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. 2015 Aug 30;24(11):1748–1755. doi: 10.1002/pro.2765

Intracellular pH modulates quinary structure

Rachel D Cohen 1, Alex J Guseman 1, Gary J Pielak 1,2,3,*
PMCID: PMC4622208  PMID: 26257390

Abstract

NMR spectroscopy can provide information about proteins in living cells. pH is an important characteristic of the intracellular environment because it modulates key protein properties such as net charge and stability. Here, we show that pH modulates quinary interactions, the weak, ubiquitous interactions between proteins and other cellular macromolecules. We use the K10H variant of the B domain of protein G (GB1, 6.2 kDa) as a pH reporter in Escherichia coli cells. By controlling the intracellular pH, we show that quinary interactions influence the quality of in-cell 15N–1H HSQC NMR spectra. At low pH, the quality is degraded because the increase in attractive interactions between E. coli proteins and GB1 slows GB1 tumbling and broadens its crosspeaks. The results demonstrate the importance of quinary interactions for furthering our understanding of protein chemistry in living cells.

Keywords: amide proton exchange, in-cell NMR, macromolecular crowding, pH, protein stability, quinary interactions

Introduction

The pH is a fundamental parameter in protein science because it influences many protein properties, including their stability and solubility.1 Traditionally, proteins were studied in simple solutions, containing salt to control the ionic strength and buffer to control the pH. However, most proteins function inside cells, where the concentration of macromolecules can exceed 300 g/L.2 In-cell NMR spectroscopy3–5 is a powerful, nonperturbing tool for studying proteins in their native environment. NMR and other techniques have been used to show that crowding modulates the stability of proteins, both enthalpically and entropically.6–9 The cellular interior introduces an additional element: quinary structure10–15 supports intracellular organization and arises from transient interactions between macromolecules that are extremely weak in buffer.

From a practical point of view, the sum of these individually weak interactions between the test protein and intracellular components can hinder the acquisition of in-cell NMR spectra of globular proteins, particularly in Escherichia coli.5,14,16 The B domain of protein G (GB1, 6.2 kDa)17,18 is one of the few globular proteins that yields high quality 15N–1H HSQC spectra in cells,19–21 making it a quintessential test protein for in-cell NMR. Here, we show that pH modulates both its stability and quinary interactions.

For proteins that undergo two-state equilibrium folding, stability is defined as the Gibb's free energy of the ensemble of unfolded forms minus that of the folded state.22 The effects of crowding can be broken down into two types of interactions: hard-core repulsions and soft (chemical) interactions.23 Hard-core effects arise from the physical volume occupied by macromolecules and can only stabilize globular proteins because the crowded environment favors the more compact, native conformation.24 Soft interactions, such as quinary interactions, can be stabilizing or destabilizing.23 For instance, charge–charge repulsions add to the effect of hard-core repulsions, shifting the equilibrium even further toward the native state. Attractive nonspecific interactions between the macromolecules and the protein favor the unfolded ensemble, because the ensemble displays more attractive surface. The net effect determines the impact on stability.

Proteins contain several ionizable side chains with pKa values between 4 and 12. Therefore, the net charge changes with pH. The pH where the net charge is zero is the isoelectric point (pI), which can be estimated from the amino acid sequence or measured by isoelectric focusing. Proteins are positively charged at pH values below the pI and negatively charged above the pI. Therefore, it is not surprising that globular protein stability is pH-dependent.25–27 This dependence is exemplified by acid denaturation, which results from the accumulation of localized positive charge on the compact native state that disperses upon unfolding.28–32 Here, we use NMR-detected amide proton exchange to quantify GB1 stability in buffer as a function of pH.

Changes in protein charge also affect quinary structure. The average pI of E. coli proteins is ∼5.33 Therefore, most E. coli proteins are negatively charged at neutral pH. Since GB1 is also negatively charged at neutral pH (pI 4.5), charge–charge repulsions facilitate acquisition of high quality in-cell spectra of 15N-enriched GB1.20,34 Here, we use a GB1 variant to report the intracellular pH and illustrate the pH dependence of quinary interactions.

Results

GB1 stability decreases with decreasing pH

We used NMR-detected amide proton exchange35,36 to quantify GB1 stability at the residue level. For globular proteins, each backbone amide participates in an equilibrium between an open (op) state and a closed (cl) state. Exchange of the amide proton for a deuteron occurs only in the open state [Eq. 1]:37,38

graphic file with name pro0024-1748-m1.jpg

The rates of opening and closing are kop and kcl, respectively. The intrinsic rate of exchange, kint, is the rate in an unstructured peptide and depends on factors such as primary structure, pH and temperature,39,40 but kint does not change in cells41 or under crowded conditions.7,42 Values of kint can be predicted using the computer program SPHERE.43 The equilibrium constant for opening, Kop, equals kop/kcl, and the free energy of opening is:

graphic file with name pro0024-1748-m2.jpg

Provided that kint is rate determining, which we know to be true for GB1,44,45 the equilibrium constant for opening is:

graphic file with name pro0024-1748-m3.jpg

The volume of each backbone amide nitrogen–amide proton crosspeak in 15N–1H HSQC spectra is proportional to its concentration. Fitting the volume–time data to an exponential decay yields kobs. In this way, we obtain information about the equilibrium thermodynamic stability of individual amide bonds.46 These individual values, however, are difficult to interpret,47 so we focus on averages, which, for our data, approximate the free energy for global unfolding.45,48

To quantify the pH-dependence of GB1 stability in buffer, we performed NMR-detected amide proton exchange experiments in a buffer comprising 20 mM citrate, 150 mM NaCl (99.9% D2O) at pH values (corrected for deuterium) of 4.4, 5.7, 6.0, 6.7, and 7.6. Spectra were serially acquired over 24 h. Data for 12 residues could be fitted at all pH values (Supporting Information Table S1). Figure 1 shows the opening free energies for each quantifiable residue at each pH. Figure 2 shows the average free energy of opening as a function of pH.

Figure 1.

Figure 1

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GB1 stability increases with increasing pH. ΔGop°’ values at 37°C for wild-type GB1 at pH 4.4 (black), 5.7 (red), 6.0 (green), 6.7 (magenta), and 7.6 (blue). The horizontal lines represent the average ΔGop°’ (Inline graphic) at each pH value.

Figure 2.

Figure 2

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Plot of average ΔG op°’ (Inline graphic) versus pH from the data in Figure 1. The error bars represent the standard deviation of the mean of the individual ΔGop°’ values. The curve is of no theoretical significance.

K10H as a pH reporter

We introduced a histidine residue at the protein surface as an in-cell pH probe.49 HSQC spectra of the original protein and the variant in buffer are shown in Supporting Information Figure S1. The titration curve of the variant is shown in Figure 3. Shift changes for both the amide proton and the backbone amide nitrogen of histidine 10 were observed. We used the pH-insensitive backbone 1H and 15N chemical shifts of W43 as an internal reference. The shifts of H10 nuclei minus those of W43 nuclei, Δ1H and Δ15N, were weighted by their gyromagnetic ratios to yield a composite chemical shift50 change:

graphic file with name pro0024-1748-m6.jpg

Figure 3.

Figure 3

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Titration curve from the composite chemical shift50 change (Δδcomp) of the H10 resonances at 37°C. The solid curves are fits to the modified Henderson–Hasselbalch equation51,52 to yield a pKa of 6.7 (Fitting the 1H and 15N data separately yield pKa values of 7.0 and 6.8, respectively (Supporting Information Fig. S2).

The data were fitted to the modified Henderson–Hasselbalch equation51,52 to give an apparent pKa of 6.7 (R2 > 0.99).

Intracellular pH can be controlled with buffer

The K10H variant can be used to monitor the pH inside E. coli cells. We compared the composite shift change of the histidine 10 resonance in cells to the titration curve (Fig. 3) to estimate the intracellular pH (Fig. 4). After the cells were washed and resuspended in a weak buffer (1 × M9 salts pH 7.4), serial acquisition of 15N–1H HSQC spectra demonstrate that the pH in E. coli cells decreased by 0.7 units, from pH 7.4 to pH 6.7, in 6 h. Similar results were observed for the T2H variant (Supporting Information Fig. S3). However, when the cells were washed and resuspended in a stronger buffer (75 mM bis–tris propane, 75 mM HEPES, 75 mM citrate, pH 7.5), the intracellular pH dropped by less than 0.3 units. We used this buffer to monitor in cell spectra acquired at several internal pH values. Higher buffer concentrations degraded the quality of the spectra, consistent with the observation that high ionic strength decreases the quality of spectra, especially when a cryogenic probe is used.53

Figure 4.

Figure 4

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Change in intracellular pH over time measured using the K10H variant in 1× M9 salts (initial pH 7.4, red) and in 75 mM bis–tris propane/75 mM HEPES/75 mM citrate (initial pH 7.5, blue). Values represent the composite corrected chemical shift.50 pH values were extrapolated from the data in Figure 3. The curve is of no theoretical significance.

Quality of the in-cell HSQC spectrum of GB1 decreases with decreasing pH

Using the three-buffer formulation described above, we acquired in-cell 15N–1H HSQC spectra of the K10H variant at pH values of 7.5, 6.0, 5.0, and in diluted lysate. At pH 7.5, relatively sharp crosspeaks were observed [Fig. 5(A)], comparable to spectra of GB1 in diluted lysate [Fig. 5(D)]. At pH 6.0, the crosspeaks broaden, and some disappear [Fig. 5B]. At pH 5.0, only a few crosspeaks are apparent [Fig. 5(C)], and these probably represent metabolites.54 When the cells from the pH 5.0 experiments are lysed, the crosspeaks reappear [Fig. 5(D)]. The disappearance of crosspeaks strongly suggests that the protein has not leaked from the cells, and control experiments on supernatants indicate that the HSQC spectra represent GB1 in cells.

Figure 5.

Figure 5

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In-cell 15N–1H HSQC spectra of K10H GB1. Cells were washed and resuspended in 75 mM bis–tris propane/HEPES/citrate at 37°C at pH 7.5 (A), 6.0 (B), and 5.0 (C). The cells from panel C were lysed and the resultant HSQC spectrum (pH 5.0) is shown (D).

Discussion

We quantified the backbone amide proton exchange rates of 12 residues that exchange via global unfolding such that ΔGop°’ ≈ ΔGden°’ and interpret the average ΔGop°’ value as the global free energy of unfolding for that condition.45,48 GB1 stability increases with increasing pH over the range studied here (Figs. 1 and 2).

Previously, the backbone NH of lysine 10 in wild-type GB1 has been used to sense the intracellular pH.15 However, both the backbone 1H and 15N resonances of K10 follow the ionization of the glutamic acid 56 side chain and exhibit an apparent pKa of 4.0.55 To create a pH probe within the physiological range, we mutated lysine 10 to histidine. The mutation should be innocuous because the side chain of K10 is solvent exposed.17 Indeed, its HSQC spectrum is nearly identical to that of wild-type GB1, except at the site of the mutation (Supporting Information Fig. S1).

We used the K10H variant to monitor the pH inside cells over time. In the absence of a strong buffer the intracellular pH decreased from ∼7.5 to ∼6.7 in 6 h. A similar acidification of the E. coli cytoplasm during in-cell NMR experiments was been observed at low buffer concentration in protein probe/protein systems comprising H50 in α-synuclein41,56 and K10 in GB1.15

Low buffer concentrations are usually used in NMR experiments because high ionic strength degrades spectral quality.53 However, as shown here and previously,15,41,56 low ionic strength buffers do not maintain a constant pH, which can affect data interpretation. We formulated a buffer to help maintain the intracellular pH without excessively degrading the quality of 15N–1H HSQC spectra. The combination of 75 mM bis–tris propane (pKa 6.8), 75 mM HEPES (pKa 7.5) and 75 mM citrate (pK1 3.1, pK2 4.8, pK3 6.4) gives a buffering range from pH 2 to nearly pH 9.

Having observed that this buffer moderates the change in intracellular pH (Fig. 4), we used it to compare in-cell HSQC spectra of the K10H variant at different pH values (Fig. 5). The crosspeaks broadened as the pH was decreased, and all but disappeared at pH 5.0. However, the crosspeaks reappeared in the diluted lysate, indicating that the protein is still folded.

Although we did not observe crosspeaks from inside cells at pH 5.0, there is ample evidence that GB1 remained folded in the cells at this pH. First, in vitro studies,48,57 including the data shown in Figures 1 and 2, show that GB1 is stable and folded well below pH 5.0. Second, amide proton exchange data for GB1 in cells under these conditions45 are similar to those observed in dilute solution.17,48 Third, E. coli in the large intestine survive both acid pH and high packing densities,58 and the cells in the NMR tube remain viable under the conditions used here.45 Fourth, if the protein was unfolded we would expect to observe the crosspeaks because disordered proteins yield high quality 15N–1H HSQC spectra due to their increased internal motion compared to globular proteins.59,60

Since GB1 appears to be folded in cells at the pH values studied, we conclude the broadening and disappearance of crosspeaks from the in-cell spectra at lower pH is the result of faster transverse relaxation caused by slower tumbling.59 The width of a crosspeak is inversely proportional to the transverse relaxation time, T2, which measures the lifetime of the coherent transverse magnetization. Slow tumbling yields shorter T2 values, giving rise to broad crosspeaks.5,61,62 This rotational motion decreases with increasing molecular weight; however, the only parameter changed in our experiments is the pH. The increase in apparent molecular weight arises from increased protein-protein interactions. That is, the accumulation of positive charge on GB1 as the intracellular pH is decreased strengthens interactions with negatively charged E. coli proteins.33 These interactions increase the effective molecular weight of GB1, thereby increasing its correlation time and degrading the quality of the HSQC spectra.

We first noted this temporal decrease in pH in cells expressing GB1 while preparing a manuscript on stability and quinary structure.15 The observation has led us to correct our data on stability in cells,45 where we assumed the intracellular pH was 7.6. Specifically, in that study, kint values were calculated for pH 7.6 when the internal pH was 5.8.15 We have recalculated the ΔGop°’ values in cells based on the correct internal pH (Fig. 6).

Figure 6.

Figure 6

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ΔGop°’ values for wild-type GB1 in buffer and cells at 37°C (PBS, 95% D2O, pH 5.8). Error bars represent the standard deviation of the mean from three trials.

This cytoplasmic acidification may also explain anecdotal observations about the variable quality of in-cell 15N–1H HSQC spectra. That is, in the absence of a strong buffer, the intracellular pH depends not only on the time between preparing the sample and acquiring the spectrum, but also the protein under investigation41,56 and even the E. coli strain used to express the protein (Supporting Information Fig. S3). An important conclusion from this work is that pH and its control are as important for in-cell NMR spectroscopy as they are for in vitro studies.

In summary, this work highlights the importance of quinary interactions in furthering our understanding of proteins inside living cells. Studies in dilute solution demonstrate that fluctuations in pH modulate protein properties; this effect grows even more complex in the dense cellular environment. The ability to maintain cells at a desired pH extends beyond NMR spectral quality and is essential to unlock the secrets of protein behavior.58,63,64

Materials and Methods

Expression, purification and NMR-detected H/D exchange

The pET-11a plasmid with the gene encoding GB1 T2Q has been described.45 The Agilent Quikchange mutagenesis kit was used with the following primers to produce the K10H variant: forward 5′ C CTG AAC GGT CAT ACC CTG AAA GGT GAA ACC ACC 3′, reverse 5′ GGT GGT TTC ACC TTT CAG GGT ATG ACC GTT CAG G 3′. The mutation was confirmed by DNA sequence analysis (GeneWiz, Research Triangle Park, NC).

Expression, purification, and NMR-detected amide proton exchange experiments were performed as described.45 A timer was initiated when lyophilized GB1 was resuspended to a final concentration of 1 mM in 500 μL of buffer prepared in 99.9% D2O. pH was corrected for the isotope effect. For glass electrodes in D2O, pH = pHread + 0.4.65 15N–1H HSQC spectra were acquired serially over 24 h on a Varian Inova 600 MHz spectrometer at 37°C. Each 20-min experiment comprised 64 increments in the 15N dimension with eight scans per increment. Data were processed via NMRPipe. Peak volumes were obtained by using NMRViewJ.66,67 Volumes were plotted as a function of time and fit to an exponential decay using SigmaPlot to yield kobs values.

Titration curve

The lyophilized K10H variant was resuspended to a final concentration of 500 μM in 50 mM bis–tris propane, 50 mM citrate, 50 mM borate, 50 mM HEPES, 5% D2O at 12 pH values (5.1, 5.5, 6.0, 6.2, 6.4, 6.7, 6.9, 7.3, 7.7, 8.2, 8.6, 8.9). HSQC spectra were acquired at 298 K on a 700 MHz Bruker Avance III HD spectrometer equipped with a Bruker TCI cryoprobe. Sweep widths were 9600 and 1950 Hz in the 1H and 15N dimensions, respectively. Each spectrum comprised 64 increments in the 15N dimension with eight scans per increment. Total acquisition time was ∼20 min per spectrum.

In-cell NMR

E. coli BL21(DE3) cells containing the plasmid encoding GB1 K10H were grown in 200 mL of M9 minimal media.45 Chloramphenicol was added to a final concentration of 50 μg/mL to halt expression, and the cells were harvested by centrifugation (1,000g, 20 min, 4°C). Buffers comprised either 1× M9 salts (48 mM Na2HPO4, 22 mM KH2PO4, 9 mM NaCl, pH 7.4) or 75 mM bis–tris propane/75 mM HEPES/75 mM citrate at various pH values. The cells were resuspended and washed with 30 mL of buffer, followed by centrifugation. After three wash/spin cycles, the pellet was resuspended in 50 μL of buffer and transferred to an NMR tube. Spectra were acquired on the 600 MHz spectrometer using sweep widths of 12,000 and 2,500 Hz in the 1H and 15N dimensions, respectively. 15N–1H HSQC spectra were acquired serially for 6 h. Each spectrum comprised 64 increments in the 15N dimension with eight scans per increment. After the experiment, the sample was centrifuged, and a spectrum of the supernatant was obtained to ensure that the signal came from protein inside cells. Experiments on the T2H variant were carried out as described above (Supporting Information Fig. S3), except that BL21(DE3) Gold cells were used.

Acknowledgments

We thank Marc ter Horst and Gregory B. Young for maintaining the spectrometers, Austin E. Smith for initiating the studies of pH in cells and Elizabeth Pielak for comments on the manuscript.

Glossary

GB1

B domain of protein G

HSQC

heteronuclear single quantum correlation

NMR

nuclear magnetic resonance

Supporting Information

Additional Supporting Information may be found in the online version of this article.

Supporting Information

pro0024-1748-sd1.pdf (303KB, pdf)

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