An Integrated Approach to Unique NMR Assignment of Methionine

Jan 23, 2017 - The strategy involves partially oxidizing the methionine and quantifying the oxidation level by both NMR and liquid chromatography−ma...
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An Integrated Approach to Unique NMR Assignment of Methionine Methyl Resonances in Proteins Fei Yu,†,§ Jing Qiao,† John Robblee, Desiree Tsao, James Anderson, and Ishan Capila* Momenta Pharmaceuticals Inc., 675 West Kendall Street, Cambridge, Massachusetts 02142, United States ABSTRACT: The application of methyl nuclear magnetic resonance (NMR) spectroscopy in protein side-chain structural studies offers unique advantages of greater peak sensitivity, even for high-molecular-weight proteins. Traditionally, the utility of methyl NMR has often been limited by the difficulty in assigning the methyl resonances. Herein, a mass spectrometry (MS)-assisted strategy to assign the methyl resonances of methionine residues is presented. The strategy involves partially oxidizing the methionine and quantifying the oxidation level by both NMR and liquid chromatography−mass spectrometry (LC-MS). The NMR assignment of methyl resonances of methionine is made by correlating the quantitative results obtained from both NMR and MS. The method has been successfully demonstrated using the proteins hen eggwhite lysozyme (HEWL) and porcine pepsin. The technique described herein can help facilitate the application of methyl NMR as a useful tool to study protein structure, dynamics, and interactions.

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further impacting protein structure, function, and stability.12,13 Furthermore, developing an understanding of mechanism and factors that influence the methionine oxidation reaction is of considerable interest in the pharmaceutical industry as methionine oxidation leads to deactivation of a protein therapeutic drug and may even cause possible immunogenic effects.14−16 Therefore, the methyl group has been used as an important probe to characterize protein structure,17,18dynamics,17,19−21 and interactions.22,23 Despite the advantages listed above, the utility of methyl NMR is diminished by the difficulty in assigning the resonances of methyl groups. In an effort to reduce the cost of the sample preparation for proteins that require expression in nonbacterial hosts, as well as to ease the complexity of the NMR spectrum, proteins studied by methyl NMR are often sparsely labeled on selected hydrophobic amino acids as opposed to universal isotopic labeling. Thus, conventional NMR assignments for protein based on backbone-directed triple resonance NMR are often not applicable. The assignment of methyl groups for sparsely labeled proteins have been made either by singleamino-acid substitutions5,24 or utilization of paramagnetic relaxation enhancement data.25,26 Both methods have their own limitations. The former method tends to be laborious and costly as multiple mutant samples are required. The second method can only be implemented when prior knowledge of the 3-D structure of the proteins is available.

MR spectroscopy is a powerful tool for determining the structure of proteins in solution. It is one of a few techniques capable of determining the three-dimensional structure of proteins at atomic resolution.1 Although NMR provides high-resolution analysis for proteins, its application is limited by the size of the protein, as high-molecular-weight proteins result in broadened line widths due to faster relaxation times. Thanks to many advances made in protein NMR since the first introduction of triple-resonance multidimensional NMR methodology, the size limit of protein for NMR studies has increased greatly in the last two decades.2−4 One of the recent advancements in protein analysis is the use of methyl NMR, wherein methyl protons from hydrophobic amino acids such as alanine, leucine, isoleucine, methionine, valine, among others, have been used as probes for structural studies on the side chain of proteins.5−7 In contrast to backbone nuclei, methyl groups on side chains possess some unique advantages. The three chemical-shift-degenerate methyl protons offer a greater intrinsic sensitivity compared to other amino acid nuclei, and their slow relaxation due to high mobility provides further sensitivity enhancement. Methionine methyl resonances, in particular, are of great use due to their low natural occurrence in proteins8 and unique chemical shift, which together often lead to well-resolved NMR spectra. Even for high-molecular-weight proteins, methionine methyl spectra are often of good quality.9 Methionine itself, an essential amino acid, functions as an antioxidant and as a key component for regulation of cellular metabolism.10 Methionine in proteins can be readily oxidized to methionine sulfoxide.11 Studies indicate the oxidation of protein surface methionine increases the protein hydrophobicity and may perturb protein folding, thus © 2017 American Chemical Society

Received: September 19, 2016 Accepted: December 23, 2016 Published: January 23, 2017 1610

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Analytical Chemistry

at 500 μL. The oxidizing reagent tBHP was added to the sample at concentration of 10% or 1% (v/v), and the sample was incubated at room temperature for 15 h. The residual tBHP was then removed with a 10 000 MWCO 0.5 mL Amicon filter. Pepsin was diluted with citrate D2O buffer (7.34 mM deuterated citrate, 105.4 mM sodium chloride, pH 5.4) to 25 mg/mL at 500 μL. The oxidizing reagent tBHP was added to the sample at concentration of 2% (v/v), and the sample was incubated at room temperature for 0, 1, and 2 h, respectively. The residual tBHP was removed with Zeba desalting column. The concentrations of the recovered samples were determined by UV at 280 nm using the calculated extinction coefficient based on the protein sequence. The concentration of HEWL and pepsin were approximately 20 mg/mL. LC-MS Sample Preparation. HEWL LC-MS sample preparation: 50 μg of HEWL at 20 μg/μL (2.5 μL) was diluted to 1 μg/μL in 6 M guanidine hydrochloride. The solution was incubated at 37 °C for 30 min on a Thermomixer at 400 rpm. Then 1 μL of 0.5 M DTT was added. Incubation was continued at 37 °C for 80 min on a Thermomixer at 400 rpm. Five microliters of 250 mM NEM was then added, and the solution was incubated at room temperature in the dark for 2 h. The solution was then buffer exchanged to 50 mM ammonium bicarbonate with the Zeba spin desalting column (7 kDa MWCO). Two micrograms of trypsin (1:25 enzyme to substrate ratio) was then added, and the digestion was carried out in the barocycler for 2 h. Then the reaction was quenched with 1 μL of formic acid. Porcine pepsin LC-MS sample preparation: The same LCMS sample preparation method shown above was used for pepsin LC-MS sample preparation, except that 2 μg Asp-N (1:25 enzyme to substrate ratio) was used as the digestion enzyme. The digestion was carried out at 37 °C for 16 h. NMR Sample Preparation. The NMR samples of HEWL and pepsin were prepared by measuring 500 μL of solution after tBHP removal and then transferring into a 5 mm NMR tube. LC-MS Analysis. The LC-MS and MS2 data for peak identification and quantification was acquired on a Q Exactive instrument coupled to a Dionex HPLC system. Five microliters of sample was injected onto an ACQUITY BEH C18 UPLC column (1.7 μm, 2.1 × 100 mm) operating at 50 °C. The flow rate was 50 μL/min. Mobile-phase A was water with 0.1% formic acid, and mobile-phase B was acetonitrile with 0.1% formic acid. Peptides were eluted using a linear gradient of 0− 50% B over 45 min. The instrument was operated in a datadependent positive ESI ion mode: the first survey MS (scan 1) was from m/z 400 to 2000 with resolving power of 70 000 and AGC target of 1 × 106 followed by the second HCD-MS2 (scan 2) with resolution power of 17 500 and AGC target of 2× 105. NMR Analysis. The heteronuclear single quantum coherence (HSQC) spectra were acquired with a Bruker 600 MHz NMR equipped with a 5 mm cryoprobe. For the HEWL sample, the HSQC spectra were collected at 25 °C with 16 scans for each 128 increment in T1 dimension. For the pepsin sample, the HSQC spectra were collected at 25 °C with 32 scans for each 128 increment in T1 dimension. X-ray Structure and Solvent Accessibility. The 3D structure of pepsin was examine using the structure obtained from the protein data bank (PDB) (5PEP).35 Solvent accessibility was calculated from the PDB coordinate files

Furthermore, even when the universally labeled samples are available, for methionine, the assignment of the methyl group can still pose a challenge as the adjacent nuclear sulfur inhibits the direct magnetization transfer from methyl group to backbone through 13C−13C J-coupling. Consequently, experiments with less sensitivity such as Nuclear Overhauser Effect Spectroscopy (NOESY) are required for assignment of methionine, and many published assignments are simply missing the chemical shift of methyl groups for methionine.27 Herein, a new MS-assisted strategy for NMR assignment of methyl groups of methionine is presented. The concept of using MS to assist NMR assignment of sparsely labeled biomolecules has been around for several years. For instance, Prestegard and co-workers have authored several publications on using MS to help assign NMR resonances such as backbone NH resonance of sparsely labeled protein,28,29 methyl groups of methylated proteins,30 and acetyl groups of oligosaccharides.31,32 The methods usually involve generating partially labeled biomolecules and measuring the isotopic enrichment level by both NMR and MS. It takes advantage of the sequence information on amino acid or saccharide obtained from MS and achieving the assignment of NMR resonances by correlating the isotopic enrichment level obtained from NMR and MS. In this study, the differential nature of oxidation of proteins by tertbutyl hydroperoxide (tBHP), a methionine-specific oxidizing agent, is exploited wherein varied levels of oxidation across the methionine residues on the protein can be achieved depending on their solvent accessibility and generate partially oxidized protein materials.33 Because the chemical shift of methyl resonances for oxidized methionine is quite different from unoxidized methionine, the oxidation level of methionine can be determined by quantifying the remaining methyl resonance of the unmodified methionine. The same oxidation level can also be measured by LC-MS at the peptide level. With the sequence information obtained by LC-MS, by correlating the oxidation level measured by both NMR and LC-MS, one can make the assignment of methyl resonances in the NMR spectrum. The method presented here was first evaluated on a model protein, hen egg-white lysozyme (HEWL). HEWL is small protein (14 kDa) with two methionine residues, and the chemical shift of methyl group for one of the methionine residues has been previously reported.34 The method was also applied to porcine pepsin (35 kDa) which has four methionine residues. Natural-abundance materials were used to demonstrate the method. The method is also applicable to other large size protein samples, and/or isotopic labeled protein samples as long as the methionine methyl resonances are well-resolved and their oxidation rates are distinct.

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METHOD AND MATERIALS The HEWL and tBHP were purchased from Sigma (St. Louis, MO). Porcine pepsin, trypsin, and Asp-N were purchased from Promega (Madison, WI). Zeba desalting spin column, dithiothreitol (DTT), and N-ethylmaleimide (NEM), were purchased from Thermo (Rockford, IL). Formic acid, water with 0.1% formic acid, and acetonitrile with 0.1% formic acid (LC-MS grade) were purchased from Fisher scientific (Faire Lawn, NJ). The 10 000 MWCO 0.5 mL Amicon filters were purchased from Millipore (Billerica, MA). Oxidation of Methionine Residues. The HEWL was diluted with citrate D2O buffer (7.34 mM deuterated citrate, 105.4 mM sodium chloride, pH 3.6) to 2.5 mM concentration 1611

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same in both techniques. To achieve incomplete oxidation and differentiate the levels of oxidation across methionine residues, the protein was oxidized using a low concentration of tBHP (1%). As shown in Figure 2, the intensities of methyl

using GETAREA. A probe radius of 1.4 Å, representing the van der Waals sphere of water, was used.

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RESULTS AND DISCUSSION Hen Egg White Lysozyme as the Model Protein. The method was first evaluated on HEWL, which contains two methionine residues, Met12 and Met105. Since the naturalabundance protein (as opposed to selectively labeled material) was used, it was necessary to first differentiate the methyl resonances of the methionine residues from those of other hydrophobic amino acids. The identification of methionine’s methyl resonances was achieved by subjecting the protein to exhaustive oxidation using high-concentration tBHP (10% v/v). Upon the reaction, two methyl resonances on the 1H−13C HSQC spectrum, resonance A (0.96 ppm/15.05 ppm ( 1 H/ 13 C)) and resonance B (−0.02 ppm/15.71 ppm (1H/13C)), disappear, suggesting that they both arise from methionine residues. The published NMR assignment of HEWL only records the chemical shift for Met105, which matches with resonance B.34 The other methionine resonance, resonance A, is thus believed to arise from Met12. Shown in Figure 1 is the methyl region of 1H−13C HSQC spectrum of HEWL wherein Met105 and Met12 are highlighted.

Figure 2. 1H−13C HSQC spectra of (a) control and (b) oxidized lysozyme.

resonances from both methionine residues decrease after 15 h oxidation. The unmodified methionine residues were quantified by integrating the remaining methyl resonances followed by normalization to the reference resonance. Oxidation of methionine may cause structural change of the protein as well as shift of resonances of nearby residues. To minimize any perturbation effect by oxidized methionine on the reference resonance, a region of the NMR spectrum was chosen as a reference, instead of a single selected resonance. Here a part of the methyl region of the NMR spectrum (0.79−0.45/ 25.09−23.04 ppm (H/C)) was integrated as a reference. The formula used to quantify the remaining methionine residues is shown below. remaining Mets (NMR) =

Metoxi × refcon × 100% Metcon × refoxi

In the formula, Metcon and Metoxi are the integral of methionine methyl resonances of control sample and oxidized sample, respectively; and refcon and refoxi are the integration of references of control and oxidized sample, respectively. The remaining nonoxidized methionine residues were also assessed by LC-MS. Three tryptic peptides including two methionine-containing peptides and one reference peptide (FESNFNTQATNR (33−44)) were targeted for quantification.

Figure 1. Methyl region of 1H−13C HSQC spectrum of hen egg white lysozyme. The methyl resonances of methionine are labeled with residue number.

With the two methionine residues identified in the 1H−13C HSQC spectrum, the approach was tested on HEWL to definitively assign these methionine methyl resonances, assuming absence of any prior literature knowledge. The approach essentially involves exposing the protein (in this case HEWL) to low concentration of the oxidizing agent and evaluating the progression of oxidation of the methionine residues over a short time course using both NMR and LC-MS. The NMR methodology will provide a rank order of the relative oxidation extent of the methionine residues. This result can then be compared with an LC-MS analysis of the same methionine containing peptides (where the position of the methionine is specifically determined) and a similar, relative extent of oxidation can be determined. While the absolute values of oxidation may not be the same across both techniques, it is expected that the rank order of the extent of change of each methionine residue over time should be the

remaining Met (MS) =

Pept(M)oxi × Pept(ref)con × 100% Pept(M)con × Pept(ref)oxi

In the formula, Pept(M)oxi and Pept(M)con stand for the integration of methionine-containing peptides from the oxidized and control samples, respectively; Pept(ref)con and Pept(ref)oxi indicate the reference peptide of control and oxidized sample, respectively. The assignment of methyl resonances of methionine was achieved by correlating the remaining unmodified methionine quantified by NMR and LC-MS. Shown in Table 1 and Figure 1612

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Analytical Chemistry Table 1. NMR Method and LC-MS Method Quantitation Results for the HEWL Remaining Unmodified Methionine after 1% tBHP Oxidation for 15 h NMR method

resonance A (0.96/15.05 ppm)

15 h LC-MS method 15 h

resonance B (−0.02/15.71 ppm)

0.80 Met12 0.76

0.70 Met105 0.69

3 is the comparison between NMR and LC-MS results. The LC-MS and NMR tests of HEWL oxidized with 1% of tBHP

Figure 4. Methyl region of 1H−13C HSQC spectrum of porcine pepsin. The four methyl resonances of methionine are labeled as Peak A−peak D.

spectrum of the starting material (0 h) is compared with the one acquired at 2 h and is shown in Figure 5. The intensities of

Figure 3. Ranking of the oxidation level measured by both NMR and MS suggests the methyl resonance A arises from Met12 and the methyl resonance B arises from Met105.

result in higher oxidation of Met 105 (by LC-MS) and resonance B (by NMR). On the basis of the ranking of the oxidation level, resonance A is assigned to methyl group of Met12, and resonance B is assigned to methyl group of Met105. This assignment agrees with the reported NMR assignment of HEWL.34 Porcine Pepsin. Following the demonstration of the method on HEWL, the method was further evaluated on porcine pepsin which has four methionine residues. Similar to HEWL, natural-abundance pepsin was used to evaluate if the approach is applicable to a protein with more than just two methionine amino acid residues. Thus, exhaustive oxidation of the protein was performed to first discriminate methionine resonances from other methyl resonances. Four resonances disappear upon the complete oxidation of pepsin, suggesting that they arise from methionine residues. Shown in Figure 4 is a portion of the methyl region of the 1H−13C HSQC spectrum for pepsin. Four methyl resonances, A, B, C, and D, were determined to be methionine signals based on their location in the characteristic HSQC region for methyl groups of methionine.7,36 To enable the complete assignment for these four methionine peaks, the protein was first subjected to partial oxidation by low-concentration tBHP (2%). Considering that there are four methionine resonances to assign, we decided to measure the oxidation level at various time points to improve the precision of the assignment. Here, the oxidation levels were monitored at three different time points using NMR and LCMS: 0, 1, and 2 h. To illustrate the different oxidation susceptibility of the methionine residues, the HSQC NMR

Figure 5. 1H−13C HSQC spectra of (a) control and (b) oxidized porcine pepsin.

the four methyl resonances all decrease upon oxidation but with different rates. After 2 h of oxidation, resonance A is no longer visible, most of resonance B disappears with a small residual peak remaining, resonance C has a mild change in peak intensity, and resonance D shows almost no change in peak intensity. The quantitation of the residual unmodified methionine resonances was carried out in a similar way to that for HEWL. The residual resonances were quantified by integrating the remaining signals followed by normalization to the reference resonance. A region of NMR methyl peaks (0.99−0.74/11.74−9.84 ppm (H/C)) was used as the reference resonance for porcine pepsin quantitation to minimize the influence of any structure changes caused by the oxidation of methionine. The same formulas used for HEWL are used for porcine pepsin NMR quantitation and then the value obtained was normalized to the initial value at 0 h. As for LC-MS, four methionine-containing peptides and one reference peptide (DRANNKVGLAPVA (314−326)) were used for quantitation, which followed the same approach described in the previous section for HEWL. The quantitative results from both NMR and LC-MS are listed in Table 2. The NMR and LC-MS results are shown in Figure 6a,b, respectively. As shown in Figure 6, the four methionine 1613

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Table 2. NMR Method and LC-MS Method Quantitation Results for the Porcine Pepsin Remaining Unmodified Methionine Treated with 2% tBHP Oxidation at 0, 1, and 2 h Time Point NMR method

resonance A (2.04/14.06 ppm)

resonance B (2.01/13.98 ppm)

resonance C (1.91/13.11 ppm)

0h 1h 2h

1.00 0.39 0.26

1.00 0.62 0.51

1.00 0.85 0.63

LC-MS method

Met289

Met245

Met199

0h 1h 2h

1.00 0.44 0.35

1.00 0.89 0.76

1.00 0.93 0.85

resonance D (1.86/14.95 ppm) 1.00 0.99 0.93 Met80 1.00 1.00 1.02

Figure 6. Ranking of the oxidation level measured by both (a) NMR and (b) MS.

Figure 7. (Left) Stereo view of X-ray crystallography of porcine pepsin (PDB 5PEP) with all methionine highlighted in red. From top to bottom: Met199, Met245, Met289, and Met80. (Right) Stereo view of X-ray crystallography of porcine pepsin (PDB 5PEP) with 90 deg counterclockwise rotation.

from 3−18% and the MS method showed a standard deviation ranging from 1−6%. Nevertheless, both NMR and LC-MS can differentiate the four methionine residues by their oxidation rate and the relative ranking of the residues in terms of oxidation rate is consistent across the repeated experiments by both techniques. Therefore, by correlating the relative ranking of the oxidation rate, the NMR assignment for the methionine resonances can be made (i.e., Met 289 for resonance A, Met 245 for resonance B, Met 199 for resonance C, and Met 80 for resonance D). As the susceptibility of oxidation for methionine is primarily determined by their surface accessibility, the X-ray structure of porcine pepsin, as shown in Figure 7, as well as the solvent accessible surface area (SASA) values for methionine side chain and percent SASA ratio, shown in Table 3, are used to confirm the observation. Analysis of the structure of pepsin35 (PDB 5PEP) has shown that Met 289 is located on the surface of the molecule, and with a SASA ratio of 45.0, it is the most exposed one among the four methionine residues. However,

residues can be differentiated by either NMR or LC-MS as they disappear at different rates. The comparison between the NMR and LC-MS results is fairly consistent, with each method showing a rank order for the oxidation rates of A > B > C > D and Met289 > Met245 > Met199 > Met80, respectively. Furthermore, the most readily oxidized (A/Met289) and least readily oxidized (D/Met80) residues show comparable reaction rates between methods. For the remaining methionine residues (B/Met245 and C/Met199), the absolute rates of oxidation measured by NMR and LC-MS are somewhat different, with the NMR method showing a greater difference in rates between (B/Met245 and C/Met199) than the LC-MS method. These results were confirmed across repeated experiments in which samples were prepared and measured independently. In each experiment, at least two technical replicates were measured, wherever possible. The variability of the method was assessed across all samples measured in both experiments. In general, the NMR methodology showed a standard deviation ranging 1614

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Table 3. SASA Value for Pepsin Methionine Residues Calculated Using GETAREA 1.1 residue

side chain

ratioa

Met 80 Met 199 Met 245 Met 289

0 4.84 44.29 71.17

0 3.1 28.0 45.0

†

(F.Y., J.Q.) These authors contributed equally to this work.

Notes

The authors declare the following competing financial interest(s): All authors are, or at some point were employees of Momenta Pharmaceuticals Inc. with stock compensation.

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ACKNOWLEDGMENTS We would like to thank Dr. Yan Yin for sharing her expertise in mass spectrometry and Paul Miller for his comments and edits to the manuscript.

a

SASA is calculated using GETAREA 1.1. 1.4 Å is used as the water probe radius. Ratio, in percentage, is the side chain area value relative to random coil Gly−X−Gly tripeptide value for each methionine residue. Residues are typically considered to be buried with percent ratio below 20, whereas those with ratio above 30 are treated as solvent exposed.

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Met 80 is found to be located on an α helix that is buried inside the protein and has a SASA ratio of 0, and therefore, it is the least exposed methionine. The LC-MS results agree well with the X-ray structure and SASA values of porcine pepsin as Met 289 presents the highest rate of oxidation and Met 80 shows the least oxidation susceptibility. Admittedly, both proteins used here have distinct methionine oxidation rates that can be discriminated by NMR and LC-MS. For proteins that have methionine residues with similar solvent accessibility, this MS-assisted NMR assignment method may be limited and would need to be examined more carefully. For instance, more time points may need to be measured, and the absolute oxidation rate, instead of a relative ranking, may need to be calculated.

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CONCLUSIONS A successful MS-assisted assignment strategy for methyl resonances of methionine in protein NMR spectroscopy has been demonstrated. The method does not require using isotopically labeled proteins and is not limited by the size of protein as long as well-resolved methionine resonances are observable, and they have distinct rates of oxidation. This makes it particularly appealing for proteins that cannot be feasibly isotopically labeled and/or for large size proteins such as monoclonal antibodies (mAbs). It opens a new avenue to assigning the methyl group of methionine residues that was long deemed difficult and could be particularly useful for application of methyl NMR as a probe to characterize protein structure, dynamics of backbone and side chain sites, and protein interactions. Furthermore, structural studies on proteins are of increasing interest especially in the evolving area of biosimilars where it is important to compare the similarity of protein structure between a biologic product and its biosimilar version. Therefore, techniques like the one described here may represent an important tool in the overall assessment of protein structural similarity of biologic products.

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REFERENCES

(1) Wuthrich, K. NMR of Proteins and Nucleic Acids; John Wiley & Sons, Inc.: Hoboken, NJ, 1986. (2) Pervushin, K.; Riek, R.; Wider, G.; Wuthrich, K. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 12366−12371. (3) Tjandra, N.; Bax, A. Science 1997, 278, 1111−1114. (4) Tolman, J. R.; Flanagan, J. M.; Kennedy, M. A.; Prestegard, J. H. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 9279−9283. (5) Sprangers, R.; Kay, L. E. Nature 2007, 445, 618−622. (6) Ollerenshaw, J. E.; Tugarinov, W.; Kay, L. E. Magn. Reson. Chem. 2003, 41, 843−852. (7) Stoffregen, M. C.; Schwer, M. M.; Renschler, F. A.; Wiesner, S. Structure 2012, 20, 573−581. (8) Brooks, D. J.; Fresco, J. R.; Lesk, A. M.; Singh, M. Mol. Biol. Evol. 2002, 19, 1645−1655. (9) Ruschak, A. M.; Kay, L. E. J. Biomol. NMR 2010, 46, 75−87. (10) Levine, R. L.; Moskovitz, J.; Stadtman, E. R. IUBMB Life 2000, 50, 301−307. (11) Nlelsen, H. K.; Loliger, J.; Hurrell, R. F. Br. J. Nutr. 1985, 53, 61−73. (12) Chao, C. C.; Ma, Y. S.; Stadtman, E. R. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 2969−2974. (13) Hershko, A.; Ciechanover, A. Prog. Nucleic Acid Res. Mol. Biol. 1986, 33, 19−56. (14) Schoneich, C.; Aced, A.; Asmus, K. D. J. Am. Chem. Soc. 1993, 115, 11376−11383. (15) Masato, A.; Kiichi, F.; Uchiyama, S. J. Pharm. Sci. 2016, 105, 623−629. (16) Chu, J. Oxidation of Methionine Residues in Protein Pharmaceuticals. Ph.D. Thesis, Massachusetts Institute of Technology, 2004. (17) Tugarinov, V.; Kay, L. E. ChemBioChem 2005, 6, 1567−1577. (18) Gardner, K. H.; Rosen, M. K.; Kay, L. E. Biochemistry 1997, 36, 1389−1401. (19) Skrynnikov, N. R.; Mulder, F. A.; Hon, B.; Dahlquist, F. W.; Kay, L. E. J. Am. Chem. Soc. 2001, 123, 4556−4566. (20) Mittermaier, A.; Kay, L. E. Science 2006, 312, 224−228. (21) Brath, U.; Akke, M.; Yang, D.; Kay, L. E.; Mulder, F. A. J. Am. Chem. Soc. 2006, 128, 5718−5727. (22) Hajduk, P. J.; Augeri, D. J.; Mack, J.; Mendoza, R.; Yang, J.; Betz, S. F.; Fesik, S. W. J. Am. Chem. Soc. 2000, 122, 7898−7904. (23) Igumenova, T. I.; Frederick, K. K.; Wand, A. Chem. Rev. 2006, 106, 1672−1699. (24) Sprangers, R.; Gribun, A.; Hwang, P. M.; Houry, W. A.; Kay, L. E. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 16678−16683. (25) John, M.; Schmitz, C.; Park, A. Y.; Dixon, N. E.; Huber, T.; Otting, G. J. Am. Chem. Soc. 2007, 129, 13749−13757. (26) Venditti, V.; Fawzi, N. L.; Clore, G. M. J. Biomol. NMR 2011, 51, 319−328. (27) Wang, S.; Horimoto, Y.; Dee, D. R.; Yada, R. Y. J. Biol. Chem. 2014, 289, 697−707. (28) Feng, L.; Orlando, R.; Prestegard, J. H. J. Am. Chem. Soc. 2004, 126, 14377−14379. (29) Feng, L.; Lee, H. S.; Prestegard, J. H. J. Biomol. NMR 2007, 38, 213−219.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 1-617-3955100. Fax: 1-617-621-0431. ORCID

Ishan Capila: 0000-0002-5036-6778 Present Address §

(F.Y.) School of Pharmacy, Yantai University, 30 Qingquan Road, Yantai, Shangdong, P.R. China, 264005 1615

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Analytical Chemistry (30) Macnaughtan, M. A.; Kane, A. M.; Prestegard, J. H. J. Am. Chem. Soc. 2005, 127, 17626−17627. (31) Yu, F.; Prestegard, J. Biophys. J. 2006, 91, 1952−1959. (32) Yu, F.; Wolff, J. J.; Amster, I. J.; Prestegard, J. H. J. Am. Chem. Soc. 2007, 129, 13288−13297. (33) Keck, R. G. Anal. Biochem. 1996, 236, 56−62. (34) Wuthrich, K. Nat. Struct. Biol. 2001, 8, 923−925. (35) Cooper, J. B.; Khan, G.; Taylor, G.; Tickle, I. J.; Blundell, T. L. J. Mol. Biol. 1990, 214, 199−222. (36) Sahakyan, A. B.; Vranken, W. F.; Cavalli, A.; Vendruscolo, M. J. Biomol. NMR 2011, 50, 331−346.

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