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1 Department of Chemistry, University of Nebraska–Lincoln, Lincoln, Nebraska 68588, USA
2 Department of Plant Pathology, University of Nebraska–Lincoln, Lincoln, Nebraska 68588, USA
Reprint requests to: Dr. David L. Smith, Department of Chemistry, University of Nebraska–Lincoln, Lincoln, Nebraska 68588-0304, USA; e-mail: dls{at}unlserve.unl.edu; fax: (402) 472-9402.
(RECEIVED January 2, 2001; FINAL REVISION March 14, 2001; ACCEPTED March 28, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps. 100101
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Keywords: Brome mosaic virus; amide hydrogen exchange; mass spectrometry; viral capsid
Abbreviations: BMV, brome mosaic virus • CCMV, cowpea chlorotic mottle virus • TBSV, tomato bushy stunt virus • CP, capsid protein • EM, electron microscopy • ESI, electrospray ionization • GdHCl, guanidine hydrochloride • HX, hydrogen exchange • HPLC, high performance liquid chromatography • MS, mass spectrometry • m/z, mass-to-charge ratio • TFA, trifluoroacetic acid
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Introduction |
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X-Ray diffraction and electron microscopy (EM) combined with image reconstruction have been the principal tools used to detect structural changes in viral capsids (Giranda et al. 1992; Mancini et al. 1997). Although NMR may be used to determine the structures of some capsid proteins as isolated subunits, it has not been suitable for high-resolution structural studies of assembled virus particles. The high-resolution structures derived from X-ray diffraction provide an important starting point for our understanding of the relation between structure and function. However, these structures provide only a static picture of the virion under non-physiological conditions. Other methods, such as ultracentrifugation (Incardona and Kaesberg 1964), limited proteolysis (Chidlow and Tremaine 1971; Lewis et al. 1998), and various spectroscopic methods (Vriend et al. 1982; Tuma and Thomas 1997; Leimkuhler et al. 2000; Perez et al. 2000), have been used to detect structural changes in viral capsids. Although the spatial resolution of these methods is low, they are essential tools for studies aimed at relating viral structure and function and are compatible with a wide range of experimental conditions.
Isotope exchange at peptide amide linkages is another method for detecting protein structural changes under diverse conditions (Englander et al. 1997; Li and Woodward 1999). Although hydrogen exchange (HX) has most often been detected by NMR, detection by mass spectrometry (MS) has become increasingly important (Smith et al. 1997; Coyle et al. 1999; Engen et al. 1999; Halgand et al. 1999). The rates at which hydrogens located at peptide amide linkages in proteins undergo isotopic exchange are highly sensitive to protein structure. Principal factors affecting hydrogen exchange rates include intramolecular hydrogen bonding and solvent accessibility. Changes in these structural features have been detected by analyzing the intact protein. Although this approach gives important information on the extent and cooperativity of structural changes, it provides no information on the locations of structural changes. However, the spatial resolution of HX MS can be substantially improved when the labeled protein is fragmented prior to mass analysis (Zhang and Smith 1993).
Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are simple plant viruses with similar capsid structures (Krol et al. 1999).Both viruses have 180 chemically identical coat protein subunits arranged to form an icosahedral surface. The capsid volume of both BMV and CCMV increases as the pH is increased from 5 to 7 (Lane 1981). Understanding the molecular basis of capsid swelling, which is a common feature of many plant viruses, is required for our understanding of capsid assembly and disassembly processes. Combining the X-ray structure of the native virus with the cryo-electron microscopy (cryo-EM) image of the swollen CCMV has provided detailed structural information on the pH-induced swelling of CCMV (Speir et al. 1995). Such information has not been reported for BMV until now.
The present study explores the limits of HX MS for detecting structural changes in viral capsids. Results of this study also establish the similarity of mechanisms responsible for pH-induced swelling in BMV and CCMV. This report includes a description of a method for separating the effects of pH on capsid structure and the intrinsic rate of amide hydrogen exchange. This approach was used to follow hydrogen exchange in intact BMV equilibrated at pH 5 and 7. The labeled virus capsid was rapidly disassembled under conditions that minimized isotope exchange. Changes in deuterium levels in the labeled capsid protein and its peptic fragments were used to identify regions undergoing structural change with expansion of the capsid.
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Results |
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A similar peptide-mapping approach was used to identify peptic fragments formed when the BMV capsid protein was digested under conditions used to quench isotope exchange (0°C, pH 2.5). Digestion with pepsin produced approximately 30 peptic fragments, which covered 99% of the BMV capsid protein sequence. Identification of these fragments was verified by CID MS/MS and C-terminal sequencing. The correct amino acid sequence of this BMV isolate and the peptic fragments used in this study are presented in Figure 1
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:
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where kex is the phenomenological rate constant for isotope exchange and t is the time the protein was exposed to D2O. The phenomenological rate constant may be expressed as follows:
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where Kunf is an equilibrium constant describing structural changes that facilitate isotope exchange, kint is the intrinsic isotope exchange rate constant, and kOH is the rate constant for base-catalyzed isotope exchange. The intrinsic exchange rate constant, kint, is given by the product of kOH and [OH-] (Bai et al. 1993). Insertion of Kunf, which may be viewed as the probability for various structural changes that enable isotope exchange in a folded polypeptide, provides the link between hydrogen exchange rates and protein structure. Structural changes enabling isotope exchange may be global unfolding or minor fluctuations that allow exchange at individual amide linkages. The goal of this research was to assess changes in Kunf as the pH was increased from 5 to 7. However, equation 2
indicates that kint at pH 7 is 100-fold greater than it is at pH 5. Substituting equation 2 into equation 1 gives equation 3:
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which indicates that pH-induced structural changes in folded proteins may be detected by hydrogen exchange if the exposure times were chosen so that the products of [OH-] and t were constant. This general approach has been used previously to compare hydrogen exchange results obtained at different temperatures (Zhang and Smith 1993 Zhang and Smith 1996).
The experimental procedure used to detect pH-induced swelling of the BMV capsid is illustrated in Figure 2. To initiate isotope exchange, solutions of equilibrated intact BMV were diluted 20-fold into two exchange buffers. The pD of these labeling solutions was either 5.43 or 7.30. Following various labeling times, the solution was acidified both to quench the isotope exchange reaction and to disassemble the virus particles. The labeled capsid protein was analyzed either as the intact protein or as its peptic fragments by HPLC ESIMS, always maintaining conditions required to quench isotope exchange. Changes in deuterium levels found following labeling at pD 5.43 or 7.30 were used to detect structural changes in the capsid. To null the dependence of kint on pD, the labeling times were chosen to keep the product of [OH-] and t constant. That is, the labeling time for pH 5.43 was 74.1 times greater than the labeling time used for pH 7.30. Incubation times used in this study are given in Table 1.
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indicate the deuterium levels when the intact virus particles were labeled at pD 7.30 for 0.33–96 min. These results, which have been adjusted for deuterium loss (11%) during the HPLC step, reflect the number of deuteriums located at peptide amide linkages. Deuterium located in the side chains, as well as at the N and C termini, was lost during HPLC. The results in Figure 3 show that the deuterium level rose sharply during the shortest labeling times, but reached a level of only 123 after labeling for 96 min at pD 7.30. Finding only 123 deuteriums in this protein that has 180 amide hydrogens (7 of 188 residues are proline) indicates that isotope exchange was incomplete on this time scale. These results show that exchange in the virus particle occurs over a time scale of several orders of magnitude, which is typical for folded proteins.
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(open data points). The time scale used to plot these results has been divided by 74.1 to adjust for the different values of kint at pD 5.43 and 7.30. Although the general behavior found for hydrogen exchange at pD 5.43 and 7.30 is similar, the deuterium levels at pD 7.30 are much greater than the deuterium levels found at pD 5.43. This difference (15%–25%) shows that the BMV capsid structures at pD 5.43 and 7.30 are substantially different. The higher level of deuterium found at pD 7.30 indicates that the structure at pD 7.30 is more open or flexible than the structure at pD 5.43. The estimated uncertainty in these measurements is represented by the size of the data points in Figure 3.
Analysis of peptic fragments of labeled capsid protein
To detect localized structural changes in the BMV coat protein, the labeled virus was disassembled into the monomer subunits, which were digested with pepsin and analyzed by directly coupled HPLC ESIMS. The digestion and HPLC were performed under conditions that minimized isotope exchange at peptide amide linkages (Bai et al. 1993). Deuterium levels in the peptic fragments were deduced from their molecular masses. The extent of artificial exchange occurring during digestion and HPLC was determined by analyzing under the same conditions BMV coat protein that either had no deuterium or was completely exchanged in D2O. Although the extent of artificial exchange depended on the amino acid sequences of the peptides, the deuterium recovery averaged over all peptic fragments was 67%.
The deuterium levels found in these peptic fragments, following labeling of the intact BMV particles in D2O at pD 5 and 7, were used to detect pH-dependent changes in the structure of the capsid protein. Mass spectra of one representative peptic fragment, including residues 123–135, are presented in Figure 4. These spectra, as well as those of all peptic fragments used in this study, exhibited a single envelope of isotopic peaks, consistent with a high level of structural homogeneity (Miranker et al. 1993; Zhang et al. 1996). That is, isotope exchange in backbone regions represented by any peptic fragment was the same for all molecules, indicating a high level of structural homogeneity in the 180 monomers comprising the BMV capsid. The deuterium levels in this peptic fragment (Fig. 4, boxes) were determined from the centroids of the envelopes of isotope peaks. Results presented in Figure 4 show how the deuterium level in the segment including residues 123–135 increased with labeling time. The labeling times, which are given next to each spectrum, were chosen so that the labeling time at pD 5.43 was a factor of 74.1 greater than the labeling time at pD 7.30 (see Table 1). To detect structural changes in the region including residues 123–135, the deuterium level found in this segment from BMV incubated for 74 min at pD 5.43 is compared with the deuterium level found in the same segment from BMV incubated for 1 min at pD 7.30. The data in Figure 4 show that the deuterium level increased from 4.9 to 7.6 when the pD was increased from 5.43 to 7.30. This increase in deuterium level indicates a general loosening of the BMV capsid protein in the region including residues 123–135.
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indicates the intermolecular distribution of deuterium in this region of the protein. Peak widths presented in Figure 4 show that the distribution is broader for labeling at pD 7.30 than it is at pD 5.43. The broader peak at the higher pD is consistent with the higher levels of deuterium found under these conditions. As discussed previously, the peak width is attributable to the number of amide linkages that are only partially deuterated (Zhang et al. 1996).
Differences in deuterium incorporation at pD 5.43 and 7.30 are presented in Figure 5 for a set of 16 segments that cover nearly the entire backbone of the BMV capsid protein. Many additional peptides with amino acid sequences overlapping with these peptides gave similar results. The spatial resolution, which is generally limited by the lengths of the peptic fragments, was enhanced in three regions by using the differences in deuterium levels found in overlapping peptides. The change in deuterium level, indicated by the Y axis in Figure 5, is expressed as the percent of the total number of amide hydrogens in each peptic fragment. As indicated by the results presented in Figure 4, the pH-induced change in deuterium levels changed with the labeling time. To simplify the discussion, only the results for labeling times where the change was greatest are presented in Figure 5. The change in the number of deuteriums found in each segment at the different pDs is given above each bar.
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Results presented in Figure 5 show that most segments (residues 1–170, 90% of the protein backbone) exhibited increased deuterium levels at pD 7, suggesting a looser or more flexible structure of the protein subunits in the swollen capsid. However, peptides corresponding to residues 171–188 in the C terminus appear to incorporate less deuterium at pH 7 than at pH 5, indicating a more compact or ordered structure in this region. Among the segments exhibiting increases in deuterium, three categories were defined based on the magnitude of the change. Moderate deuterium changes (less than 10%) were found for segments including residues 48–61, 91–108, and 166–170, indicating minimal structural changes. Intermediate increases (10%–20%) were found for segments including residues 1–47, 62–69, 109–118, and 152–165, indicating more substantial structural changes. Large increases in deuterium level were found in segments including residues 70–90 and 118–151, indicating extensive structural changes. These three categories of exchange are indicated by shading in Figure 5.
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Discussion |
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. Although all 180 subunits have nominally identical structures, symmetry considerations lead to designation of three groups, A, B, and C. The A and B groups make up the hexamers, and the C groups make up the pentamers. The icosahedral threefold (quasi-sixfold) and fivefold axes of the hexamers and pentamers are illustrated in Figure 6 as triangles and pentagons, respectively. Points of twofold symmetry arising from dimers are indicated as ovals. The threefold axis at the center of the icosahedral asymmetric unit (central triangle in Fig. 6) is not exact (quasi-threefold axis) because of the different rotation axes outside of its local environment.
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). The N- and C-terminal arms of the capsid subunits interweave into a network to stabilize the capsid. For example, six N-terminal short ß-strands cluster into a parallel ß-tube structure, which stabilizes the hexameric units. The extended C-terminal arm (residues 180–190) appears to interact with the adjacent twofold related subunit by penetrating into a gap formed by the N-terminal arm and the ß-barrel core.
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Our most detailed structural information on the swelling of bromoviruses comes from fitting the 28-Å resolution cryo-EM density map of the swollen capsid with the high-resolution atomic model of individual subunits of CCMV coat protein (Speir et al. 1995). This model indicates the presence of large holes at the center of the quasiequivalent trimer (Fig. 6, triangle at the trimer interface). Putative calcium-binding sites (small circles in Fig. 6) consisting of clusters of aspartic and glutamic acid residues were identified in the protein trimer interface. These acidic residues were assumed to be protonated at pH 5 but deprotonated at pH 7, creating electrostatic repulsion that triggered expansion of the capsid. However, pentamers and hexamers retain their shapes, and the dimeric protein–protein interactions remain largely intact in the swollen capsid. One of the goals of the present study was to determine whether a similar model may be used to explain acid-induced expansion of the BMV capsid.
Structural changes in BMV capsid monomers
To locate structural changes within BMV capsid subunits, the amino acid sequences of the CCMV and BMV coat protein were aligned. The high sequence identity (70%) of the proteins permits alignment with minimal ambiguity, indicating that the subunit structure of BMV may be represented by the atomic coordinates for CCMV. A similar scheme has been used to locate modified residues in the capsid of a BMV mutant (Flasinski et al. 1997).
Specific regions of the BMV backbone are colored in Figure 7a to indicate four categories of change in the deuterium levels found when the pH was increased from 5 to 7 (see Fig. 5). Segments exhibiting the largest increases (>20%) are colored red, and segments with intermediate (10%–20%) or minimal increases (<10%) in deuterium are indicated by yellow and green, respectively. Regions labeled blue in Figure 7a had decreased levels of deuterium at pH 7. The color scheme in Figure 7a offers a qualitative, low-resolution view of structural changes in various regions of BMV coat protein as the pH was increased from 5 to 7. The strong dependence of amide hydrogen exchange rates on intramolecular hydrogen bonding and access to the solvent suggests that the increased hydrogen exchange rates correlate with expansion of the viral capsid. The widespread distribution of regions exhibiting increased hydrogen exchange indicates that expansion of the capsid at the higher pH involves loosening of the structure along much of the capsid protein backbone.
Regions exhibiting different levels of hydrogen exchange can be related to specific structural elements. For example, the largest increases in deuterium (shown in red) occurred in backbone segments including three 
-helices (CD, most of EF and GH), two ß-strands (ßF and ßG), and loops that connect them. Intermediate changes in deuterium levels were found in the extended N-terminal arm including part of ßC, ßH, the HI loop, and part of EF (shown in yellow). The yellow ß-barrel segments were found adjacent to the red regions, indicating that the larger structural changes in the red areas caused weakening of the ß-barrel fold. However, finding only small changes in several ß-strands (green regions including ßB, ßD, ßE, and ßI) shows that the major part of the canonical antiparallel ß-barrel core remained essentially intact in the expanded form of the capsid.
The increased deuterium level in the N terminus may reflect weakening of the ß-hexameric structure that stabilizes the hexamer. Modeling of X-ray and electron microscopy results indicates that the parallel ß-tube is partially disrupted in swollen CCMV (Speir et al. 1995). In addition, proteolysis experiments of swollen BMV produced primarily N-terminal peptides, suggesting increased exposure of the N terminus in the expanded virus (Chidlow and Tremaine 1971; Agrawal and Tremaine 1972). However, the expanded BMV capsid, though loosely structured, is still an assembled form, suggesting that the protein–protein hexameric interaction is retained in the swollen virus.
Two fragments in the C terminus including residues 171–188 (blue in Fig. 7) exhibited decreased levels of deuterium at pH 7. This decrease in hydrogen exchange suggests tightening of structure in the vicinity of the C terminus as the virion expands. The CCMV crystal structure indicates that the C terminus (residues 181–190) is clamped between the N-terminal arm (residues 36–54) and the ß-strand wall (ßB-I-D-G) of the adjacent twofold related protein subunit. This inter-subunit interaction may be the principal factor responsible for the high stability of dimers. A closer look at the exchange data from the clamping site also supports strong dimer interactions. The clamping site consists of a ß-strand wall (ßB-I-D-G) and the N-terminal arm. The ßB, ßI, and ßD strands (shown in green in Fig. 7) showed minimal deuterium change, indicating the stability of the ß-wall structure. The deuterium level in the peptide segment containing the ßB strand decreased slightly (see residues 48–61 in Fig. 5). Although the deuterium level in the long N-terminal segment increased with swelling of the virus, it is possible that exchange in some regions within this segment decreased with capsid expansion.
The present hydrogen exchange results suggest that this inter-subunit interaction remains strong in the expanded virus. This view is consistent with results obtained by other methods. Cryo-EM of swollen CCMV indicates a strong protein–protein dimeric interaction (Speir et al. 1995). Increased structure within the N-terminal segment with virion expansion also agrees with an X-ray study of the swollen state of tomato bushy stunt virus (TBSV), which suggests formation of new secondary structural elements in the N terminus in a similar pH-induced swelling process (Robinson and Harrison 1982). This persistent interaction of the C terminus of one subunit with a pocket in an adjacent subunit is consistent with the high stability of the dimer in solution after disassembly of the swollen virus (Bancroft 1970).
Structural changes in the BMV capsid trimer interface
Both cryo-EM studies of CCMV (Speir et al. 1995) and crystallographic studies of swollen TBSV (Robinson and Harrison 1982) indicate that large structural changes occur in the trimer interface with swelling. This region is indicated by the triangle joining two hexamers and one pentamer in Figure 6. To examine the relation between the present hydrogen exchange results and structural changes in the trimer interface of BMV, a ribbon plot of the three subunits (A, B, and C) comprising this interface is presented in Figure 7b. The quasi-threefold axis is located at the center of Figure 7b. The color scheme, which is the same as used in Figure 7a, shows how the deuterium levels changed with swelling of the BMV capsid. This presentation shows that regions with the largest increases of deuterium surround the trimer interface, indicating that weakening of protein–protein interactions in the trimer interface is an important part of the swelling and possibly disassembly processes in BMV.
Insight on the general mechanism for pH-induced swelling of BMV and related plant viruses may be traced to early titration experiments, which suggested that a group of acidic residues (Asp and Glue) had anomalously high pKas (Bancroft 1970). The abnormally high pKa of these carboxyl groups probably reflects the clustering effect of acidic groups and the presence of phosphate residues from RNA in the vicinity. The X-ray crystal structure of CCMV indicates clusters of carboxyl groups between neighboring subunits in the capsid quasiequivalent trimer interface (Speir et al. 1995). These carboxyl clusters, each containing three acidic residues (two Glu and one Asp) and two Glns, were modeled as calcium-binding sites. Aligning the sequence of BMV and CCMV coat proteins also led us to similar carboxyl clusters (Glu 81, Glu 85, Asp 149, and Gln 155) in the trimer interface. It is noteworthy that the helical structures containing these carboxyl groups are also the regions with the largest deuterium increases (Fig. 7b). This observation with the titration results points to a swelling model in which deprotonation increases the number of carboxylate anions that repel each other, displacing near-by structural units. Finding the largest increases in hydrogen exchange in the trimer interface indicates that a similar mechanism may be responsible for swelling of BMV.
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This study points to a potentially important role for HX MS in studies of viral capsids. Although X-ray crystallography and cryo-EM with image reconstruction continue to be the principal tools for high-resolution structural studies of viruses, HX MS will be particularly useful for studying virions under a wide range of physiological and nonphysiological conditions. Potential applications of hydrogen exchange and mass spectrometry are numerous. For example, some antiviral agents bind to specific regions of viral capsids, blocking certain steps in the virus life cycle such as uncoating or cell attachment (Badger et al. 1988). Binding of antiviral agents normally induces conformational changes in the binding pocket as well as other areas of the coat protein. The present results suggest that HX MS may be ideal for determining the fingerprint of a particular type of drug binding and may prove useful for drug screening.
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Peptide mapping using trypsin, Lys C, and pepsin
Trypsin digestion of BMV coat protein was performed at 20°C (0.05 M phosphate buffer at pH 7.5) with an enzyme/substrate (E/S) weight-to-weight ratio of 1/50. Digestion time was only 10 min to limit the digestion in the N terminus, which would otherwise be cut into many tiny pieces because it is rich in arginine and lysine residues. Digestion with Lys C was performed at 37°C and pH 7.5 for 2.5 h (E/S = 1/50). Pepsin digestion was performed for 5 min (E/S = 1/1) using conditions required to minimize hydrogen exchange (0°C, 0.1 M phosphate buffer at pH 2.4). Peptides in the digests were fractionated by HPLC (C4 column, 4.6 x 250 mm, 0%–60% acetonitrile in 60 min, 0.1 % TFA) and collected for analysis by ESIMS (Finnigan LCQ ion trap). Peptides were identified by a combination of molecular mass analysis, collision-induced dissociation (CID) MS/MS, and C-terminal sequencing using carboxypeptidase B and Y. These measurements were made with a Finnigan LCQ ion trap mass spectrometer.
Hydrogen exchange/mass spectrometry
The general procedure used to detect structural changes in the BMV capsid by hydrogen exchange is illustrated in Figure 2. Intact virus particles (30 mg/mL) were equilibrated overnight in H2O buffer (100 mM phosphate at pH 5.1 or 7.5) prior to 20-fold dilution into D2O buffer (10 mM phosphate at pD 5.43 or 7.30) to initiate HD exchange. Reported pH and pD values were taken directly from the meter without correction. Exchange times used for labeling at pD 7.30 spanned the range from 20 sec to 1.5 h. To cancel the effect of pH on the intrinsic rate of exchange, the exchange time at pD 5.43 was 74.1 times greater than the exchange time at pD 7.30. Exchange times used in this study are given in Table 1. A typical error of measuring pH in this study was 0.02, which would cause deviations in exchange rate constants of less than 5%. Following incubation in D2O, an aliquot of the labeled BMV was mixed 1 : 1 with the D2O quench buffer (0°C, 0.2 M phosphate at pD 2.4, 6 M GdHCl) to quench isotope exchange. The GdHCl accelerated disassembly of the BMV capsid.
Intact labeled BMV coat protein was analyzed with a Finnigan LCQ mass spectrometer coupled to microbore reverse phase HPLC (C4, 1 x 50 mm; 0°C; 30%–70% acetonitrile in 4–5 min; 0.05% TFA; 40 µL/min). Approximately 30 µL (400 pmoles) of sample were injected. Because protic solvents were used for HPLC, labile deuterium in side chains and N- or C-terminal ends was replaced with protium (Englander et al. 1985; Zhang and Smith 1993). Deuterium back exchange from amide linkages was minimized by cooling the chromatographic system to 0°C, maintaining a pH of 2.4, and using a steep elution gradient. Two control samples were analyzed under the same conditions to assess the extent of exchange occurring during analysis, despite maintaining quench conditions. Results for these control samples, undeuterated protein (m0%), and totally exchanged protein (m100%) were used to adjust for the artificial exchange that occurred during analysis (Zhang and Smith 1993). The undeuterated control sample was prepared by diluting a solution of BMV into D2O buffer under quench conditions (0°C at pH 2.4). The completely exchanged control sample was prepared by diluting a solution of BMV into D2O phosphate buffer at pD 2.4 and incubating for 48 h at 37°C.
To investigate hydrogen exchange in different regions of the BMV capsid protein, the labeled virus was diluted twofold (0.1 M phosphate buffer at pH 2.4), digested with pepsin (E/S = 2/1 w/w, 5 min, 0°C), and analyzed by HPLC ESI MS. The peptides were separated by reversed phase chromatography (C18 1 x 50 mm; 0°C; 2%–60% acetonitrile in 6 min; 0.05% TFA; 40 µL/min). Adjustments for a small amount of deuterium loss during digestion and HPLC were made using results obtained for each of the control samples, as described above. This artificial loss of deuterium was 15%–20% in most peptides. The MS analyses were performed with a Micromass Autospec magnetic sector mass spectrometer equipped with a focal plane detector and standard ESI interface. Data were processed by centroiding isotopic distributions corresponding to the +1, +2, or +3 charge states of each deuterated peptide.
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Acknowledgments |
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. The authors also thank Y.M. She and K. Standing for sharing unpublished results on the sequence of BMV. Part of the manuscript was prepared while D.L. Smith was on sabbatical leave at the Institute for Structural Biology in Grenoble, France. This work was supported by the National Institutes of Health (RO1 GM40384) and the Nebraska Center for Mass Spectrometry. 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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References |
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