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Quantifying protection in disordered proteins using millisecond hydrogen exchange-mass spectrometry and peptic reference peptides Mohammed A. Al-Naqshabandi, and David D Weis Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01312 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 7, 2017

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Biochemistry

Quantifying protection in disordered proteins using millisecond hydrogen exchange-mass spectrometry and peptic reference peptides By-lines Mohammed A. Al-Naqshabandi† and David D. Weis* Department of Chemistry, University of Kansas †

Department of General Sciences, Soran University, Soran, Kurdistan Region, Iraq

*Correspondence to: David D. Weis, Department of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence KS, 66045 USA +1-785-864-1377 (voice) +1-785-864-5396 (facsimile) [email protected] Funding Source Statement Financial support to M.A-N. from the Higher Committee for Education Development in Iraq and a National Science Foundation CAREER award to D.D.W. (MCB-1149538) are acknowledged.

1 ACS Paragon Plus Environment

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ABBREVIATIONS ACTR, residues 1023-1093 of the activator for thyroid and retinoid receptors (UniProt NCOA3_HUMAN); CBP, residues 2059-2117 of the CREB binding protein (UniProt CBP_MOUSE); HX, amide hydrogen exchange; IDP, intrinsically disordered protein; LC, liquid chromatography; MS, mass spectrometry; NMR, nuclear magnetic resonance


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ABSTRACT The extent and location of transient structure in intrinsically disordered proteins (IDPs) provide valuable insights into their conformational ensembles and can lead to a better understanding of coupled binding and folding. Millisecond amide hydrogen exchange (HX) can provide such information, but it is difficult to quantify the degree of transient structuring. One reason is that transiently disordered proteins undergo HX at rates only slightly slower than the rate of amide HX by an unstructured random coil, the chemical HX rate. In this work, we evaluate several different methods to obtain an accurate model for the chemical HX rate suitable for millisecond hydrogen exchange-mass spectrometry (HX-MS) analysis of disordered proteins: (1) calculations using the method of Englander [Bai, et al., Proteins 1993, 17, 75-86], (2) measurement of HX in the presence of 6 M urea or 3 M guanidinium chloride, and (3) measurement of HX by peptide fragments derived directly from the proteins of interest. First, using unstructured model peptides and disordered domains of the activator for thyroid and retinoid receptors (ACTR) and the CREB binding protein (CBP) as the model IDPs, we show that the Englander method has slight inaccuracies that lead to under-estimation of the chemical exchange rate. Second, HX-MS measurements of model peptides show that HX rates are changed dramatically by high concentrations of denaturant. Third, we find that measurements of HX by reference peptides from the proteins of interest provides the most accurate approach for quantifying the extent of transient structure in disordered proteins by millisecond HX-MS.


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Intrinsically disordered proteins (IDPs) have regions that are highly flexible and lack stable secondary or tertiary structure.1-6 There is considerable interest in IDPs due to their important roles in many biological processes and functions. The biological functions include transcriptional regulation, cellular signaling, post-translational modification, and cellular recognition.7-9 IDPs are associated principally with functions that require protein-protein interactions such as in cellular signaling.7 Although many studies have shown that IDPs participate in functional interactions, less is known about the structural details of the interactions. Many IDPs use highly flexible regions to mediate protein-protein interaction through the phenomenon of coupled folding and binding.10-12 In this type of interaction, folding of a disordered protein occurs upon engagement with its binding partner. Two modes of binding have been proposed: conformational selection and induced folding. In conformational selection, transiently folded states, that exist within the conformational ensemble, are selected by the binding partner to allow binding. In induced folding, the conformational flexibility of the IDP eases the interaction with its binding partner, ultimately leading to the folding of the disordered protein.13, 14 More detailed information is required to understand the complexity of mechanisms of protein-protein interaction involving disordered proteins. Obtaining localized information about the presence or absence of transiently folded conformers of IDPs in the free state can be very important to provide support for one proposed mode of binding over the other. Many techniques have been employed to provide information about transient structure in IDPs.14-19 NMR and amide hydrogen exchange (HX) mass spectrometry are among the techniques that can measure the degree of protein structure with high spatial resolution. The rate of HX is highly dependent on hydrogen bonding and solvent accessibility, both related to the conformational flexibility of the protein backbone.20, 21 Highly flexible regions will exchange


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more rapidly while rigid regions exchange more slowly.22 In the case of IDPs, millisecond HX is required because most of the exchange occurs on this timescale at room temperature at nearneutral pH.23-26 Classically, HX measurements have been interpreted in terms of the protection factor, defined as kref kobs where k denotes the rate constant for exchange by an unstructured reference state (ref) and observed (obs) experimentally. Under standard biophysical conditions (pH 7, 300 K), k ref ≈ 1 s −1 . The magnitude of protection can be used to estimate the extent of structure. In well-folded proteins, protection factors can be as high as 108.27 Thus for well-folded proteins, the apparent degree of structure is readily apparent from the observed HX kinetics alone. In the case of transiently-structured regions in proteins, however, where the protection factors of less than 10 are anticipated, protection from HX is only detectable if an accurate estimate of kref , the rate of HX by an unstructured reference state, is available.28 The unprotected reference state must represent the fastest possible exchange in order to quantify the degree of protection. Without an unprotected reference state, distinguishing amongst subtle differences in hydrogen exchange kinetics in transiently structured regions is problematic. Generally, the chemical exchange rate, calculated using an empirical formula developed by Englander based on unstructured model peptides,29, 30 is used to model HX by an unprotected reference state. Using chemical exchange as a reference state here is the key to measuring protection. Any inaccuracy in the rate of HX by the unprotected reference state would underestimate or overestimate the degree of protection. In some cases however, we24 and others31-33 have observed that the HX rates predicted by modelled chemical exchange were slower than the measured HX rate in unstructured peptides and proteins. These observations suggest that the modeled chemical exchange may not be sufficiently accurate to be used to


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estimate protection in transiently structured IDPs where the differences between the HX rate of the IDP and a truly unstructured random coil will be small. In this work, we explore three different approaches to model HX by an unstructured random coil to obtain a reference state for quantifying the degree of transient structure in IDPs. The approaches for modeling unstructured HX are (1) chemical exchange calculation based on the Englander method, (2) measurement of HX under strongly denaturing conditions (6 M urea and 3 M guanidinium chloride), and (3) measurement of HX by peptic peptides obtained by digesting the protein of interest. In an ideal case, the rate of exchange by the reference would be the same as the exchange by an unstructured peptide. In the first case, we compared hydrogen exchange by free peptides with calculations based on the Englander method. In the second case, for HX in the presence of denaturing agents, we compared the rates of HX by free peptides to their rates of HX under strongly denaturing conditions. In the third case, using predigested peptic reference peptides, we compared HX rates obtained from model IDPs with HX rates by isolated predigested peptides. The intrinsically disordered interaction domains of the activator for thyroid and retinoid receptors (ACTR) and the CREB binding protein (CBP) were used as the model IDPs. ACTR is a near-random coil that has some residual helicity11, 34, 35 while CBP is a molten globule that transiently becomes unstructured.23, 24, 36, 37 By correlating our HX measurements with previous data from these model systems, we have found that predigested peptic peptides provide accurate models of HX by unstructured random coil conformations that enables the use of msec HX to map transiently-structured elements in intrinsically disordered proteins.


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MATERIALS AND METHODS Protein expression and purification ACTR1023-1093 (UniProt NCOA3_HUMAN) and CBP2059-2117 (UniProt CBP_MOUSE) domains were co-expressed from a pET22B co-expression vector11 in Escherichia coli BL21 (DE3) and separately purified as described previously.23 The proteins were stored at –80 °C. Prior to use, stocks were dialyzed against 20 mM sodium citrate and 100 mM NaCl buffer (pH 6.5) for 2 hours followed by overnight dialysis at 4 °C (Slide-A-Lyzer MINI, 2000 Da cutoff, Thermo Fisher Scientific, Waltham, MA). The ACTR and CBP stock concentrations, 18.8 µM and 17.2 µM, respectively, were determined using a bicinchoninic acid assay standardized with bovine serum albumin (Thermo Fisher Scientific, Waltham, MA). Preparation of peptic reference peptides ACTR was concentrated by lyophilizing 3 mL of ACTR stock (Labconco, Freezone model 7670521, Kansas City, MO, USA) followed by reconstitution in 1 mL of ultrapure water. The concentrated stock was dialyzed against citrate buffer (pH 6.5) two times for two hours each followed by overnight dialysis at 4 °C (Slide-A-Lyzer MINI 2000 Da cutoff, Thermo Fisher Scientific, Waltham, MA). The concentration of the resulting ACTR stock was 45.2 µM. ACTR peptic peptides were prepared by diluting 50 µL of ACTR to 2.2 mL with 0.1% formic acid and 150 µL of stock CBP was diluted to 2.4 mL with 0.1% formic acid. The samples were passed through an immobilized pepsin column, described in the LC-MS section, using a syringe pump at a flow rate 50 µL min−1 at room temperature. The digested samples were vacuum dried for 1 hour at 40 °C using a Labconco CentriVap Concentrator (model 780016) combined with Freezone cold trap. The dried samples were reconstituted in 50 µL of citrate buffer (pH 6.5), frozen, and stored at –80 °C.


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Hydrogen exchange labeling The pH values of all deuterium-containing solutions (pD) are reported after applying the glass electrode isotope effect.38 Millisecond hydrogen exchange was conducted using a homebuilt quench-flow device, described previously,24 with some improvements in syringe types and fittings. To prevent leaks at high flow rate, syringes with luer lock tips were replaced with ChemSeal syringes with 1/4-28 thread tip (Hamilton, Reno, NV). All three syringes were connected to three lines of fused silica capillaries with an adapter assembly (P-627 female 10-32 coned to female 1/4-28 thread configuration, IDEX, Oak Harbor, WA). NanoTight tubing sleeves (F-242) and fittings (F-300) (IDEX, Oak Harbor, WA) for the assembly were used to connect the silica capillaries and the assembly. ACTR and CBP were labeled in separate experiments using the quench-flow device. The ACTR (8 µM) and CBP (8 µM) proteins were compared with their reference peptides ACTR (3 µM) and CBP (3 µM), respectively. All stocks were spiked with internal standard peptides FKPGI (GenScript, Piscataway, NJ) and YPI (Anaspec, Freemont, CA) used here to confirm the consistency of labeling conditions. The samples, in H2O buffer (20 mM sodium citrate, 100 mM NaCl, pH 6.5), were labeled with a 5fold excess of D2O buffer (20 mM sodium citrate, 100 mM NaCl, pD 6.5). According to the applied flow rates, the labeling time ranged between 42 and 3500 milliseconds. The labeled samples were then quenched at a 5:6 volume ratio with quench buffer (200 mM sodium phosphate, pH 2.3). The quenched samples were immediately flash-frozen with liquid nitrogen and held at –80°C until analysis. Fully deuterated controls were labeled manually with a 5-fold excess of D2O buffer for 18 hours and then quenched at a 5:6 volume ratio with the quenching buffer. For undeuterated controls, the samples were manually diluted 5-fold with H2O buffer


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instead of D2O buffer and followed the steps for quenching and flash-freezing the samples. All HX labeling was carried out at room temperature (23±1°C) in triplicate for each labeling time. HX by YPI, a slowly exchanging model peptide was measured by labeled for between 20 and 4000 sec using an H/D-X PAL robot (LEAP Technologies, Carrboro, NC). Labeling and quenching used the same conditions as the quench-flow experiments: labeling with a 5:1 dilution in 20 mM sodium citrate, 100 mM NaCl, pD 6.5 and quenching with a 5:6 dilution in 200 mM sodium phosphate, pH 2.3. Chemical exchange kinetics The rate of chemical exchange by amide hydrogens was calculated using a sum of exponentials for each amide in a peptide to give the extent of deuteration as a function of labeling time, D ( t ) n

(

D ( t ) = ∑ 1 − e − ki t i=3

)

(1)

where n represents the number of residues in the peptide and ki is the rate constant of chemical exchange30 for residue i. As shown in equation (1), the value of i begins with 3 to discount fast back-exchange at the first amide ( i = 2 ) and the presence of a primary amine instead of an amide at the N-terminus ( i = 1 ). The rate constant is zero for proline residues because there is no amide hydrogen. To calculate chemical exchange, we used an Excel spreadsheet adapted from a spreadsheet provided by the Englander Lab (available online at http://hx2.med.upenn.edu/) that calculates chemical HX based on empirically derived formulas.30, 39 Chemical exchange was calculated at pD 6.5 at 22 °C. Since HX on the msec timescale does not report on slowlyexchanging residues, the conventional method for correcting for back-exchange, based on measurement of a heavily deuterated sample, cannot be applied.40 Thus, we normalized the


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modeled chemical exchange from equation (1) to the ratio between the measured mass increase of quench-flow labeled samples at the longest HX time, ∆mQF ( tmax ) , and the measured extent of chemical exchange predicted at the longest HX time, D ( tmax ) :

∆mch ( t ) = ∆mQF ( tmax )

D (t ) D ( tmax )

(2)

thus ∆mch ( t ) is the calculated chemical exchange adjusted to the level of back-exchange. LC-MS analysis ACTR peptic peptides were assigned using MS data from a time-of-flight mass spectrometer (Agilent 6220) and MS/MS data from a linear ion trap (Thermo LTQ-XL), as described previously.35 CBP peptides were assigned using a quadrupole time-of-flight mass spectrometer (Agilent 6530). MS/MS spectra of the CBP peptides were assigned using MassHunter Qualitative Analysis (version B.07.00). Each MS/MS spectrum was examined to validate the assignment and to identify potential in-source fragmented peptides based on coelution and a common C-terminus. All LC-MS analysis of labeled sample was carried out on a time-of-flight mass spectrometer (Agilent 6220) combined with HPLC (Agilent 1200 series). Individual quenched, frozen samples were thawed by hand for 3 minutes immediately before injection into a refrigerated column compartment built in-house.41 The temperature of the compartment was maintained at 0 °C. The LC mobile phases were 0.1% formic acid in water (A) and 90% acetonitrile/10% water/0.1% formic acid (B). After injection, the protein samples were digested online using an immobilized pepsin column (2.1 mm × 100 mm) prepared in-house42, 43 at 200 µL/min mobile phase A. The peptides were trapped and desalted over 4 min on a C12 trap (Jupiter Proteo, 4 µm, 90 Å, Phenomenex, Torrance, CA) packed in custom 1.5 × 5 mm OPTI10 ACS Paragon Plus Environment

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LYNX II cartridges (Optimize Technologies, Apple Valley, Minnesota). The resulting peptides were then separated on a C18 column (Zorbax 300SB, 3.5 µm, 1 × 50 mm, Agilent, Santa Clara, CA) using a 6 min 5-60% B gradient for ACTR peptides and a 6 min 5-45% B gradient for CBP peptides at 50 µL min−1. All mass spectra were measured in positive mode with an electrospray capillary voltage of 4000 V, a drying gas flow 10 L min−1, a fragmentor of 150 V, and a desolvation temperature of 325 °C. HD Examiner (version 2.0, Sierra Analytics, Modesto, CA) was used for initial data analysis. The time required to reach 50% deuteration ( t50% ) for each peptide in both states of proteins was used for kinetic analysis of HX.35 For the t50% determination, the deuterium uptake values were normalized to 100% using the totally deuterated controls. Then the t50% values were determined using linear interpolation between the two points that spanned 50% exchange. We determined a protection ratio as

protection ratio =

t50% ( protein ) t50% ( reference )

(3)

Although the protection ratio resembles a protection factor, the protection ratio is obtained from an empirical determination of peptide-averaged HX kinetics. The ratio is useful for quantifying protection, but thermodynamic interpretations that are applied to protection factors may not be applicable. Since there were many overlapping peptides for ACTR, we also calculated the residue-resolved protection ratios for ACTR. The residue-resolved protection ratios, obtained by weighted residue-by-residue averaging of the overlapping peptide values were determined as described previously.35 Two ACTR segments, 1051-1054 and 1058-1061, were excluded because they did not reach 50% exchange.


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RESULTS Calculated chemical exchange as a reference state To evaluate the accuracy of chemical exchange estimated by the Englander method28, 29 for modelling HX by the unstructured reference state, we compared HX data from unstructured peptides with exchange kinetics calculated according to equation (1). Two representative deuterium uptake plots for peptic reference peptides from ACTR (residues 1023-1032) and CBP (residues 2059-2068), obtained by pepsin digestion before labeling, are shown in Figure 1. The reference peptides from ACTR and CBP exchange faster than predicted by the Englander method. This discrepancy is present for all peptic peptides from ACTR and CBP (see Figure S1).


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Figure 1. HX by unstructured peptides is faster than HX predicted by chemical exchange calculations. Measured and predicted deuterium uptake curves from the N-terminal peptic peptides of (A) ACTR (residues 1023-1032), (B) CBP (residues 2059-2068), and unstructured model peptides (C) FKPGI and (D) YPI are shown. Open symbols represent HX-MS measurements with the error bars denoting one standard deviation from three technical replicates. The dashed lines indicate calculated chemical exchange, as detailed in the Materials and Methods section. The horizontal dashed lines represent the maximum deuterium uptake measured in a highly deuterated control. The maximum on the vertical scale indicates the theoretical maximum exchange based on 1:5 dilution during HX. A complete set of deuterium uptake curves for all of the reference peptides is shown in Supporting Figure S1.


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To determine if the differences in the exchange that we observed were specific to ACTR and CBP peptides, we also measured exchange by two model peptides, YPI and FKPGI. These model peptides were designed to minimize helical propensity. The peptides are short and they contain a helix-breaking proline and other residues with low helix-forming propensity.44 YPI has only one exchangeable amide hydrogen, at isoleucine. This amide exchanges slowly and can be accurately described by a single first-order rate constant.45 In the case of FKPGI, the amide hydrogen at lysine undergoes rapid back-exchange. Thus FKPGI has two amides that undergo measurable exchange, at isoleucine and glycine, with slow and fast rates, respectively.30 We compared the HX data of the model peptide FKPGI with the calculated exchange on the millisecond timescale. As shown in Figure 1C, HX by FKPGI is also faster than the calculated chemical exchange. To confirm that the differences in the exchange are not related to our quench-flow system, we used conventional HX labeling for YPI deuteration on a longer labeling timescale, as shown in Figure 1D. As with all of the millisecond HX measurements, measured exchange by YPI was also faster than exchange predicted by the chemical exchange calculation. These results indicate that chemical exchange using the Englander method may not be sufficiently accurate to model the unprotected reference state for regions of proteins that are only slightly protected or not protected at all. Chemically denatured protein as a reference state An alternative to the Englander method to estimate HX by the unprotected reference state would be to measure HX in the presence of denaturants. Unlike the chemical exchange calculation, measurements in denaturants are based on direct experimental measurements. However high concentrations of denaturant can alter chemical exchange rates. Urea can form


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hydrogen bonds with NH group of peptides leading to protection from HX.46 Guanidinium is known to have some impact on the acid-catalyzed peptide hydrogen exchange46 and high concentrations of guanidinium also substantially change the ionic strength of the labeling solution, a factor known to alter rates of chemical exchange.30 Indeed, relative to HX in the absence of denaturants, the YPI model peptide exchanged 1.5-fold more quickly in 3 M guanidinium chloride and 1.9-fold more slowly in 6 M urea (see Supporting Information, Figure S2, Table S2). Peptic peptides as a reference state An alternative empirical approach to obtain HX kinetics by an unprotected reference state is to first digest the protein of interest with pepsin and then subsequently label the resultant reference peptides. We expect that the peptic reference peptides are unstructured because they are derived from intrinsically disordered proteins that have a low abundance of hydrophobic residues and a high abundance of polar and charged residues.47, 48 These characteristics makes it difficult for the reference peptides to become structurally protected. In this approach, the rate of chemical exchange is thus estimated based on an experimental determination of HX by the reference peptides. The reference peptides and the intact protein are labeled under identical conditions to facilitate the comparison between their HX kinetics. Use of identical labeling conditions eliminates any effects arising from different chemical exchange rates due to differences in the solution conditions. We validated this approach by using two model IDPs, ACTR and CBP, where transient structure has been extensively mapped at single residue resolution by NMR.34, 36, 49 We used offline pepsin digestion of ACTR and CBP to obtain the reference peptides, as described in the Materials and Methods section. We used millisecond HX labeling of intact ACTR and CBP and


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their reference peptides. HX kinetics by FKPGI and YPI, added as internal standards, confirms that the two states had consistent labeling (see Supporting Figure S3). Figure 2 shows representative deuterium uptake curves comparing HX by segments of ACTR and CBP with their corresponding reference peptides. A complete set of deuterium uptake data for ACTR and CBP in both states is provided in Figures S3 and S4, respectively.


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Figure 2. The reference peptides undergo HX at rates equal to or greater than their corresponding segments in the intact proteins revealing regions of transient structure. Representative deuterium uptake curves from ACTR (A) the unstructured Nterminal segment (residues 1023-1035), (B) a transiently-structured region (residues 10511057) and from transiently structured regions in CBP (C) residues 2059-2068 and (D) residues 2102-2117. HX by the reference peptides is shown by the open circles and HX by the protein segments by the filled triangles. The error bars denote one standard deviation from three technical replicates. The horizontal dashed lines represent the maximum deuterium uptake measured in a highly deuterated control. The maximum on the vertical scale indicates the theoretical maximum exchange based on 1:5 dilution during HX. Complete sets of deuterium uptake curves are shown in Supporting Figures S3 and S4.


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There are extensive NMR data characterizing residual structure in ACTR in both the isolated form and in complex with CBP. 11, 34, 36, 37, 49 On the basis of the NMR data, ACTR has the characteristics of an unstructured random coil that transiently folds in the regions that also form helices when it binds to CBP. Both the N- and C-terminal tails of ACTR are disordered, as established by NMR secondary chemical shift measurements. Because ACTR has both unstructured and transiently structured regions, it is a good model to determine the accuracy of the reference peptide approach. The most useful starting points for validation are the unstructured regions, where the rate of HX should be equal for the protein segments and the reference peptides. The N-terminal ACTR 1023-1035 segment exchanged at the same rate as its reference peptide (see Figure 2A), as expected in an unstructured region. In contrast, HX by ACTR in the 1051-1057 segment was substantially slower than its reference peptide (see Figure 2B) consistent with the presence of transient helicity in this region. CBP, based on the NMR data, is a molten globule that transiently occupies an unstructured state. As a molten globular protein, CBP has well-defined secondary structure such that its structure has been solved by NMR.36 There is a significant slowing of HX in the N-terminal segment 2059-2068 relative to the reference peptide (see Figure 2C). The degree of protection against exchange is less in the Cterminal CBP segment 2102-2117 (see Figure 2D). To quantify the differences in HX rates between the segments in the intact protein and the reference peptides, we used an empirical estimate of the half-time for HX, t50% , to calculate an empirical protection ratio as described by equation (3). A protection ratio of unity indicates that there is no protection, meaning that the segment exchanges at the same rate as its corresponding reference peptide. If there is protection from exchange, then the protection ratio would become greater than one. Here, we avoid the use of the term protection factor. The protection that we


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quantify represents an average of all residues in a given segment; the protection ratio should not be confused with single residue protection factors. We have aligned the protection ratios with the regions of ACTR and CBP where transient helicity has been detected by NMR, as shown in Figures 3A and 4A. (The protection ratios for all peptides are tabulate in Supporting Table S1.) Two ACTR segments, 1051-1054 and 1058-1061 (shown in black in Figure 3A), were excluded from residue-averaged protection ratio calculation because they did not reach 50% exchange (see Table S1). These two peptides (RALL, HTLL) are short segments that end with leucine residues that slow chemical exchange of the amide by the side chain steric effect.30

Figure 3. Protection from HX in ACTR correlates with transient structure detected by NMR measurements. (A) Protection ratios for each peptic segment of ACTR are shown aligned with the sequence and the regions of ACTR that become helical when ACTR binds to CBP (shown as rectangles) and regions that were not resolved (dashed line).11 The protection ratios are indicated by the blue color scale with darker shades indicating greater protection. Regions marked in white denote residues that do not have measurable HX in a given peptide segment due either to rapid back-exchange or absence of an exchangeable amide hydrogen. Two peptides in the middle region of ACTR, colored in black, are not reported (NR) and not included in subsequent analysis because they did not reach 50% exchange. The protected regions align well with (B) transient structure determined by NMR secondary chemical shifts 19 ACS Paragon Plus Environment

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of the backbone carbonyl carbon (C′).34 (C) Residue-resolved HX protection measurements correlate well with the NMR secondary chemical shifts. The horizontal dashed line in (C) crosses the vertical axis at a value of 1 indicating no protection.

Figure 4. Protection from HX in CBP correlates with transient structure detected in NMR measurements. (A) Protection ratios for each peptic segment of CBP are shown aligned with the sequence and the regions of CBP that become helical when CBP binds to ACTR (shown as rectangles).11 The protection ratios are indicated by the blue color scale with darker shades indicating greater protection. One peptide, colored in black, was excluded from the calculations because it did not reach 50% exchange. The protected regions align well with (B) transient structure measurement determined by NMR secondary chemical shifts of the backbone α carbon ( C α ).49 The secondary chemical shift data were obtained using a plot digitizer available online at http://arohatgi.info/WebPlotDigitizer/ For the segments from the N- and C-termini of ACTR, residues 1023-1044 and 10871093, there were no or only slight differences in HX, leading to protection ratios of less than 1.1. Between these unstructured tails, in the middle region of ACTR, 1038-1069, several segments were significantly protected from HX. Among these segments, the most strongly protected, with a protection ratio of 11, is 1044-1054. This region represents the highest degree of residual structure in ACTR and aligns well with the largest secondary chemical shifts detected by NMR (see Figure 3B). In general, there is good agreement between the protection ratios obtained from millisecond HX and NMR secondary chemical shifts for measuring residual structure in ACTR.


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Biochemistry

The degree of overlap between the peptic peptides of ACTR makes it possible to estimate protection ratios at near single-residue resolution, using a weighted residue-by-residue average of the segment-averaged protection ratios35 as shown in Figure 3C. In general, there is good correlation between the residue-resolved protection ratio values and the NMR secondary chemical shifts shown in Figure 3B. The region of strongest protection, 1047-1052, is displaced somewhat towards the N-terminal side when compared with the NMR data. The reason for this displacement is the small number of segments accounted for with the residue-averaged protection ratio calculation. There is only one segment that spans residues 1045-1051 leading to decreased spatial resolution in this region. Absence of protection in both the N- and C-terminal tails is also in good agreement with the NMR measurements. On the basis of NMR measurements, CBP is best classified as a molten globule: CBP has well-defined secondary and tertiary structure such that its structure in the free state has been solved based on NMR, but CBP also transiently unfolds.23, 36, 49 Based on the HX measurements, CBP is generally more protected than ACTR as shown by the protection ratios in Figure 4A. There was substantial protection from exchange in the N-terminal region of the protein, particularly in residues 2059-2068. This segment spans three residues of a region with the most intense values of NMR secondary chemical shift as shown in Figure 4B.49 The most protected regions of CBP have protection ratios of 9-11 and span the region from 2061 to 2097. Although the spatial resolution of the peptic peptides is not as good as with ACTR, the more strongly protected regions still correlate well with stronger secondary chemical shifts. There are some discrepancies in the N-terminal region because there are not enough segments that cover only the unstructured residues of CBP, 2059-2067. The C-terminal region of CBP was the least protected, with protection ratios ranging from 1 to 3. These data are in a good agreement with NMR data in


Biochemistry

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the helix 3 region. Notably, the C-terminal segment of CBP, 2110-2118, is unprotected and also has no transient structure based on the NMR measurements. The 2069-2072 segment in CBP did not reach 50% exchange on the timescale of measurements. This segment, QDLL, has bulky leucine residues at the end of the segment which is known to slow chemical exchange by steric protection of the amide hydrogen.30 A protection ratios was not calculated for this segment. For CBP, we only used segment-averaged protection ratio because the number of peptides for CBP was limited which makes the residue averaging calculation unreliable. DISCUSSION Understanding the mechanism of coupled binding and folding by IDPs requires a detailed understanding about the unbound conformational ensembles. In particular, evidence of transiently-folded conformers under native conditions would support a conformational selection model for coupled binding and folding.10, 50 Previous work showed that millisecond HX can provide vital information about IDPs and their interactions,23, 24, 26, 28, 35, 51 but it is difficult to detect transient structure using HX kinetics without an accurate model for HX by an unprotected reference state. Our goal in this study was to develop a method that can be used to quantify the degree of transient structure in IDPs. For this purpose, we have evaluated theoretical and experimental options to identify the most accurate method to model HX by an unstructured polypeptide. The accepted approach to obtain HX kinetics for an unstructured polypeptide is based on Englander’s method29, 30 to estimate chemical exchange rates based on model peptides. We observed that ACTR and CBP and two model peptides, FKPGI and YPI, all exchanged significantly faster than the calculated chemical exchange rate (see Figure 1 and Figure S2). The effect cannot be attributed to an artifact of the quench-flow HX device because YPI exchange


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Biochemistry

was measured using standard HX methods on the seconds-to-hours timescale (see Figure 1D). Previously, Mori and co-workers found that some amides in disordered ∆131∆ staphylococcal nuclease exchanged faster or slower than predicted by the Englander model.33 Similarly, Del Mar et al. reported that many segments across regions of α-synuclein, an intrinsically disordered protein, exchange at rates faster than the calculated chemical exchange rate.31 Based on our own observations and previous work, it appears that chemical exchange estimated using Englander’s method may not be accurate enough to measure subtle protection in IDPs. In the Englander model, the chemical exchange calculations are based on measured HX rates obtained using model dipeptides, oligopeptides, and polypeptides at high ionic strength (0.5 M KCl).29, 30 The estimate accounts for inductive, electrostatic, and steric effects of the two adjacent side chains, but longer range effects that might also be present are not accounted for. So in many cases, this calculation may not be reliable for different solution conditions, for example, low ionic strength or high concentrations of additives such as guanidinium and urea in buffers. Some other factors need to be accounted for in the HX. In our labeling, we have used 1:5 dilution, leaving ~17 atom% H. Under these conditions, the rate of chemical exchange at nearneutral pH will depend on the concentrations of both forms of hydroxide, OH– and OD–. Errors in the determination of these concentrations will lead to errors in the estimated rate of chemical exchange. Accurate determination of the concentrations of OH– and OD– in mixed isotopic waters is a complicated problem involving autoprotolysis of H2O, HDO, and D2O52, 53 and also isotope effects at the pH electrode.38, 54 Overall, these factors may have led to the apparent acceleration of the chemical exchange for ACTR, CBP, and the model peptides. These issues suggest that chemical exchange rates estimated by Englander’s method could be in error by approximately a factor of two. In the case of measuring protection in well-folded globular


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proteins, where the protection factors range from 103 to 108, this error in chemical exchange would be negligible. Hence, chemical exchange estimates are suitable for determining protection in such cases. In the case of an IDP, however, the errors could lead to substantial under- or overestimation of the extent of transient structure formation. We turned to an alternative empirical approach to measuring chemical exchange: HX measurements on predigested peptides of the protein-of-interest. The approach is based on the assumption that proteolysis of an IDP will yield peptide segments that are unable to retain structure in isolation from the full-length protein. Based on this assumption, measurement of HX in the predigested peptide is a direct measurement of chemical exchange by amide hydrogens in an unstructured polypeptide. Direct validation of this assumption is impractical: it would require structural characterization of each individual peptide by methods such as NMR or far UV circular dichroism. Instead, we used two IDPs that exhibit very different degrees of transient structure to validate the approach. Both proteins have unstructured tails that serve as internal controls. An advantage of the use of predigested peptides is that the peptides can be labeled using the same experimental conditions as the intact protein. Protection ratios obtained using the reference peptides correlated well with NMR-derived secondary chemical shifts, a measure of transient structure. Based on this good correlation, we conclude that measurement of HX in reference peptides obtained by predigestion of the protein of interest is the most suitable method for estimating protection in HX-MS measurements of disordered proteins. Because any difference in the composition of the buffer may result in differences in the exchange rate, one of the major advantages of using peptic reference peptide as a reference is that there is no need to change the composition of the buffers. The other advantage is that no synthetic peptide standard is needed. This approach offers a simple, easy to apply, and reliable empirical unprotected


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Biochemistry

reference. There is clearly no need to change buffer composition by adding a chemical denaturant. Data correction due to altered buffer composition is also not required making the method less susceptible to HX miscalculations. The approach can give new insight into the analysis of transient structure in other IDPs. However, this approach may not work for all kinds of proteins because the isolated peptide might form secondary structure. If the reference becomes protected in this way, then it can no longer serve as a reference peptide. The formation of structure in the reference peptides is more likely to be a problem with well-folded globular proteins because they have a much stronger propensity to fold. In such cases, peptic peptides in a low denaturant concentration might be a better choice. ACKNOWLEDGEMENTS We thank Agilent Technologies for an equipment loan. Financial support to M.A-N. from the Higher Committee for Education Development in Iraq and a National Science Foundation CAREER award to D.D.W. (MCB-1149538) are gratefully acknowledged. SUPPORTING INFORMATION Table S1 listing assignments of peptic peptides, their t 50% values, and their protection ratios; Figure S1 showing measured and calculated exchange for peptic peptides; a description of results obtained from msec HX measurements of YPI in denaturants comprised of Figure S2 showing HX kinetics of YPI in urea and guanidinium hydrochloride and Table S2 listing kinetic parameters from an analysis of Figure S2; Figure S3 showing measured exchange for internal standard peptides; Figure S4 showing measured exchange for all ACTR segments and their corresponding reference peptides; and Figure S5 showing measured exchange for all CBP segments and their corresponding reference peptides


Biochemistry

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