphorylation (2Johnson L.N. Lowe E.D. Noble M.E. Owen D.J. FEBS Lett. 1998; 430: 1-11Crossref PubMed Scopus (184) Google Scholar), second messengers such as cAMP and Ca2+, fatty acylation, protein-protein and domain-domain interactions, and localization through scaffolding and adaptor proteins (3Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1444) Google Scholar, 4Newlon M.G. Roy M. Morikis D. Hausken Z.E. Coghlan V. Scott J.D. Jennings P.A. Nat. Struct. Biol. 1999; 6: 222-227Crossref PubMed Scopus (184) Google Scholar). These processes ensure that the correct protein kinase is activated or repressed at the appropriate time and at the correct location in the cell. Indeed, mutations in protein kinases that alter their regulation are frequently linked to disease (5Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. Parslow T.G. Weiss A. Science. 1994; 264: 1599-1601Crossref PubMed Scopus (436) Google Scholar, 6Clauser E. Leconte I. Auzan C. Horm. Res. 1992; 38: 5-12Crossref PubMed Scopus (10) Google Scholar, 7Elder M.E. Lin D. Clever J. Chan A.C. Hope T.J. Weiss A. Parslow T.G. Science. 1994; 264: 1596-1599Crossref PubMed Scopus (433) Google Scholar, 8Rawlings D.J. Saffran D.C. Tsukada S. Largaespada D.A. Grimaldi J.C. Cohen L. Mohr R.N. Bazan J.F. Howard M. Copeland N.G. Jenkins N.A. Witte O.N. Science. 1993; 261: 358-361Crossref PubMed Scopus (781) Google Scholar, 9Thomas J.D. Sideras P. Smith C.I. Vorechovsky I. Chapman V. Paul W.E. Science. 1993; 261: 355-358Crossref PubMed Scopus (578) Google Scholar, 10Robertson S.C. Tynan J. Donoghue D.J. Trends Genet. 2000; 16: 265-271Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Protein kinases possess a well conserved core composed of a small ATP binding domain and a larger substrate binding domain as exemplified in Fig. 1 for the catalytic subunit (C-subunit)1 of protein kinase A (PKA) (11Knighton D.R. Zheng J.H. Ten Eyck L.F. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (814) Google Scholar, 12Knighton D.R. Zheng J.H. Ten Eyck L.F. Ashford V.A. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 407-414Crossref PubMed Scopus (1467) Google Scholar). The active site lies between these two domains with ATP embedded deep within the pocket and the substrate fixed toward the periphery. Protein kinases are conformationally dynamic, and several movements within the core kinase structure have been observed. For example, PKA has been crystallized in both open and closed (Fig.1) forms that differ primarily by domain rotation (13Zheng J. Knighton D.R. Xuong N.H. Taylor S.S. Sowadski J.M. Ten Eyck L.F. Protein Sci. 1993; 2: 1559-1573Crossref PubMed Scopus (284) Google Scholar). Small angle x-ray scattering methods suggest these conformational dynamics may also occur in solution (14Olah G.A. Mitchell R.D. Sosnick T.R. Walsh D.A. Trewhella J. Biochemistry. 1993; 32: 3649-3657Crossref PubMed Scopus (73) Google Scholar). In addition, phosphorylation of the activation loop segment in Cdk2 and the kinase domain of insulin receptor kinase lowers B factors and causes an ordering of this region (15De Bondt H.L. Rosenblatt J. Jancarik J. Jones H.D. Morgan D.O. Kim S.H. Nature. 1993; 363: 595-602Crossref PubMed Scopus (834) Google Scholar, 16Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1216) Google Scholar, 17Russo A.A. Jeffrey P.D. Pavletich N.P. Nat. Struct. Biol. 1996; 3: 696-700Crossref PubMed Scopus (505) Google Scholar). While it is unlikely that the activation loop serves the universal function of an autoinhibitor (18Adams J.A. McGlone M.L. Gibson R. Taylor S.S. Biochemistry. 1995; 34: 2447-2454Crossref PubMed Scopus (134) Google Scholar,19Saylor P. Hanna E. Adams J.A. Biochemistry. 1998; 37: 17875-17881Crossref PubMed Scopus (17) Google Scholar), loop motion has been linked to other interesting changes in structure that may have a general role in regulation. For protein kinase structures that have been solved in both phosphorylated (active) and dephosphorylated (inactive) states, the ordering of the activation loop upon phosphorylation results in a notable shift in helix C (16Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1216) Google Scholar,20Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E.J. Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 21Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (787) Google Scholar). This is thought to be a key element in kinase activation, since this movement places a conserved glutamate in this helix (Glu91 in PKA) within hydrogen bonding distance of a conserved lysine (Lys72 in PKA) residue in the active site. This lysine chelates either the β or the α/β phosphates of ATP and upon mutation results in a low activity mutant (22Carrera A.C. Alexandrov K. Roberts T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 442-446Crossref PubMed Scopus (164) Google Scholar, 23Gibbs C.S. Zoller M.J. J. Biol. Chem. 1991; 266: 8923-8931Abstract Full Text PDF PubMed Google Scholar, 24Robinson M.J. Harkins P.C. Zhang J. Baer R. Haycock J.W. Cobb M.H. Goldsmith E.J. Biochemistry. 1996; 35: 5641-5646Crossref PubMed Scopus (129) Google Scholar). Based upon these findings, it is thought that protein kinase regulation through activation loop phosphorylation is linked to the formation of this essential glutamate-lysine dyad. Detailed kinetic studies of the paradigm protein kinase, PKA, reveal that conformational changes not only are part of normal catalysis but also may be slow relative to turnover and provide a means of regulating enzyme function. Two conformational changes (one before and one after the phosphoryl transfer step) partially controlkcat in wild-type PKA under physiological concentrations of magnesium (25Shaffer J. Adams J.A. Biochemistry. 1999; 38: 12072-12079Crossref PubMed Scopus (57) Google Scholar, 26Shaffer J. Adams J.A. Biochemistry. 1999; 38: 5572-5581Crossref PubMed Scopus (45) Google Scholar). At least one of these steps appears to be linked to nucleotide binding. Stopped-flow experiments performed on a fluorescently labeled mutant of PKA demonstrate that turnover is partially limited by a conformational change occurring after the phosphoryl transfer step (27Lew J. Taylor S.S. Adams J.A. Biochemistry. 1997; 36: 6717-6724Crossref PubMed Scopus (68) Google Scholar). That some of these structural changes are linked to nucleotide binding is further supported by stopped-flow binding studies using fluorescently labeled mant derivatives of ATP and ADP (28Ni Q. Shaffer J. Adams J.A. Protein Sci. 2000; 9: 1818-1827Crossref PubMed Scopus (41) Google Scholar). The binding of mant-ADP is accompanied by slow conformational changes that are close in value to the turnover rate. In this paper, we now describe structural methods for attaining a molecular description of these conformational changes. By applying hydrogen-deuterium (H-D) exchange techniques coupled with MALDI-TOF mass spectrometric detection (29Mandell J.G. Falick A.M. Komives E.A. Anal. Chem. 1998; 70: 3987-3995Crossref PubMed Scopus (185) Google Scholar), we have shown that four distinct polypeptide regions in PKA alter their protection from amide proton exchange upon ADP binding. Two of these regions are located in the active site, while two are distal to the nucleotide pocket. The latter regions contain part of the C-terminal tail and helix C, the secondary structural element containing one essential member of the glutamate-lysine dyad, namely Glu91. The data suggest that when nucleotides bind to PKA, helix C changes conformation, perhaps causing formation or strengthening of the dyad, and the closed form of the enzyme is stabilized. PD10 columns for buffer exchange were obtained from Amersham Pharmacia Biotech. Dipotassium-ADP was obtained from ICN Biomedicals Inc. D2O (99.9% deuterium) was obtained from ISOTEC Inc. Pepsin immobilized on 6% beaded agarose was obtained from Pierce. Trifluoroacetic acid and acetonitrile were obtained from Fisher and were of peptide synthesis grade and optima grade, respectively. α-Cyano-4-hydroxycinnamic acid was obtained from Aldrich and recrystallized once from ethanol. ATP, Mops, lactate dehydrogenase, pyruvate kinase, reduced nicotinamide adenine dinucleotide (NADH), and phosphoenolpyruvate were purchased from Sigma. The substrate peptide, LRRASLG (Kemptide), was synthesized at the USC Microchemical Core Facility using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by C-18 reverse phase HPLC. The C-subunit of murine PKA was expressed in E. coli and purified as previously described (30Herberg F.W. Bell S.M. Taylor S.S. Protein Eng. 1993; 6: 771-777Crossref PubMed Scopus (110) Google Scholar). Isozyme I, the first isoform eluting from the cation exchange column, was used for all experiments. The buffer was changed to 100 mm KPi, pH 7.0, 5 mm 2-mercaptoethanol on a PD10 column, and the protein was concentrated to 192 μm. The protein was stored at 4 °C and used in the experiments without lyophilizing. The activity of the C-subunit was measured using a spectrophotometric coupled enzyme assay (31Cook P.F. Neville Jr., M.E. Vrana K.E. Hartl F.T. Roskoski Jr., R. Biochemistry. 1982; 21: 5794-5799Crossref PubMed Scopus (348) Google Scholar). The C-subunit was preequilibrated with 1 mm ATP and 11 mm MgCl2 in 100 mm Mops (pH 7), and the reaction was initiated with 0.5 mm Kemptide. All exchange mixtures for the C-subunit contained the following: 16 μm C-subunit, 40 mm KPi, 50 mm KCl, and 10 mm MgCl2. Final pH* was 6.9, and final percentage of D2O was 87.5%. Exchange experiments performed in the presence of nucleotide included 1 mm ADP and 11 mm MgCl2. The C-subunit was preequilibrated with ADP in H2O before starting the deuterium exchange by diluting into D2O. The D2O mixtures were prepared as follows (numbers are per 12-μl aliquot). The amounts of 100 mm KPi and 4 m KCl needed were mixed, dried in a Speedvac, dissolved in D2O, and mixed with D2O solutions of ADP and MgCl2. The final volume was 10.5 μl, and it contained 36 mm KPi, 57 mm KCl, and either 10 mm MgCl2 or 1 mm ADP and 11 mm MgCl2. The H2O mixtures (1.5 μl per 12-μl aliquot) contained 128 μm C-subunit, 67 mm KPi, and either 10 mmMgCl2 or 1 mm ADP and 11 mmMgCl2. The deuterium exchange was initiated by combining the H2O and D2O solutions. The solutions were incubated at 20 °C. At various times a 12-μl aliquot was added to an ice-cold tube containing 36 μl of 0.19% trifluoroacetic acid and 25 μl of pepsin bead slurry (previously washed two times in 1 ml of cold 0.05% trifluoroacetic acid). This brought the pH* of the C-subunit solution down to 2.5 and quenched the deuterium exchange. The mixture was incubated on ice with occasional mixing for 5 min to facilitate pepsin proteolysis of the C-subunit. The mixture was then centrifuged for 20 s at 12,000 × g at 4 °C to remove the pepsin beads, and the solution was divided in aliquots and frozen in liquid N2. The samples were stored at −80 °C until MALDI-TOF MS analysis. The mixtures for deuterium exchanging C-subunit at pH* 5.9 were prepared similarly to the pH* 6.9 samples. A predetermined amount of phosphoric acid that would bring the solution to pH* 5.9 was added together with KCl and KPi before drying in the Speedvac. The concentration of trifluoroacetic acid used to quench the reaction was adjusted accordingly to reach pH* 2.5. In-exchange of deuterium under quench conditions was measured by adding the protein solution directly to a mix of the D2O solution, quench solution, and pepsin and performing the remaining procedure as normal. This sample corresponds to time point 0. The back-exchange occurring during the procedure was measured essentially as in Ref. 32Resing K.A. Ahn N.G. Biochemistry. 1998; 37: 463-475Crossref PubMed Scopus (68) Google Scholar by using a previously pepsin-digested protein sample, drying it completely, redissolving it in labeling buffer, and incubating at 20 °C for 1 h to achieve complete exchange of backbone amide protons for deuterium. To assess the amount of label lost during sample workup (back-exchange) the deuterated samples were treated to quench and MALDI-TOF MS analysis as described above. This control measures, for each peptide, the maximal experimental mass that corresponds to a fully exchanged peptide. MALDI-TOF MS was performed essentially as described previously (29Mandell J.G. Falick A.M. Komives E.A. Anal. Chem. 1998; 70: 3987-3995Crossref PubMed Scopus (185) Google Scholar), under which conditions the H-D exchange is kept at a minimal rate. Samples were kept on finely crushed dry ice, and target plates were kept at 4 °C. The matrix solution consisted of 5 mg/ml α-cyano-4-hydroxycinnamic acid in 1:1:1 acetonitrile, ethanol, 0.52% trifluoroacetic acid (final pH 2.0). The samples were thawed quickly, and 5 μl were mixed with a 5-μl aliquot of 4 °C matrix solution. One μl was spotted on the target plate at 4 °C and dried in 1.5 min under moderate vacuum. Mass spectra were acquired on a PerSeptive Biosystems Voyager DE STR MALDI-TOF. Data were acquired at a 2-GHz sampling rate, 100,000 data channels, with a 20,000-V accelerating voltage, 78% grid voltage, and 0.012% guide wire voltage and using delayed extraction with a 100-ns pulse delay. 256 scans were averaged in ∼3 min. The mass spectra were calibrated in the software GRAMS using the 1194.6485 and 1793.9704 mass peptides. The spectra were then base line-corrected, and centroids of each peak were determined using in-house software (29Mandell J.G. Falick A.M. Komives E.A. Anal. Chem. 1998; 70: 3987-3995Crossref PubMed Scopus (185) Google Scholar). The number of deuteriums in-exchanged at time t was calculated as in Ref. 33Zhang Z. Smith D.L. Protein Sci. 1993; 2: 522-531Crossref PubMed Scopus (902) Google Scholar using Equation1, D(t)=m(t)−m(0)m(100)−m(0)×NEquation 1 where m(t) is the observed centroid mass of a peptide at time point t, m(0) is the observed mass at time point 0 (in-exchange control), m(100) is the observed mass of a fully exchanged digest (with consideration to back-exchange; see above), and N is the total number of peptide amide protons in the peptide. The data presented here originate from a single incubation experiment. Similar results have been obtained in four previous experiments. H-D exchange reactions are exquisitely sensitive to changes in structure and dynamics that accompany protein folding, ligand binding, or formation of protein-protein interactions. H-D exchange in small proteins can be monitored at the residue-specific level by combining H-D exchange with NMR detection. In the case of PKA, a 40-kDa protein, we utilize a medium resolution method that gives region-specific information by combining H-D exchange, pepsin fragmentation, and MALDI-TOF MS detection. A schematic for this technique is displayed in Fig. 2. The C-subunit, preequilibrated with or without the nucleotide, is incubated in D2O up to 3 h. After designated time periods, the exchange is quenched at pH* 2.5, and the protein is digested with pepsin. During the exchange reaction, the nucleotide concentration is ∼100-fold higher than the Ki for ADP (25Shaffer J. Adams J.A. Biochemistry. 1999; 38: 12072-12079Crossref PubMed Scopus (57) Google Scholar), assuring more than 99% binding at all times. In addition, the observed association rate for ADP is estimated to be 2000 s− 1 (34Adams J.A. Taylor S.S. Biochemistry. 1992; 31: 8516-8522Crossref PubMed Scopus (159) Google Scholar), whereas the average intrinsic H-D exchange rate is slower (3.4 and 0.34 s− 1 at 20 °C and pH* 6.9 and 5.9, respectively (35Bai Y. Milne J.S. Mayne L. Englander S.W. Proteins. 1993; 17: 75-86Crossref PubMed Scopus (1770) Google Scholar)), ensuring that H-D exchange does not outcompete the ligand binding reaction. The peptides appearing in the mass spectrum after pepsin fragmentation (Fig. 3) have been assigned to the amino acid sequence of the C-subunit. They cover ∼65% of the primary structure (29Mandell J.G. Falick A.M. Komives E.A. Anal. Chem. 1998; 70: 3987-3995Crossref PubMed Scopus (185) Google Scholar). Due to the nature of our exchange protocol, we monitored a subset of these peptides (TableI). The incorporation of deuterium necessarily broadens the peaks (Fig. 3, lower panel) with resulting lower signal-to-noise ratio. Three sets of peaks separated only by 3–6 mass units in the H2O samples became, upon deuteration, predominantly overlapping and not suitable for analysis (data not shown).Table IPeptides and structural elements analyzed by H-D exchangeStructure coveredPeptide 1-aPeptide sequencing and assignment can be found in Ref. 29. K+ indicates the peptide was observed as a potassium adduct.Mass MH+ 1-bCalculated mass for monoisotopic MH+ peak in H2O.N 1-cN denotes the total number of backbone amides in the peptide not including prolines.Exchange protection with ADP bound 1-dRegions found to experience exchange protection with ADP after 3 h of exchange in D2O. By definition a protected region displays one mass unit or more difference after this time period.pH* 6.9pH* 5.9Helix A and loop27–401643.876012NoNoHelix A and loop27–40, K+12Noβ1 and Gly-rich loop41–541584.802413NoYesβ1 and Gly-rich loop44–541194.648510NoYesβ3, helix B66–832113.232317NoNoHelix C and loop92–1001088.65828YesYesHelix D, loop, helix E133–1451628.888611NoNoCatalytic loop163–1721260.69548YesYesCatalytic loop164–1741373.77959YesYesLoop and helix F212–2211167.57999NoLoop and helix F212–221, K+9NoNoLoop and helix G237–2501708.895312NoNoHelix G and loop246–2611907.054514NoNoHelix G and loop247–2611793.970413NoNoHelix G, loop, helix H247–2642083.061316NoNoHelix J and extended turn303–3262676.451720NoYesHelix J and extended turn303–3272823.520121NoYesHelix J and extended turn305–3262492.330518No1-a Peptide sequencing and assignment can be found in Ref. 29Mandell J.G. Falick A.M. Komives E.A. Anal. Chem. 1998; 70: 3987-3995Crossref PubMed Scopus (185) Google Scholar. K+ indicates the peptide was observed as a potassium adduct.1-b Calculated mass for monoisotopic MH+ peak in H2O.1-c N denotes the total number of backbone amides in the peptide not including prolines.1-d Regions found to experience exchange protection with ADP after 3 h of exchange in D2O. By definition a protected region displays one mass unit or more difference after this time period. Open table in a new tab The average mass of each peptide was determined by integrating over the full envelope of peaks (29Mandell J.G. Falick A.M. Komives E.A. Anal. Chem. 1998; 70: 3987-3995Crossref PubMed Scopus (185) Google Scholar), and the mass was converted into number of in-exchanged deuteriums using Equation 1. The in- and back-exchange controls set the zero and infinite time points for D(t). The in-exchange controls ranged from 13–20% (16% average), whereas the back-exchange controls ranged from 73 to 91% (83% average) for the specific peptides. Since each peptide fragment can contain a number of ligand-sensitive exchangeable protons, with different intrinsic exchange rates, a visual inspection of the curves was used to evaluate the data. The C-subunit is not stable for more than a day under our experimental conditions, which excludes measurement of the slowest exchanging protons that are only exposed with the global unfolding of the protein. For deuterium in-exchange experiments (Fig. 2), an apparent equilibrium is generally observed after 100–200 min (Fig.4), as has also been reported for other systems (32Resing K.A. Ahn N.G. Biochemistry. 1998; 37: 463-475Crossref PubMed Scopus (68) Google Scholar, 33Zhang Z. Smith D.L. Protein Sci. 1993; 2: 522-531Crossref PubMed Scopus (902) Google Scholar, 36Wang F. Blanchard J.S. Tang X.J. Biochemistry. 1997; 36: 3755-3759Crossref PubMed Scopus (44) Google Scholar, 37Wang F. Miles R.W. Kicska G. Nieves E. Schramm V.L. Angeletti R.H. Protein Sci. 2000; 9: 1660-1668Crossref PubMed Scopus (48) Google Scholar). Thus, the focus of this study is the specific identification of structural elements that display differences in exchange when the C-subunit has ADP bound. The extent of deuteration of each peptide probe was followed as a function of time at pH* 6.9. Fig. 4 is a presentation of several probes typical of the results obtained. Over the time course of the exchange experiments, the masses of the probes tend to increase in a biphasic manner. The presence of ADP either has no effect or protects amide protons from H-D exchange compared with the apoenzyme, as is evident in the decreased level of label incorporation. The amide protons within probes covering residues 92–100 and 164–174 are clearly protected from H-D exchange over a 3-h period in the presence of ADP (Fig. 4, A and B, and Table I). In comparison, many probes are largely unaffected by nucleotide within experimental error (Fig. 4, C and D, and TableI). At pH* 6.9, a total of three probes covering two regions in the C-subunit displays altered solvent accessibility in the presence of nucleotide. The results for all probes are summarized in Table I. Given the experimental error in the technique, we designate a probe as protected if the mass is 1 or more units lower compared with the free C-subunit after 3 h of exchange. The previously described experiments were performed at pH* 6.9, where the average half-life for exchange of an amide proton is ∼200 ms (35Bai Y. Milne J.S. Mayne L. Englander S.W. Proteins. 1993; 17: 75-86Crossref PubMed Scopus (1770) Google Scholar). We repeated the experiments with the pH* of the H-D exchange reaction lowered by 1 unit, which decreases the intrinsic H-D exchange rate by 10-fold. Thus, more subtle differences in amide exchange protection can be detected over the same time period. Lowering the pH value further is not possible due to instability of the C-subunit at pH* values below 5.5. Fig. 5 displays several exchange studies at pH* 5.9. As shown in Fig. 5 E and summarized in Table I, exchange protection by ADP is observed at pH* 5.9 for all probes that are also protected by the nucleotide at pH* 6.9 (i.e. probes encompassing residues 92–100, 163–172, and 164–174 are protected from exchange by ADP at pH* 5.9 and 6.9). Most, but not all, other regions displayed no difference in H-D exchange in the presence or absence of nucleotide at pH* 5.9 (Fig. 5 F, Table I). However, at the lower pH, additional nucleotide-protected regions are apparent. As shown in Fig. 5, A–D, two new regions of the protein, covered by a total of four probes, are protected from exchange by ADP. These probes include residues 41–54, 44–54, 303–326, and 303–327 (Table I). These additional regions displaying exchange protection with nucleotide at pH* 5.9 are not likely to be due to changes in structure at low pH for several reasons. The steady-state kinetic parameters, Km for ATP andkcat, are identical over a wide pH range, which includes the two pH values of this study (38Yoon M.Y. Cook P.F. Biochemistry. 1987; 26: 4118-4125Crossref PubMed Scopus (74) Google Scholar). The rate of phosphoryl transfer, measured in pre-steady-state kinetic studies, is constant from pH 6 to 9 (39Zhou J. Adams J.A. Biochemistry. 1997; 36: 2977-2984Crossref PubMed Scopus (80) Google Scholar). Finally, PKA does not undergo a time-dependent inactivation within the time frame of our experiments. The specific activity of the C-subunit was followed at pH* 5.9 and 6.9 over 3 h, and it was found that the C-subunit did not lose more than 5% of its original activity (data not shown). Probing hydrogen bonding and structural dynamics in proteins with hydrogen exchange was initiated by Linderstrøm-Lang nearly 50 years ago (40Linderstrøm-Lang K. Neuberger A. Symposium on Protein Structure. Methuen, London1958: 23-34Google Scholar). 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• W1615954049 created "2016-06-24" @default.
• W1615954049 creator A5036584204 @default.
• W1615954049 creator A5053680151 @default.
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• W1615954049 date "2001-04-01" @default.
• W1615954049 modified "2023-10-14" @default.
• W1615954049 title "Structural Characterization of Protein Kinase A as a Function of Nucleotide Binding" @default.
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• W1615954049 doi "https://doi.org/10.1074/jbc.m011543200" @default.
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