Spatially and kinetically resolved changes in the conformational dynamics of the Hsp90 chaperone machine
2009; Springer Nature; Volume: 28; Issue: 5 Linguagem: Inglês
10.1038/emboj.2008.306
ISSN1460-2075
AutoresC. Graf, Marta Stankiewicz-Kosyl, Günter Krämer, Matthias P. Mayer,
Tópico(s)Enzyme Structure and Function
ResumoArticle22 January 2009free access Spatially and kinetically resolved changes in the conformational dynamics of the Hsp90 chaperone machine Christian Graf Christian Graf Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Marta Stankiewicz Marta Stankiewicz Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Günter Kramer Günter Kramer Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Matthias P Mayer Corresponding Author Matthias P Mayer Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Christian Graf Christian Graf Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Marta Stankiewicz Marta Stankiewicz Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Günter Kramer Günter Kramer Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Matthias P Mayer Corresponding Author Matthias P Mayer Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Author Information Christian Graf1, Marta Stankiewicz1, Günter Kramer1 and Matthias P Mayer 1 1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany *Corresponding author. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany. Tel.: +49 6221 546829; Fax: +49 6221 545894; E-mail: [email protected] The EMBO Journal (2009)28:602-613https://doi.org/10.1038/emboj.2008.306 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The highly conserved 90 kDa heat shock protein (Hsp90) chaperones use ATP to regulate the stability and activity of many signalling molecules like protein kinases and transcription factors. Studies using crystallography, electron microscopy and small-angle X-ray scattering yielded controversial results for the conformational states that these dimeric multidomain proteins assume while progressing through the ATPase cycle. To better understand the molecular mechanism of Hsp90 proteins, we studied the conformational dynamics of the Escherichia coli homologue HtpG in solution using amide hydrogen exchange mass spectrometry (HX-MS) and fluorescence spectroscopy. A conformation-sensitive fluorescent probe allowed to elucidate the ATPase cycle of HtpG. Continuous-labelling and pulse-labelling HX-MS experiments revealed major ATP-induced conformational changes throughout the protein that do not occur simultaneously, but progress surprisingly slow from the immediate nucleotide-binding site towards the N terminus and the middle domain. The conversion between the different conformational states is rate limiting for ATP hydrolysis, and the nucleotide-coordinating residue, Glu34, is important for the rate constant of conversion. Our findings, for the first time, allow to kinetically resolve changes in the conformational dynamics of individual structural elements of Hsp90. Introduction The 90 kDa heat shock proteins (Hsp90) are essential chaperones in all eukaryotes. Assisted by a large number of cochaperones, Hsp90s control the stability and activity of some 200 client proteins, which are mostly regulatory components of signal transduction pathways, such as receptors, protein kinases and transcription factors (Mayer and Bukau, 1999; Wegele et al, 2004; Pearl et al, 2008). The Hsp90 family of proteins is highly conserved, for example, Escherichia coli and human members share 40% sequence identity. Hsp90 proteins consist of an N-terminal nucleotide-binding domain (NBD), a middle domain (MD) and a C-terminal dimerization domain (DD). Structural information exists for both individual domains and nearly full-length Hsp90 variants in complex with different nucleotides and inhibitors (Stebbins et al, 1997; Prodromou et al, 1997b; Meyer et al, 2003; Harris et al, 2004; Huai et al, 2005; Ali et al, 2006 reviewed in Pearl and Prodromou, 2006; Shiau et al, 2006). ATP binding and hydrolysis is essential for the chaperone activity of Hsp90 in vivo (Obermann et al, 1998; Panaretou et al, 1998). On the basis of the structure of the related GHKL-ATPase DNA gyrase B and biochemical data, the ATPase cycle of Hsp90 is proposed to involve several conformational changes. These include an ATP-dependent docking of NBD and MD of the same monomer, and a dimerization of the two NBDs in the Hsp90 dimer, which results in a ring-shaped structure (Pearl and Prodromou, 2002; Richter et al, 2002; Immormino et al, 2004; Ali et al, 2006). The crystal structure of yeast Hsp90 in complex with AMPPNP and Sba1 and recent biochemical studies are consistent with this proposed catalytic mechanism (Ali et al, 2006; Cunningham et al, 2008; Vaughan et al, 2009). However, a crystallographic and electron microscopy study on HtpG presented evidence for strikingly different conformations depending on the presence or absence of AMPPNP or ADP, none of which fits the conformation proposed to be necessary for catalysis (Shiau et al, 2006). In this publication, a highly compact ADP-bound state was proposed, which could, however, not be observed by small-angle X-ray scattering (SAXS) in another investigation (Krukenberg et al, 2008). The SAXS data of HtpG were interpreted in terms of multiple coexisting conformations. To analyse the conformational dynamics of Hsp90 during progression through the ATPase cycle, we employed amide hydrogen-1H/2H-exchange (HX) mass spectrometry (MS) and fluorescence spectroscopy. We focused on the bacterial Hsp90 homologue HtpG because it is the most simple representative of the Hsp90 family and does not seem to be regulated by cochaperones. In this study, we resolved the changes in conformational dynamics in space and time. We show that ATP binding induces a series of conformational changes to attain a tense pre-hydrolysis state with greatly reduced secondary structure flexibility. Results Deuteron incorporation into full-length HtpG To analyse the conformational flexibility of HtpG, we performed continuous-labelling HX-MS experiments as described previously (Rist et al, 2003, 2006) and shown schematically in Figure 1A. With these experiments, global stability and different conformational states of a protein can be analysed. Figure 1.HX kinetics of the full-length HtpG and localization of fast and slow exchanging regions. (A) Scheme of the continuous labelling HX experiment. HX is initiated by diluting the equilibrated sample 1:20 into D2O buffer (pD 7.4). After different incubation times at 30°C, temperature is lowered to 0°C and pH to 2.5 by addition of ice-cold phosphate buffer (quench buffer). The reaction is subsequently analysed by HPLC-MS. For the analysis of overall exchange rate constants (B, C) full-length proteins are desalted on a reversed-phase trap column and eluted over an analytical column into the mass spectrometer. For localization of fast and slow exchanging regions (D) peptic fragments of the protein are generated online by including a column with immobilized pepsin in front of the trap column. (B) Numbers of deuterons incorporated into full-length E. coli HtpG in the absence of nucleotides (black circles) and in the presence of ADP (blue diamonds), ATP (red triangles) and AMPPNP (cyan squares). The y-axis to the right indicates the percentage exchange of total number of exchangeable amide hydrogens. The solid lines are fits of a tri-exponential equation to the data to guide the eye. (C) Deuteron incorporation into full-length HtpG-E34A variant in the absence of nucleotides (open black circles) and in the presence of ATP (open red triangles). (D) Localization of fast and slow exchanging regions of nucleotide-free HtpG. Secondary structure representation of nucleotide-free HtpG (PDB ID code 2IOQ) with segments corresponding to peptic fragments coloured according to the relative deuteron incorporation into the nucleotide-free protein after 10 s (left panel) and 10 min (right panel) in D2O as indicated in the figure. Numbers indicate segments mentioned in the text. NBD, nucleotide-binding domain; MD, middle domain; DD, dimerization domain. N and C terminus are indicated. Download figure Download PowerPoint Apo-HtpG exchanged about 38% of its exchangeable amide hydrogens within 10 s and about 60% within 1 h in D2O (Figure 1B). Addition of ADP only induced a small protection (ca. 7 amide hydrogens). However, in the presence of ATP solvent, accessibility of HtpG was strongly reduced as compared with apo-HtpG, with almost 50 protected amide hydrogens. This protection slowly decreased to the level of the apo state by 1200 s (Figure 1B; for a detailed discussion see Supplementary data part 1). These data indicate that the HtpG state with least amide hydrogens accessible can only be reached through addition of ATP but not of ADP, which seems to be at odds with a structural study on HtpG (Shiau et al, 2006). However, these experiments do not exclude the alternative interpretation that the solvent protected ATP-induced state is a post-hydrolysis conformation. We considered this possibility because the generation of the ATP-induced protected state required a pre-incubation of HtpG with ATP that was much longer than we had expected for a protein with high nucleotide association rate constants (see below). The ATPase cycle of HtpG The issue, whether the highly protected state was an ATP-bound state or a post-hydrolysis state, could be resolved by determining the rate-limiting step of the ATPase cycle. To this end, we measured the ATPase rate constant of HtpG under steady state and single turnover conditions. The velocity of ATP hydrolysis of HtpG increased with increasing ATP concentrations, according to normal Michaelis–Menten kinetics, resulting in a kcat of 0.011±0.001 s−1 (τ=90 s) and a KM of 250±82 μM at 30°C (Supplementary Figure 1). Under single turnover conditions, the amount of ATP decreased with single exponential kinetics resulting in a hydrolysis rate constant khyd of 0.0077±0.0003 s−1, which is smaller but close to the steady state ATPase rate constant. These results show that product release is not rate limiting in the ATPase cycle of HtpG. Therefore, in the presence of high concentrations of ATP, the majority of HtpG molecules is in the ATP-bound state. As an independent confirmation of these results, we repeated the HX experiments in the presence of the non-hydrolysable ATP analogue AMPPNP, which was devoid of any detectable amounts of ATP (Figure 1B). Here, we also observed a strong protection of about 35 amide hydrogens as compared with 7 in the presence of ADP and 47 in the presence of ATP. Strikingly, in the presence of AMPPNP, we observed the same protection throughout the entire kinetics up to 3600 s incubation time, which contrasts with the loss of protection observed after 1200 s in the presence of ATP. We made the same observation when using an HtpG variant with Glu34 replaced by Ala (HtpG-E34A; constructed in analogy to the hydrolysis-deficient yeast Hsp82–E33A (Obermann et al, 1998; Panaretou et al, 1998)) (Figure 1C), which in our hands hydrolyses ATP (τ=1071 s) about 10-times more slowly than wild-type HtpG at 30°C. However, at very long time-points, the deuteron incorporation curve for HtpG-E34A in the presence of ATP also approaches the exchange curve for apo-HtpG-E34A (data not shown). These data indicate that the loss of protection in the presence of ATP is directly linked to ATP hydrolysis. Taken together, our results show that (1) ATP hydrolysis is the rate-limiting step in the ATPase cycle of HtpG, (2) the ATP-bound state is the state with least solvent accessible amide hydrogens and (3) the ADP state shows only little protection as compared with the nucleotide-free state suggesting similar conformations. Henceforth, we call the most solvent protected ATP-bound state ‘tensed (T) state’ and the nucleotide-free state ‘relaxed (R) state’. Localization of fast and slow exchanging regions in nucleotide-free HtpG To localize the regions within HtpG that contain slow and fast exchanging amide hydrogens, we digested the protein after the continuous-labelling HX reaction under quench conditions, using a column with immobilized pepsin in our HPLC setup as described previously (Rist et al, 2003). Such experiments allow the identification of structural elements in HtpG that are tightly folded in solution or that are in a rapid unfolding-refolding equilibrium. The regularly observed peptic peptides of HtpG had an average length of 14.5 residues and covered 88% of HtpG's sequence (Supplementary Figure 2). Figure 1D shows secondary structure representations of apo-HtpG coloured according to the deuteration level of the analysed peptic fragments when the nucleotide-free protein was incubated for 10 s or 10 min in D2O. Our HX data of HtpG free in solution are in good agreement with the published crystal structure of apo-HtpG (PDB entry code 2IOQ). Two exceptions are regions of the NBD that exhibited a high degree of solvent accessibility with almost complete deuteration within 10 s. These regions are the very N terminus (residues 2–19, all numbers in the text refer to the number of backbone amides within a peptide therefore omitting the first residue of the peptide, which does not contain an amide) and the so-called ATP-lid (residues 90–98 and 121–127; the ATP-lid was originally defined in yeast Hsp82 by Pearl and coworker as residues 95–125 (Prodromou et al, 1997a)). The crystal structure indicates a secondary structure with a significant number of hydrogen bonds for these regions (Figure 1D and Supplementary Figure 2). However, these regions have a relatively high b-factor denoting high degrees of positional variation consistent with our data. Localization of nucleotide-induced changes in HtpG To localize nucleotide-induced conformational changes, we compared deuteron incorporation into different segments of HtpG in the absence of nucleotide and in the presence of ADP, ATP and AMPPNP (Figure 2). The addition of ADP only had small effects on the incorporation of deuterons. For example, amide hydrogens were protected in the nucleotide-binding site (residues 90–98) and in the N-terminal segment (residues 2–19). In addition, a small but significant deprotection was detected in specific segments in the NBD (residues 108–119), the MD (residues 279–299 and 401–416) and almost the entire DD. Figure 2.Nucleotide dependent changes in HtpG. (A) Difference plots of deuterons incorporated in the presence of ADP (left panel), ATP (middle panel) and AMPPNP (right panel) minus deuterons incorporated in the absence of nucleotides into HtpG segments after 30 s in D2O. The domain structure of HtpG and numbers of the corresponding segments are given at the left of the panels. The data are the average of three independent experiments. The gray area indicates the average standard error. Bars within the gray area are not significantly different from zero. Bars to the left indicate nucleotide-induced protection, bars to the right indicate nucleotide-induced increase in solvent accessibility, that is, opening of the structure. (B) Nucleotide-dependent HX kinetics of selected segments of HtpG. Secondary structure representation of the crystal structure of full-length E. coli HtpG in complex with ADP (PDB ID code 2IOP; only one dimer shown with protomers in different shades of gray). Segments with more than 1.5 amide hydrogens protected in the presence of ATP at time-point 30 s are coloured in dark blue and the HX kinetics for these segments in the absence of nucleotides (black circles) and the presence of ADP (open squares) and ATP (black triangles) and for HtpG-E34A in the presence of ATP (open triangles) are shown. The solid lines are fits of a bi-exponential equation to the data to guide the eye. For this fit, the total amplitude was fixed to the total number of exchangeable amide hydrogens, and the rate constant for the first phase was estimated from the theoretical exchange rate constant in the unprotected peptide. Segments with 0.5 to 1.5 amide hydrogens protected at 30 s are coloured in cyan. Segments deprotected upon ATP binding are coloured in orange. Download figure Download PowerPoint In the presence of ATP, we observed, in seven segments, a large protection (up to 7 amide hydrogens) compared with the nucleotide-free state (Figure 2A, middle panel; Figure 2B, dark blue segments): the N-terminal segments 2–19 and 21–31, the ATP-lid segments 90–98 and 121–127, the last helix of the NBD (residues 192–206) and the loop in the MD (residues 319–334 and 336–359) that contains the catalytic Arg336 (homologous to Arg380 in yeast Hsp82) (for detailed discussion see Supplementary data part 2). We detected a small but significant protection in additional segments scattered throughout the entire protein (Figure 2B, cyan segments). Two segments in the MD exhibited slightly more exchange in the presence of ATP than in the nucleotide-free state (Figure 2B, orange segments). Thus, our data show that ATP induces conformational changes not only in the NBD but throughout the HtpG protein leading to a significant loss of conformational dynamics. The kinetics of deuteron incorporation in all of the highly protected segments in the presence of ATP were nearly identical with an average rate constant of 0.0038±0.0005 s−1 (Figure 2B). This is fully consistent with ATP hydrolysis being rate limiting for HX in the ATP-bound state. As expected from the data for the full-length protein, the mutant protein, HtpG-E34A, exhibited a similar protection in the presence of ATP and this protection was observed over a much longer time interval (Figure 2B). Consistent with the hypothesis that ATP hydrolysis is rate limiting for HX, a bimodal distribution of the isotope peaks was observed in several peptides (Supplementary Figure 3). Bimodal isotopic peak distributions are indicative of the coexistence of two different conformational states. These two conformational states are the ATP-bound T state, in which the segments are largely protected from HX, and the ADP-bound and nucleotide-free R state with higher HX rate constants. In the presence of AMPPNP, the same changes in deuteron incorporation occurred as in the ATP-bound state, which confirms that the T state is the pre-hydrolysis state (Figure 2A). The magnitude of the observed protection was slightly reduced compared with the ATP-bound state, consistent with our HX data for full-length HtpG. Some of the observed ATP-induced changes in amide hydrogen protection could be caused by an N-terminal dimerization, which was shown to occur in yeast Hsp82 upon ATP or AMPPNP binding (Prodromou et al, 2000; Ali et al, 2006). To test this hypothesis, we constructed a C-terminal truncation variant of HtpG (HtpGΔC containing residues 1–496), which lacks the DD and, consequently, is monomeric (Krukenberg et al, 2008). For reasons explained in detail below this truncation variant contained, in addition, the Glu12 to Cys amino acid replacement (HtpGΔC-E12C). This amino acid replacement did not change the conformational dynamics of the protein significantly as the full-length HtpG-E12C incorporated deuterons with the same kinetics as wild-type HtpG (data not shown). As verified by size exclusion chromatography in the absence or continuous presence of ATP and site-specific cross-linking HtpGΔC-E12C was monomeric independent of the nucleotide bound (Supplementary Figure 4 and data not shown). Comparison of deuteron incorporation into HtpGΔC-E12C in the presence or absence of ATP revealed the same degree of protection as observed for full-length HtpG, indicating that N-terminal dimerization is not responsible for the protection of amide hydrogens on addition of ATP (Supplementary Figure 5A). This result seems similar to the biochemical data for human Hsp90, the ATPase activity of which was shown not to be dependent on N-terminal dimerization (McLaughlin et al, 2004). Taken together, significant nucleotide-dependent changes of the conformational dynamics of HtpG were observed in all three domains of the protein. Our data not only show the existence of an R and a T state but also show which structural elements of the protein are more tightly folded in the T state. This T state is not dependent on N-terminal dimerization because it can be reached in a monomeric truncation variant of HtpG. Kinetics of R to T transition As mentioned above, we noticed that the T state was only observed after a pre-incubation with ATP. To explore this observation in more detail, we measured the kinetics of R-to-T transition using pulse-labelling HX experiments as schematically illustrated in Figure 3A. HtpG and HtpG-E34A were incubated for different time intervals with ATP and then pulse-labelled for 10 s with D2O. As the HX reaction times were always identical, changes in the numbers of incorporated deuterons are because of the ATP-induced transition from the R to the T state. In the absence of ATP or at the shortest time-point after addition of ATP, only a single mass peak is observed (Figure 3B and C). However, the mass of pulse-labelled HtpG after the shortest incubation with ATP was already smaller than the mass of pulse-labelled apo-HtpG, indicating a conformational transition that happens within 10 s. At later time-points, a second species with a still lower mass appeared and became dominant. These data strongly suggest a two-phase transition: a rapid transition between the fast exchanging R state to a slightly protected intermediate (I) state and a subsequent slow transition to the considerably protected T state. The second transition is much slower in HtpG-E34A (Figure 3B, right panel). We fitted an equation for two Gaussian peaks to the data and determined the relative amount of I and T states (Figure 3D). The average I to T conversion rate constant for wild-type HtpG and mutant HtpG-E34A were 0.01 s−1 and 0.0006 s−1, respectively, which are within the error of the experiment identical to the ATP hydrolysis rate constants showing that the conformational changes are rate-limiting for ATP hydrolysis under these conditions. Figure 3.Kinetics of the ATP-induced R to T transition of HtpG. (A) Scheme of the experiment. (B, C) Deconvoluted mass spectra of wild-type HtpG (B) and HtpG-E34A (C) before incubation in D2O (bottom curve), after 10 s in D2O in the absence of nucleotides (apo; R state) or after different pre-incubation times with ATP (as indicated to the left; 10-fold different scale between A and B) followed by pulse-labelling for 10 s in D2O. Dashed gray lines indicate the maximum of the mass peaks after 10 s pre-incubation in the presence of ATP (I state) and after 600 s (B) and 3600 s (C) pre-incubation in ATP (T state) as designated. The masses for R, I and T states were 73 158±3, 73 151±1 and 73 106±1 for wild-type HtpG and 73 100±3.5, 73 086±3 and 73 046±6 for HtpG-E34A, respectively. (D) Relative amount of T state versus the pre-incubation time in ATP. A first-order rate equation was fitted to the data (solid line) to give the I to T conversion rate constants of 0.01 s−1 and 0.0006 s−1 for wild-type HtpG and HtpG-E34A, respectively. Download figure Download PowerPoint We also analysed the R to T transition for the monomeric HtpGΔC-E12C (Supplementary Figure 5B). Similar to the full-length proteins, the HtpGΔC-E12C converted rapidly into an I state on addition of ATP, and then very slowly into the T state. The rate constant for this conversion was much lower than for full-length HtpG, suggesting that the ATPase activity of this fragment is considerably lower than for the full-length protein as has been reported for the analogous fragment in yeast Hsp82 (Richter et al, 2001). Direct measurement of the ATPase rate constant for HtpGΔC-E12C yielded a value of 0.0018 s−1, confirming this hypothesis. Together, these data show that the R to T transition occurs in at least two steps with a first, fast step equilibrating within 10 s and a second, slow step, the rate constant of which is decreased dramatically either by the single amino acid replacement of Glu34 to Ala or by the elimination of dimerization. Spatial resolution of the R to T transition Our HX-MS data on peptide level revealed ATP-dependent conformational changes not only in regions close to the nucleotide-binding site but also in regions far distant in space. This raises the question of whether all ATP-induced conformational changes occur simultaneously in a concerted fashion or whether they occur in an ordered succession of events. In the first scenario, the protein would oscillate continuously between an open and a closed state with ATP-binding trapping the closed state. In the second scenario, ATP would bind to the open state and trigger local conformational changes that are subsequently propagated throughout the protein. To distinguish between these two scenarios, we performed spatially resolved pulse-labelling HX experiments by including peptic digestion under quench conditions. The comparison of the mass spectra of selected peptic peptides of HtpG in the nucleotide-free state with 10 s of incubation with ATP already revealed a significant shift of the centre of the isotopic peak distribution towards lower m/z values most notably in peptides 90–98 and 121–127, and also in peptides 2–19 and 336–359 (Figure 4A). These data locate the conformational changes that lead to the I state. At subsequent time-points, the isotope peaks in most spectra formed a clear bimodal distribution showing the simultaneous presence of two HtpG states with different solvent accessibility. The more accessible state slowly converts into a more protected state. From the intensities of the highest peaks in the two states, we determined the rate constants for the I to T transition for each of the peptides analysed. It was interesting to note that the rate constant of I to T conversion was different in the analysed segments (Figure 4B). The transition rate constant was highest in the ATP-lid (segments 90–98 and 121–127) and decreased towards the N-terminal segment, 2–19, and the MD segments, 319–334 and 336–359, which contains the catalytic Arg336. For the HtpG-E34A variant protein, we observed the same phenomenon, although with overall much lower rate constants (Figure 4C and Supplementary Figure 6) as expected from the data for the I to T conversion of the full-length protein. However, the transition from the R to the I state occurred for HtpG-E34A, also within 10 s as a close analysis of the data for the earliest ATP pre-incubation time-points revealed. These data confirm that the R to T transition occurs in two phases: a fast phase, which cannot be resolved by HX, and a slow phase. As the first phase not only affects the ATP-lid region but also the N terminus and segments in the MD, it is a global transition to an intermediate state (I) that may happen in a concerted fashion. The major second phase, the transition from the I state to the T state, does not occur in all segments simultaneously but in sequential steps. The I to T conversion in the MD segments had the same rate constant as the single turnover ATPase rate constant, indicating that this conformational change is rate limiting for γ-phosphate cleavage. Figure 4.Spatially resolved kinetics of R to T transition. (A) Representative mass spectra (m/z scale) of selected peptic peptides of wild-type HtpG before incubation in D2O (bottom curve; monoisotopic mass-to-charge ratio and charge state are given), after 10 s in D2O in the absence of nucleotides (apo) or after pre-incubation for 10 to 300 s with ATP (as indicated to the left) followed by pulse-labelling for 10 s in D2O. Dotted gray lines indicate the highest peak after 10 s pre-incubation in ATP followed by 10 s HX (I state, black circle) and the highest peak after 300 s pre-incubation in ATP followed by 10 s HX (T state, black square). The numbers above the spectra refer to the corresponding backbone amides. The actual peptic peptide contains one additional N-terminal residue. (B, C) I to T transition rate constants for selected peptides of wild-type HtpG (B) and HtpG-E34A (C) calculated as described in Materials and methods. The bars represent the mean of three independent determinations and standard error of mean. Download figure Download PowerPoint Conformational changes in HtpG followed by fluorescence To follow the kinetics of the conformational changes with better temporal resolution, we introduced a cysteine for fluorescent labelling at position 12 in HtpG, which is in the middle of a segment that strongly changes amide hydrogen accessibility on addition of ATP. HtpG-E12C was labelled with the environmental sensitive fluorophor, Hilyte Fluor™ 488 (HtpGFL). The presence of ATP but not of ADP induced a time-dependent decrease in fluorescence (Figure 5A). Addition of a large excess of ADP to reactions of HtpGFL, already equilibrated with ATP, led to an increase in fluorescence (Figure 5B). Close inspection of the fluorescence traces after the addition of ATP revealed
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