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1 Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA
2 Molecular Cell and Biology Laboratory, The Salk Institute, La Jolla, California 92037, USA
(RECEIVED December 6, 2005; FINAL REVISION June 4, 2006; ACCEPTED July 18, 2006)
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Abstract |
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10 sec. Tip LBD1 binding did not cause gross structural changes in Lck SH3 but globally stabilized the domain and reduced the rate of partial unfolding by a factor of five. The region of partial unfolding in Lck SH3 was found to be similar to that identified for other SH3 domains that partially unfold. Although the sequence conservation between Lck SH3 and other closely related SH3 domains is high, the dynamics do not appear to be conserved. Keywords: hydrogen exchange; mass spectrometry; Src-family kinase; EX1 kinetics; protein unfolding
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsElectronic supplemental materialAcknowledgmentsReferences |
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The tyrosine kinase interacting protein (Tip), produced by subgroup C strains of the Herpesvirus saimiri (HVS), binds to and activates Lck. HVS, a 
2-herpesvirus, induces rapid and fatal T-cell lymphoma in many primate species (Fickenscher and Fleckenstein 2001) and is also capable of transforming human T-cells in vitro (Biesinger et al. 1992). In contrast with HVS subgroups A and B, the HVS subgroup C viral genome contains the genes stpC and tip (Ensser et al. 2003). These genes are not required for propagation of the virus but are responsible for oncogenecity (Duboise et al. 1998). HVS-transformed T-cells express the 24–29 kDa Tip protein (Geck et al. 1990, 1991; Biesinger et al. 1995), which binds to and activates Lck both in vitro and in vivo (Biesinger et al. 1995; Jung et al. 1995; Wiese et al. 1996; Hartley et al. 1999, 2000; Kjellen et al. 2002). Tip consists (from N to C termini) of a glutamate-rich region, one or two serine-rich regions (depending on the HVS strain), Lck binding elements referred to as ligand binding domains 2 and 1 (LBD2 and LBD1), and a hydrophobic membrane anchor (Ensser et al. 2003). Together, LBD1 and LBD2 are responsible for binding and activating Lck: LBD1 (also referred to as SH3B) binds to the SH3 domain of Lck (Jung et al. 1995; Hartley et al. 1999) while LBD2 (also referred to as CSKH) binds to the kinase domain of Lck (Hartley et al. 2000). Both LBD1 and LBD2 can independently bind to and activate Lck, but the activation is maximized when both are present (Hartley et al. 2000). In addition to LBD1 and LBD2, it appears that phosphorylated Y127 (HVS C488 numbering) may bind to the Lck SH2 domain (Bauer et al. 2004).
Little is known about Tip secondary and tertiary structure. LBD1 forms a type II polyproline helix (Schweimer et al. 2002) and binds to the SH3 domain of Lck in a prototypical polyproline class II helix/SH3 binding interaction (Mayer 2001). The binding of a proline-rich region of a viral protein to the SH3 domain of a non-receptor tyrosine kinase is not limited to the Tip/Lck interaction. Other examples of such association include the binding to and activation of Hck SH3 by HIV Nef (Lee et al. 1995; Saksela et al. 1995; Briggs et al. 1997; Moarefi et al. 1997; Briggs et al. 2000) and the binding between SH3 domains of several Src family kinases (Lyn, Hck, Src, Fyn, and Yes) and the Herpesvirus ateles protein Tio (Albrecht et al. 1999). Viral proteins such as Tip, Nef, and Tio are believed to activate their SFK targets by displacing the SH3 domain from its down-regulatory interaction with the SH2-kinase linker, thereby activating the kinase (Mayer 2001).
The interaction between Tip LBD1 peptide and Lck SH3 has been investigated by a combination of NMR and molecular modeling (Schweimer et al. 2002; Bauer et al. 2004). Surprisingly, the structure of Lck SH3 in complex with Tip LBD1 is quite similar to the structure of free Lck SH3 (Bauer et al. 2004). Changes in chemical shift were most significant in the RT loop, n-Src loop, and the 
-hairpin, all in close proximity to the modeled binding site (Bauer et al. 2004). Smaller changes in chemical shift were also observed in regions distant from the binding site. It has been hypothesized that the Tip LBD1/Lck SH3 interaction may alter the dynamics of Lck SH3 rather than its conformation.
Changes in the dynamics of an SH3 domain as the result of ligand binding have been observed before. Using hydrogen exchange (HX) and mass spectrometry (MS), we have previously shown that dynamics in the Hck SH3 domain are significantly slowed by ligand binding (Engen et al. 1997; Gmeiner et al. 2001; Hochrein et al. 2006). The SH3 domain of Hck undergoes partial unfolding under physiological conditions with a half-life of 20 min. However, in the presence of a peptide from the HIV Nef protein, the rate of localized unfolding decreases threefold. Interestingly, the secondary structural elements of Hck SH3 involved in the partial unfolding are not directly part of the Nef binding site but are in positions where their unfolding would likely alter the conformation of the Nef binding site (Engen et al. 1997). Given that Hck and Lck are both SFKs and that they both interact with viral proteins that displace the SH3 domain from its down-regulatory position, we speculated that perhaps Lck SH3 underwent partial unfolding that could be modulated by Tip binding. Our data show that indeed Lck SH3 partially unfolds under physiological conditions, albeit with a much faster rate than Hck. The structural elements involved in the unfolding were the same as those involved in Hck SH3 and other SH3 domain unfolding reactions. When Tip LBD1 was incubated with Lck, the rate of partial unfolding decreased significantly and the overall dynamics of the entire SH3 domain were decreased.
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Results |
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H/D exchange mass spectrometry and protein dynamics
The theory of hydrogen/deuterium exchange has been reviewed extensively (Woodward et al. 1982; Englander and Kallenbach 1984), as has its application to mass spectrometry to probe the structure and dynamics of proteins (Smith et al. 1997; Hoofnagle et al. 2003; Wales and Engen 2006a). Briefly, when a protein is incubated in D2O, backbone amide hydrogens in the protein can exchange with deuterium in the solvent. The rate of exchange depends on a number of factors (i.e., pH, ionic strength, solvent accessibility, extent of hydrogen bonding) with solvent accessibility and hydrogen bonding being the factors determined by the protein itself. For example, amide hydrogens on the surface of a protein are replaced by deuterium more rapidly than those buried in the hydrophobic interior. Similarly, amide hydrogens that are not hydrogen bonded will exchange more rapidly than amide hydrogens that are hydrogen bonded. Deuterium uptake, therefore, can be used as a probe of solvent exposure and hydrogen bonding within proteins. More importantly, changes in these parameters, as might accompany ligand binding or dynamic structural fluctuations, can be revealed by hydrogen exchange measurements.
Hydrogen exchange primarily occurs via protein fluctuations in solution (Englander and Kallenbach 1984). The fluctuations (or openings) may range from highly localized (exposing a single residue at a time to deuterium) to more global in nature (exposing many residues simultaneously). Such protein fluctuations occur at a given rate, depending on the protein and the structural environment. The kinetics of hydrogen exchange provide qualitative details about these dynamics. The protein refolding (or closing) rate is normally fast relative to the exchange rate (EX2 kinetics). When the protein refolding (or closing) rate is slow relative to the exchange rate, the unfolded regions become completely deuterated before refolding can occur (EX1 kinetics). In EX2 kinetics the unfolded regions quickly refold before deuterium exchange can occur. Therefore, many unfolding events must occur before the protein population becomes completely deuterated. EX1 kinetics in the absence of denaturants are rare (Englander and Kallenbach 1984).
HX MS can be used to distinguish between EX1 and EX2 kinetics (Miranker et al. 1993; Engen and Smith 2000). In EX2 kinetics, a single population that gains mass over time will be observed. In contrast, EX1 kinetics results in mass spectra that show a bimodal isotope pattern: an undeuterated (lower mass) population and a deuterated (higher mass) population. Over time, the intensity of the undeuterated population decreases, while the intensity of the deuterated population increases, indicating the transition of the population from the folded state to the unfolded state. Although the protein is in continuous equilibrium between the open and closed states, the transition appears unidirectional because a large excess of D2O (>95%) forces the labeling reaction in one direction. Both EX1 and EX2 kinetics can be observed in the same protein if the EX1 kinetics involve only a part of the protein.
To obtain information about protein dynamics, a protein is incubated in D2O and mass spectrometry used to determine the extent and location of deuterium uptake (Zhang and Smith 1993). After a period of exchange, the labeling reaction is quenched by reducing the pH to 2.6 and the temperature to 0°C, thereby slowing the exchange rate by five orders of magnitude (Engen and Smith 2000). Deuterium incorporation can be localized to small regions of the protein by treating labeled protein with pepsin and measuring the amount of deuterium incorporation in each of the peptides. When tertiary structure information is available, the structural location(s) of the deuterium incorporation can be ascertained.
Dynamics in intact Lck SH3
Unbound Lck SH3 was first analyzed with HX MS to determine if its dynamics were similar to other SH3 domains and to provide a reference point for comparison with bound Lck SH3. Lck SH3 was labeled with D2O for between 3 and 1000 sec. A 10-fold molar excess of angiotensin II, a peptide that does not bind to Lck SH3, was added during labeling as a control for non-specific binding. Deuterium incorporation into Lck SH3 in the presence of angiotensin II was the same as in neat Lck SH3 (data not shown). Mass spectra of the isotopic envelope of the +5 charge state of Lck SH3 show a continuous increase in mass over the course of the exchange reaction (Fig. 1A). The deuterium uptake curve (Fig. 2) shows a rapid increase in deuterium that reaches a maximum of 58 Da in 200 sec. The continuous increase in mass is indicative of EX2 kinetics overall and suggests that Lck SH3 is highly dynamic in solution as the domain is almost totally labeled with deuterium after only 200 sec in D2O. This result is in contrast to other SH3 domains (including Hck) which require as much as 10,000 sec to become nearly completely deuterated (Hochrein et al. 2006; Wales and Engen 2006b).
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10 sec. In theory, the peak width maximum will be reached when the two EX1 populations (folded and unfolded protein) are at equal intensity, giving a measure of the half-life of unfolding. Thus, for unbound Lck SH3, the half-life of unfolding is 10 sec. Because the two populations cannot be resolved in the mass spectra, the unfolding must involve only a part of the SH3 domain. Hence, the unfolding observed by EX1 kinetics is considered partial unfolding. Based on fitting two Gaussian distributions with a width equal to the isotope envelope of a peak from a deuterium labeling time outside that of the EX1 kinetics event (Engen et al. 1997; Engen and Smith 2000; Gmeiner et al. 2001; Hochrein et al. 2006; Wales and Engen 2006b; Weis et al. 2006), it is possible to estimate the number of residues involved in the unfolding, even though the two populations are not resolved in the mass spectra. Such an analysis is shown in the inset to Figure 1A for the spectrum at 10 sec, the widest isotopic envelope. The two Gaussians have a separation of 1.45 in the m/z domain, which corresponds to 7.3 Da in the mass domain. This value does not consider back-exchange during analysis (see Materials and Methods). When back-exchange is taken into account, between eight and 10 residues are involved in the partial unfolding event.
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50 sec of labeling rather than 10 sec for unbound SH3. As shown in Figure 3, the peak width maximum (i.e., the half-life of folded SH3) is 50 sec for bound Lck SH3 versus 10 sec for unbound Lck SH3. Thus the rate of local unfolding is five times slower for LBD1 bound Lck SH3 than for unbound Lck SH3. It can be concluded, therefore, that both EX2 and EX1 kinetics are hindered in the presence of the Tip LBD1 ligand.
Localizing partial unfolding in Lck SH3
To localize the unfolding to small regions within the SH3 domain, Lck SH3 was labeled as described above and digested with pepsin to produce peptides of 11–23 residues in length. Five peptides covering 84.7% of the expressed protein and 96.6% of the Lck SH3 sequence were identified (see Fig. 4). Peptic peptides that displayed a similar EX1 kinetic signature to that of the intact protein were of special interest as they allowed the region of partial unfolding to be narrowed (recall that based on analysis of the intact protein, only 8–10 residues participate in partial unfolding). An example of the peptic peptide data is shown in Figure 5 for the peptide covering Lck SH3 residues 44–61. The mass-to-charge ratio of the isotopic envelope for this peptide increases continuously, reaching complete deuteration in 200 sec. The distribution was narrow at times other than near the 10-sec time point, a behavior closely resembling that of the intact Lck SH3 (see Fig. 1A). The presence of EX1 kinetics behavior at the same time as displayed for the intact protein indicates that this region of the protein participates in the partial unfolding event. Spectra of two other fragments (12–34, 16–33) displayed similar behavior (see Supplemental Material).
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8–10 residues. The differences in peak width of the EX1-containing peptides can be used to estimate how many residues in each peptide participate in the partial unfolding. The difference between the peak width (Fig. 7) in the non-EX1 time points (those at the base of the peak in the width graph) and the width in the midst of the unfolding event (time points near the apex of the peak in the width graph) gives an estimate of the number of residues involved in each region (see also Supplemental Material for the raw spectra that show which time points have EX1 kinetic profiles). For example, in the 44–61 peptide, the increase in peak width is approximately four residues for unbound Lck SH3 and 2.5 residues for bound SH3. As these values are not adjusted for back-exchange the true numbers may be as high as five and three, respectively. After applying the same method to the 16–33 peptide, it appears that there are approximately three to four residues in the 16–33 peptide and four to five residues in the 44–61 region that are involved in partial unfolding. There may be one residue participating in the 34–43 region while no residues are involved in the 62–72 region. The unfolding region in Hck SH3 was in a similar location (Fig. 8B) (Engen et al. 1997), as was that of the Lyn and 
-spectrin SH3 domains (Wales and Engen 2006b).
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsElectronic supplemental materialAcknowledgmentsReferences |
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10 sec. This unfolding rate is in stark contrast to that observed for Hck SH3 (17 min) (Engen et al. 1997) and in the middle of the range observed for a battery of other SH3 domains (range, 1 sec to 1 h) (Wales and Engen 2006b). When Tip LBD1 binds to Lck SH3, the partial unfolding behavior persists but is slowed fivefold. The effects of the interaction between Lck SH3 and Tip LBD1 are not limited merely to the Tip LBD1 binding site (Schweimer et al. 2002; Bauer et al. 2004), but instead the domain appears to be globally stabilized. Similar behavior has been observed for other interactions between SH3 domains and their ligands, for example the interaction between the Sos peptide and the N-terminal drk SH3 domain (Zhang and Forman-Kay 1997). Although the dynamics of Lck SH3 are slowed in the presence of Tip LBD1, there is no evidence of a gross conformational change in the SH3 domain. A large conformational change would be evident if some regions of Lck SH3 showed either much more or much less deuterium incorporation in the presence of Tip LBD1 in the initial or at later time points. Since the H/D exchange curves for all regions (with the exception of the C-terminal tail) of Lck SH3 show exactly the same amount of initial deuterium incorporation and the same final amount of incorporation, there is no evidence that any of the regions of Lck SH3 undergo a change in their solvent exposure or hydrogen bonding upon Tip LBD1 binding. Instead, the rate of incorporation was altered, implying a change in dynamics rather than overall conformation. These results are consistent with an NMR investigation of this interaction in which no differences were observed between the solution structures of Lck SH3 and Lck SH3 in the presence of a Tip LBD1 peptide (Schweimer et al. 2002). Similar behavior has also been observed in the complex between Tip LBD1 peptide and Lyn SH3 (Bauer et al. 2005).
Peptic digestion after deuterium exchange narrowed the region of partial unfolding to two parts of the domain: the RT-loop and the C–D strands (see Fig. 8A). Analysis of other SH3 domains (Engen et al. 1997; Wales and Engen 2006b) has shown that partial unfolding occurs in nearly the same location in Hck SH3, Lyn SH3, and -spectrin SH3. There is something unique about these regions of the SH3 domain, and Lck SH3 is no exception. It remains unclear if a functional role can be attributed to the unfolding and if the location has any relation to a functional outcome. Because the viral protein Tip slows the unfolding upon binding, as does the viral protein Nef when bound to the Hck SH3 domain (Engen et al. 1997; Hochrein et al. 2006), it may be that reduced dynamics in the SH3 domain favors association with the viral protein over intramolecular interactions in the down-regulated full-length Lck kinase, thereby preferentially maintaining the active state when SH3 is bound to the viral protein. Experiments are in progress to measure the dynamics of Lck SH3 in full-length, down-regulated Lck to determine if this speculation is correct.
A valuable outcome of this work is the development of a binding assay for the Lck SH3 domain that can be used in a multitude of contexts. The degree of Lck SH3 binding can be determined by monitoring the decrease in the rate of partial unfolding in bound versus unbound states. Such a measurement may be performed with the entire Lck SH3 domain or any of the peptides involved in the unfolding event. Based on monitoring the dynamics of Lck SH3, we will be able to tell if the SH3 domain associates with its natural linker in various states of activation in the full-length kinase including those which involve interactions with the Tip protein.
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Materials and methods |
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6 kDa. The identity and purity of the Lck SH3 protein were confirmed by electrospray mass spectrometry (observed mass, 7928.9 Da; theoretical mass, 7928.7 Da). The protein was lyophilized and stored at –80°C. Prior to use in HX MS experiments, lyophilized Lck SH3 was reconstituted in water, exchanged into TSD buffer (25 mM Tris, 100 mM NaCl, 3 mM DTT, H2O at pH 7.5) using a 5 mL HiTrap desalting column (Amersham) at 4°C, and concentrated to 190–250 pmol/µL using a Centricon YM-3 spin concentrator (Amicon) at 4°C. The protein concentration was determined by a modified Bradford assay (Bradford 1976) (BioRad 500–0006).
Peptide preparation
Tip LBD1 peptide (Ace-ATWDPGMPTPPLPPRPANLG-NH2), encompassing residues 168–187 of HVS C488 Tip, was synthesized by Sigma-Genosys at 93% purity. Angiotensin II peptide (DRVYIHPF), used here as a negative control, was obtained as a lyophilized powder at 99% purity from Sigma. Both peptides were used as supplied.
Deuterium labeling
H/D exchange methods are similar to methods previously described (Engen et al. 1997, 1999; Engen 2003; Hochrein et al. 2006; Wales and Engen 2006b). Briefly, 8–10 nmol of Lck SH3 were mixed with a 10-fold molar excess of Tip LBD1 or angiotensin II and allowed to equilibrate for 1–3 h in TSD buffer at ambient temperature. Small aliquots (1–2 µL) containing 350 pmol of Lck SH3 and 3500 pmol of peptide were rapidly diluted 20-fold with deuterium-labeling buffer (25 mM Tris, 100 mM NaCl, 3 mM DTT, D2O at pH 7.5, 21°C) to initiate H/D exchange. Undeuterated samples were prepared by substitution of TSD buffer for the deuterium-labeling buffer. After time intervals of 3–1000 sec, the H/D exchange reaction was quenched by addition of an equal volume of quench buffer (200 mM potassium phosphate, H2O at pH 2.6). Each sample was immediately frozen on liquid nitrogen and stored at –80°C until analysis. To minimize variability two independent sets of samples were prepared using the same peptide stock solutions and buffers. All samples in a given set were prepared concurrently and analyzed in a single day as described below.
Intact protein analysis
Each hydrogen exchange sample was rapidly thawed at 0°C, desalted, and concentrated by HPLC prior to mass analysis. HPLC was carried out with a Shimadzu HPLC system with water and acetonitrile (both containing 0.05% TFA) as the mobile phases. The protein was desalted and concentrated on a 5 µL protein trap (Michrom BioResources) using 15% B at 200 µL/min for 3 min and 50 µL/min for 30 sec (this step also removed the Tip LBD1 or angiotensin II peptide). The desalted protein was directed at 50 µL/min with a 2 min 15%–65% B gradient into a Waters QTOF2 mass spectrometer for mass analysis. To minimize deuterium back-exchange, the sample loop, injection valve, and trap were placed in an ice bath (Zhang and Smith 1993). Myoglobin was infused into the mass spectrometer after each sample for mass calibration. The relative deuterium level was determined by subtracting the mass of undeuterated Lck SH3 from the mass of deuterated Lck SH3 at each exchange time point. No correction was made for deuterium back-exchange (Zhang and Smith 1993) and all values are therefore reported as relative deuterium levels. The average amount of back-exchange using the experimental setup just described was 12%–15% based on analysis of highly deuterated protein standards. The width of the Lck SH3 isotopic distribution was determined at 20% peak height using the +5 charge state and converted from m/z to molecular mass.
Peptic peptide analysis
To localize deuterium exchange within the Lck SH3 domain, labeled and quenched protein was digested with pepsin in solution prior to analysis. The post-digestion analysis was similar to that described above for intact protein. Immediately after thawing, the protein sample was digested at 0°C for 5 min with porcine pepsin (1:60,000 grade, Sigma) at a 1:1 (w/w) ratio. The sample was then injected onto a C18 column (Magic C18, 1.0 mm x 50 mm, 5 µm particles, 200 Å pore, Michrom BioResources). The peptic peptides were desalted and concentrated with 5% acetonitrile for 1 min at 100 µL/min and 30 sec at 50 µL/min and then eluted at 50 µL/min with a 5%–60% acetonitrile gradient over five minutes into the same mass spectrometer as was described for intact protein analysis. In addition to placing the loop, valve, and column on ice, the mobile phases were cooled by passing them through stainless steel coils immersed in the ice bath. The average mass of each peptide was determined by measuring the centroid of the isotopic distribution of the most easily detected charge state. The relative deuterium level was determined as described in the intact protein analysis section. The average amount of back-exchange using the experimental setup described was 18%–20% based on analysis of highly deuterated peptide standards. As pepsin is a non-specific protease, the peptic peptides were identified in a separate experiment. Lck SH3 was digested with pepsin as described above, the peptides were collected at the column outlet and identified offline by MS/MS. Prior to analysis, the peptides were concentrated to near dryness on a rotary concentrator (ThermoSavant SpeedVac), and dissolved in water/acetonitrile/formic acid (1:1:0.2%, v/v/v). The peptides were then infused into a Waters QTOF2 mass spectrometer through a Nanospray ESI probe and subjected to MS/MS analysis.
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Electronic supplemental material |
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Footnotes |
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4 The Barnett Institute, Northeastern University, Boston, MA 02115, USA.
Supplemental material: see www.proteinscience.org
Reprint requests to: John R. Engen, The Barnett Institute, 341 Mugar Life Sciences, Northeastern University, 360 Huntington Ave., Boston, MA 02115, USA; e-mail: j.engen{at}neu.edu; fax: (617) 373-2855.
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.052016406.
Abbreviations: HVS, Herpesvirus saimiri; Tip, tyrosine kinase interacting protein from HVS; LBD1, ligand binding domain 1 from Tip; Lck, lymphocytic cell kinase; SH3, Src homology 3; HX MS, hydrogen exchange/mass spectrometry.
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References |
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= 0.807 in the m/z domain) as the 1000 sec unbound +5 isotopic envelope and are centered at 1593.45 and 1594.90 in the m/z domain.
) or Tip LBD1 (•). The Lck SH3:peptide ratio was the same as in 
