Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions: CHARACTERIZATION BY HYDROGEN EXCHANGE MASS SPECTROMETRY AND NMR

Abstract

The susceptibility of the cellular prion protein (PrP(C)) to convert to an alternative misfolded conformation (PrP(Sc)), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23-231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4.

Keywords: hydrogen-deuterium exchange; mass spectrometry (MS); nuclear magnetic resonance (NMR); partially unfolded forms; prion; protein dynamic.

FIGURE 1.

Misfolding of moPrP at pH 4 and at pH 5.5. a shows the far-UV CD spectra of moPrP at pH 4 and pH 5.5 at 0 h (solid line and dashed line) and at 3 weeks (short-dashed line and dotted line) of misfolding at 25 °C, respectively. Misfolding was initiated by the addition of 150 mm NaCl. b shows the kinetics of misfolding at pH 4 (circles) and at pH 5.5 (squares) at 37 °C. Misfolding was monitored by the measurement of the CD signal at 216 nm. The error bars represent standard deviations from three independent experiments. The solid lines are exponential fits through the data. An earlier study has shown that the time course of misfolding as monitored by CD is the same as the time course of oligomerization as measured by size exclusion chromatography (32, 35). MRE, mean residue ellipticity; deg, degrees.

FIGURE 2.

Native state HDX of moPrP at pD 4 and 25 °C monitored by mass spectrometry. a shows the progressive increase in m/z values of the 27th chargestate of the protein at 0 s, 5 min, and 160 min of exchange in deuteration buffer along with the m/z value for 100% deuterated moPrP. The short-dashed lines indicate the centroid of the peak. b shows the mass spectra of three peptide fragments obtained from the protein at different times of exchange along with the controls of protonated (0%) and deuterated (100%) peptide fragments. Peptide fragments 127–132, 154–167, and 205–212 correspond to β1, the loop linking α1 and α2 (inclusive of β2), and the central segment of α3, respectively. The black short-dashed lines indicate the centroid m/z of the given peptide.

FIGURE 3.

HDX-MS of backbone amide hydrogens of moPrP in deuterated solvent at 25 °C and pD 4. The percent hydrogen retention versus time data for five peptide fragments, 149–153, 155–162, 190–197, 204–224, and 205–212, in the presence of 0 (black circles), 1 (red circles), and 2 m (green circles) urea are shown (a, c, e, g, and i). The solid lines in black, red, and green are the exponential decay fits to the 0, 1, and 2 m urea data, respectively. The dependences of the rates of HDX on urea concentration for the five segments are shown (b, d, f, h, and j). For segment 155–162, both urea concentration-independent rates (squares), which correspond to intrinsic exchange rates, and urea concentration-dependent rates (circles) of HDX are shown. Segment 204–224 yields two urea concentration-dependent HDX rates as shown in h (squares and circles). The error bars represent standard deviations from three independent experiments.

FIGURE 4.

Native state HDX of moPrP at pD 4 and 25 °C monitored by NMR. a–d show the progressive decrease in ¹H-¹⁵N cross-peak intensities of a few representative residues, Asn¹⁵², Tyr¹⁵⁴, Arg¹⁵⁵, Tyr¹⁵⁶, Asp¹⁷⁷, Asn¹⁸⁰, Asp²⁰¹, Arg²⁰⁷, and Gln²¹¹, at 0 s, 10 min, 1 h, and 4 h of HDX, respectively.

FIGURE 5.

HDX-NMR of backbone amide hydrogens of moPrP in deuterated solvent at 25 °C and pD 4. a–h show the change in percent hydrogen occupancy with increasing times of exchange for residues Tyr¹²⁷, Ile¹³⁸, Met¹⁵³,Val¹⁶⁰, Asn¹⁸⁰, Lys²⁰⁴, Gln²¹¹, and Gln²¹⁶. The solid line through each curve represents an exponential decay fit through the data.

FIGURE 6.

Residue-wise values of ΔG_op at pD 4 and 25 °C. ΔG_op values were obtained from HDX-NMR measurements in the absence of denaturant as shown in a. The dashed line represents the free energy of global unfolding of moPrP at pH 4, at 25 °C (5). The secondary structure of the protein is depicted at the top where arrows indicate β-strands, rectangles indicate helices, and solid lines connecting two secondary structural units indicate loops. b maps the residues for which ΔG_op values were obtained from HDX-NMR, and average m_op values were obtained from HDX-MS studies, onto the structure of the C-terminal domain of moPrP (Protein Data Bank code 1AG2). White corresponds to residues undergoing denaturant-independent exchange, whereas blue, orange, and red colors correspond to residues with average m_op values of exchange of 0.4 ± 0.03, 0.8 ± 0.1, and 1.1 ± 0.08 kcal mol⁻¹ m⁻¹.

FIGURE 7.

Denaturant dependences of ΔG_op for different segments of moPrP at pD 4 and 25 °C. a–l show the denaturant dependences of ΔG_op of the different sequence segments (circles). The dashed lines through the data are linear least square fits to the data, and the slopes yield the m_op values for the different segments. The solid line represents the dependence of the global stability (ΔG_U) on denaturant concentration; its slope is given by m_U (5). In the case of segment 204–224 that spans α3, two protection factors corresponding to different regions of the segment are obtained, resulting in two distinct ΔG_op values as shown in j (squares and circles).The dashed and dotted lines are linear least square fits to the data. The error bars represent standard deviations from three independent experiments.

FIGURE 8.

Defining partially unfolded forms of moPrP. ΔG_op and m_op values are shown for different segments of the protein. The ΔG_op values shown are values determined in the absence of denaturant. ΔG_op and m_op values are shown for segments 127–132 (red circle), 133–148 (green circle), 149–153 (blue circle), 154–167 (yellow circle), 182–196 (dark blue circle), 190–197 (dark blue square), 197–201 (dark blue diamond), 197–206 (dark cyan circle), 204–224 (purple circle), 205–212 (purple square), and 217–223 (purple diamond). The black filled circle corresponds to ΔG_U and m_U. On the basis of m_op values, sequence segments were classified into two distinct groups, PUF1 and PUF2. PUF1 corresponds to sequence segments having an m_op value of 0.4 ± 0.03 kcal mol⁻¹ m⁻¹, and PUF2 corresponds to those having an m_op value of 0.8 ± 0.1 kcal mol⁻¹ m⁻¹. PUF1 segments have a ΔG_op of 2.2 ± 0.2 kcal mol⁻¹, and PUF2 segments have a ΔG_op of 3.1 ± 0.5 kcal mol⁻¹.

FIGURE 9.

CD spectra and ANS binding of moPrP at pH 4 and at pH 7. a shows the CD spectra of moPrP at pH 7 (black solid line) and pH 4 (black dotted line) at 25 °C. b and c show the fluorescence spectra of 2 μm moPrP at 25 °C in the presence of 20 μm ANS at pH 4 and pH 7, respectively. The solid and the dotted lines are the ANS fluorescence spectra in the presence and absence of moPrP. MRE, mean residue ellipticity; deg, degrees.