Amide hydrogen exchange shows that malate dehydrogenase is a folded monomer at pH 5

Abstract

Although there is general agreement that native mitochondrial malate dehydrogenase (MDH) exists as a dimer at pH 7, its aggregation state at pH 5 is less certain. The present amide hydrogen exchange study was performed to determine whether MDH remains a dimer at pH 5. To detect pH-induced changes in solvent accessibility, MDH was exposed to D₂O at pH 5 or 7, then fragmented with pepsin into peptides that were analyzed by mass spectrometry. Even after adjustments for the effect of pH on the intrinsic rate of hydrogen exchange, large increases in deuterium levels were found at pH 5 only in peptic fragments derived from the subunit binding surface of MDH. In parallel experiments, elevated deuterium levels were also found in the same regions of MDH monomer trapped inside a mutant form of the chaperonin GroEL. This selective increase in hydrogen exchange rates, which was attributed to increased solvent accessibility of these regions, provides new evidence that MDH is a monomer at pH 5.

Results of an early study of malate dehydrogenase (MDH) showed that this enzyme is a homodimer (Devenyi et al. 1966). Later studies showed that the dimer dissociates (K_D ≈ 200 nM) into monomers at low concentrations of MDH (Shore and Chakrabarti 1976; Bleile et al. 1977). However, other groups have reported finding no evidence for dissociation of the monomer (Frieden et al. 1978; Jaenicke et al. 1979; Sanchez et al. 1998). Their results indicate that the dissociation constant for the MDH dimer is less than 3 nM. Additional studies have shown that dissociation of MDH depends on pH. Results from size-exclusion chromatography (Bleile et al. 1977; Wood et al. 1978), analytical ultracentrifugation (Hodges et al. 1977), and intrinsic fluorescence (Wood et al. 1981) indicate that MDH dissociates to monomers at pH 5. However, results of a recent fluorescence polarization study indicate that MDH does not dissociate at pH 5 (Sanchez et al. 1998).

Resolution of these opposing views of the quaternary structure of MDH is important for our understanding of the mechanism of its function and for studies in which it has been used as a model to study the role of subunit interactions in enzymology (Wood et al. 1981; Steffan and McAlister-Henn 1991; Chen and Smith 2000). To help resolve this controversy, hydrogen exchange and mass spectrometry were used in the present study to investigate the quaternary structure of MDH at pH 5.0. X-ray crystallography shows that the two subunits in MDH have extensive contacts with each other (Gleason et al. 1994), which indicates that hydrogen exchange may be a highly specific method for distinguishing between monomeric and dimeric forms of MDH (Hvidt and Nielsen 1966; Englander and Kallenbach 1984; Mandell et al. 1998). In this report, we present evidence that MDH is a folded monomer at pH 5.

Results and Discussion

The aggregation state of MDH at pH 5 was studied by comparing the hydrogen exchange kinetics in intact MDH at pH 5 and 7. Hydrogen exchange into native proteins normally proceeds via the EX2 mechanism (Li and Woodward 1999), in which the rate constant for exchange at any amide linkage, k_ex, can be expressed as

1

In this expression, K is the unfolding equilibrium constant, α is the probability of exchange from the folded protein, and k₂ is the rate constant for exchange from unfolded polypeptides (Woodward et al. 1982; Li and Woodward 1999). Both K and α depend on protein structure, whereas k₂ is independent of protein structure but depends on pH (Bai et al. 1993). Because hydrogen exchange is primarily base-catalyzed at pH 5 and 7, its rate is proportional to the concentration of hydroxyl ions in the solution. If the structure and dynamics of MDH remain relatively unchanged when the pH is decreased from 7 to 5, the exchange rate is expected to be 100 times slower at pH 5 (Bai et al. 1993). To compensate for the slower exchange rate at low pH, labeling times used at pH 5 in these experiments were 100 times longer than that at pH 7 (Smith et al. 1997). A similar approach has been used to study the thermal unfolding of proteins (Zhang and Smith 1993; Liu and Smith 1994).

To locate the regions in MDH that show pH-dependent structural changes, the labeled protein was cleaved into small fragments by pepsin after quenching the isotope labeling reaction with acid (Englander et al. 1985; Zhang and Smith 1993). Deuterium levels of these fragments were determined by directly coupled HPLC-MS (Zhang and Smith 1993; Smith et al. 1997). A set of 24 peptic fragments covering 94% of the MDH backbone was used in this study (Table 1).

Table 1.

Hydrogen exchange results for peptic fragments of MDH incubated in D2O at pH 5.0 or 7.0

			Change in deuterium
Residues in peptic fragments	Number of amide hydrogensa	Increase in number of exposed residuesb	pH 5–pH 7c	Trapped monomer–dimerd
1–18	16	3	1.4	0.3
1–19	17	4	1.3	0.5
19–31	11	2	1.9	1.2
20–31	10	1	1.9	1.1
32–41	8	4	5.7	2.6
42–49	7	8	5.1	N.D.e
42–66	22	9	14.1	10.0
71–89	15	0	0.3	−0.3
90–97	7	0	0	0.1
101–112	10	0	0.1	−0.4
115–129	12	0	−0.1	−0.1
131–148	16	0	0.4	−0.5
149–156	7	5	0.1	0.1
150–156	6	5	0	0
157–199	37	3	−0.2	−0.6
200–212	12	2	1.6	0.4
213–227	14	8	5.4	5.1
228–234	6	2	0.1	−0.1
228–236	8	2	0	0
235–250	15	0	0.2	0.0
254–269	13	0	0.3	−0.4
269–291	21	0	−0.2	−0.5
270–291	20	0	0.4	−0.4
292–308	15	0	−0.2	−0.3

^a Number of peptide amide hydrogens in each peptic fragment.

^b Increase in the number of residues with surface areas increasing by more than 1 Ų when dimers dissociate to monomers.

^c Differences in deuterium levels found in peptic fragments from MDH after labeling at pH 5 for 100 sec or pH 7 for 1 sec. Values listed have been adjusted for loss of deuterium during analysis (Zhang and Smith 1993).

^d Differences in deuterium levels found in peptic fragments from MDH monomer trapped inside a mutant GroEL and fragments from native dimers, both labeled in D₂O for 1 sec at pH 8.0 (Chen and Smith 2000).

^e Peptic fragment was not detected in this particular experiment.

In most peptic fragments, the deuterium levels were similar when MDH was incubated at pH 5 or 7, after adjusting the exchange time for the change in pH. Results for the segment including residues 269–291 (Fig. 1A ▶) illustrate this behavior. The deuterium level in this segment, which has 21 amide hydrogens, increased from 2.8 to 5.0 when intact MDH was incubated for 1–16 sec at pH 7. Similar levels of deuterium (mean difference for five time points = 0.17 deuteriums) were found in the same fragment when derived from MDH incubated at pH 5 for 100–1600 sec. Under these exchange conditions, unfolded polypeptides undergo complete exchange at pH 7.0 within 1–5 sec (Bai et al. 1993). Thus, only the most rapidly exchanging amide hydrogens located on the surface of folded MDH underwent appreciable exchange under the conditions used in these experiments. Exchange in most regions of the MDH backbone was incomplete, even for the longest labeling time, indicating that MDH was folded at both pH 5 and 7.

Fig. 1.

Deuterium levels found in four peptic fragments of MDH following incubation of the intact protein in D₂O at pH 5 (open circles) or 7 (filled circles). The incubation times have been offset to account for the pH dependence of hydrogen exchange rates in unfolded polypeptides. Results for peptic fragments including residues 269–291, 42–49, and 213–227 are presented in panels A–D.

Other segments, such as the one including residues 42–49, had substantially higher deuterium levels when the MDH was labeled at pH 5. Although this segment contained fewer than 0.2 deuteriums after labeling at pH 7 for 2 sec, hydrogen at all seven amide linkages in this segment was replaced with deuterium after labeling at pH 5 for the equivalent time of 200 sec (Fig. 1B ▶). Large increases in deuterium content were also detected for segments including residues 32–41 (Fig. 1C ▶), 213–227 (Fig. 1D ▶), and 42–66 (data not shown) when MDH was incubated at pH 5. The changes in deuterium level measured in all 24 peptic fragments derived from MDH incubated at pH 5 and 7 for 100 or 1 sec, respectively, are summarized in Table 1. Smaller but detectable differences were observed for regions including residues 1–31 and 200–212.

Interestingly, all of the regions that showed increased deuterium uptake are located on or near the subunit binding surface (red and blue in Fig. 2 ▶) and contain at least one residue that is more exposed to the solvent in the monomer (Table 1). For example, Ala 42 and Ser 45 make contact with the other subunit through main chain interactions. The side chains of Asp 43 and His 46 also contact the other subunit through salt bridges (Gleason et al. 1994). Consequently, all eight residues in the segment including residues 42–49 have a larger solvent-accessible surface area in the monomeric state. These results indicate that the solvent accessibility of the subunit interface was much greater at pH 5. Other parts of the molecule, however, had similar deuterium uptake at pH 5 and 7 (Table 1), indicating that environments of the fast-exchanging amide hydrogens remained largely unchanged in regions other than the subunit interface. The selective increase in solvent access at pH 5 was interpreted as evidence that MDH exists primarily as a monomer at this pH.

Fig. 2.

Dimeric structure of MDH (Gleason et al. 1994). Regions showing the largest increases in deuterium (increase > 5 deuterium for 100sec labeling) are colored red; regions showing smaller increases in deuterium (1 < increase > 5) are colored blue. All of one subunit is colored yellow.

Results of a recent study of MDH folding inside the cavity of the chaperonin GroEL confirm this interpretation (Chen and Smith 2000). Unfolded MDH, which is a well-described substrate for GroEL (Miller et al. 1993; Peralta et al. 1994; Ranson et al. 1997), binds to the chaperonin as a monomer. With the assistance of GroEL, MDH may fold to a monomer, which forms a dimer on release into solution. A single-ring mutant of GroEL, SR1, also assists the folding of MDH but does not release it into the solution. Therefore, the end product of the folding reaction is a folded monomer trapped inside the SR1 cavity (Rye et al. 1997). In our study of SR1-assisted folding of MDH (Chen and Smith 2000), MDH was pulse-labeled with D₂O (pH 8.0 for 1 sec) in either its native state (dimer) or folded but trapped inside SR1. Differences in deuterium levels in each peptic fragment from that study are compared with results from the present study in Table 1. All but two regions (residues 1–19 and 200–212) that had increased deuterium uptake at pH 5 also had increased uptake when MDH was labeled inside the SR1 cavity. These results indicate that MDH folded inside the SR1 cavity and MDH at pH 5 have similar structures and that MDH is a monomer at pH 5.

Not all of the segments known to lie on the subunit binding surface showed increased deuterium levels at pH 5 or when trapped inside SR1. To understand this behavior, it is important to note that hydrogen exchange rates in folded proteins often span a range of 10⁸. The protection against hydrogen exchange arises from a combination of decreased solvent accessibility and hydrogen bonding, characteristics of folded proteins. Either of these factors could give rise to significant hydrogen exchange protection for amide hydrogens located in the MDH subunit binding surface. For example, amide hydrogens in the middle of a helix usually have slow exchange kinetics, even when they are on the surface of a protein. This explains why hydrogen exchange in the segment including residues 150–156 did not appear to change in the present experiments even though five of the six residues in this segment have increased surface exposure in the monomer (Table 1). Hydrogen exchange in this segment is very slow because it is located in the center of a long α-helix. Similarly, segments including residues 228–234 and 228–236 are located in the middle of a long helix. Part of this region is near the subunit interface and would therefore be expected to exchange faster in the monomer than the dimer. However, this change was not detected because of the short labeling times used in these experiments.

This study shows that the environment of the amide hydrogens located at the subunit interface of MDH changed substantially at pH 5 without global unfolding of the molecule. Moreover, the exchange kinetics of the protein at this pH resembled that of a folded monomer trapped inside the cavity of SR1. These results show that MDH is a folded monomer at pH 5. This conclusion is also supported by size-exclusion chromatography, which showed that MDH at pH 7 has an apparent molecular mass of 60 kD, whereas MDH equilibrated at pH 5 has an apparent molecular mass of 30 kD (Chen and Smith 2000). Similar results have been reported previously (Bleile et al. 1977; Wood et al. 1978). Finding that MDH dissociates to monomers at pH 5 is also consistent with a study in which His 46, which is located in the subunit interface, was mutated to Leu (Steffan and McAlister-Henn 1991). The reason for the discrepancy between these results and those found using fluorescence polarization is not evident (Sanchez et al. 1998). However, it is noted that isotope exchange is less likely to disturb the structure of folded proteins than fluorescein labeling.

The present study also shows how hydrogen exchange, when combined with protein fragmentation and mass spectrometry, can provide information on solvent accessibility of specific regions in a protein. Although NMR has been used extensively to detect hydrogen exchange in proteins, it generally is not suited to measuring the most rapidly exchanging amide hydrogens in proteins (Chamberlain and Marqusee 2000). This application of hydrogen exchange and mass spectrometry is of general significance because it shows how this approach can be used to study protein–protein and protein–ligand interactions. In addition to its use in identifying binding surfaces, the change in deuterium levels found in certain segments of the subunit binding surfaces with protein concentration might be used to determine equilibrium binding constants.

Materials and methods

Isotope labeling

Malate dehydrogenase (Boehringer Mannheim) was purified by ultrafiltration. For labeling at pH 7.0, MDH (30 μM) in 20 mM Hepes/H₂O at pH 7.0 was injected into a flow quench system consisting of two syringe pumps (Harvard Apparatus) and two mixers (Upchurch). Isotope labeling was initiated in the first mixer with a 13.5-fold dilution of 20 mM Hepes/D₂O at pH 7.0, and terminated in the second mixer with 600 mM phosphate to give a final pH of 2.5. The labeling times were 1, 2, 4, 8, and 16 sec. For labeling at pH 5.0, MDH was incubated with 20 mM sodium acetate/H₂O at pH 5.0 at a final protein concentration of 30 μM. After 30 min, it was diluted 13.5-fold into 20 mM sodium acetate/D₂ at pH 5.0 for 100, 200, 400, 800, and 1600 sec. Isotope labeling was terminated by decreasing the pH to 2.5. Samples were frozen at −70°C until further analysis.

MDH digestion and fragment analysis

The isotopically labeled MDH was digested with pepsin at a 1 : 1 mass ratio for 1.5 min at 0°C. The peptic fragments were fractionated on a C4 column (Vydac, 50 ×1 mm) operating at 40 μL/min at 0°C. Both phases, H₂O and acetonitrile, contained 0.05% trifluoroacetic acid. The acetonitrile gradient was 2%–37% in 9 min. The eluate was detected online with a Micromass AutoSpec mass spectrometer equipped with an electrospray source and a focal plane detector. The separation step was performed in protiated solvents, thereby removing deuterium from side chains and amino/carboxyl termini that exchange much faster than amide linkages (Englander et al. 1985; Bai et al. 1993). As a result, an increase in molecular weight of the peptides was a direct measure of deuteration at peptide linkages. Nondeuterated as well as completely exchanged control samples were analyzed to adjust for deuterium back-exchange during analysis (Zhang and Smith 1993). The nondeuterated control was prepared by diluting MDH into the labeling buffer premixed with the quenching buffer. The completely exchanged MDH was prepared by incubating MDH in KH₂PO₄/ D₂O at pH 2.5 for 16 h.