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Nitroxide Labeling of Proteins and the Determination of Paramagnetic Relaxation Derived Distance Restraints for NMR Studies

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Journal of Molecular Biology
Mar 2016



Site-specific attachment of paramagnetic spin labels to biomolecules causes distance-dependent line-broadening effects, which can be exploited to study the structure and dynamics of these molecules in solution. This protocol describes how to attach nitroxide spin labels to proteins and how to collect and analyze NMR data using these labeled samples. We also explain how to derive distance restraints for paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) studies.

Keywords: Paramagnetic relaxation enhancement (顺磁弛豫增强), Large protein NMR (大蛋白质NMR), Nuclear magnetic resonance spectroscopy (核磁共振光谱法), Nitroxide spin label (硝基氧自旋标记), Distance restraints (距离约束)


This protocol describes how to attach nitroxide spin labels to proteins and how the modified proteins can be employed to derive distance restraints using paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) methods. Site-specific attachment of paramagnetic spin labels to proteins enhances the transverse relaxation rates of nearby nuclei leading to line-broadening effects that can be used to derive distance restraints (Battiste and Wagner, 2000; Iwahara et al., 2004; Clore and Iwahara, 2009). PRE-derived distance restraints have been used to characterize the structures of various molecules, including amongst others membrane proteins (Roosild et al., 2005), multi-domain proteins displaying inter-domain dynamics (Sjodt et al., 2016), single domain proteins (Battiste and Wagner, 2000), protein-DNA complexes (Clore and Iwahara, 2009), transient protein-protein interactions (Tang et al., 2007; Villareal et al., 2011), and intrinsically disordered proteins (Bertoncini et al., 2005). Two approaches are generally used to obtain PRE-derived distance restraints using proteins that are labeled with paramagnetic probes. The first method quantitatively measures the probe’s effects on the transverse relaxation rates of nearby protein nuclei by determining Γ2 (described in detail by Iwahara and Clore) (Iwahara et al., 2004; Clore and Iwahara, 2009). The second method is less quantitative, but in practice it is easier to implement. It was originally employed by Battiste and Wagner, and measures the probe’s effects by comparing cross-peak intensity ratios in diamagnetic- and paramagnetic-spectra of the labeled protein (Battiste and Wagner, 2000). The nitroxide spin label MTSL is frequently used as a paramagnetic probe in PRE-NMR studies of proteins because it is readily attached via a disulfide bond to cysteine residues, and also relatively small, inexpensive, and commercially available. The aim of this protocol is to describe how to attach MTSL nitroxide spin labels to proteins and how to derive PRE-distance restraints following the approach described by Battiste and Wagner (2000).

Materials and Reagents

  1. Amber vial
  2. Desalting spin column, for example, a ZebaTM spin desalting column, 7k MWCO, 2 ml (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 89889 )
  3. 15 ml conical test tube
  4. Aluminum foil
  5. A centrifugal filter, such as Amicon Ultra-15 centrifugal filter (EMD Millipore, catalog number: UFC900308 )
  6. Standard 5 mm NMR tube (SP Industries, catalog number: 535-PP-7 )
  7. 2D [1H-15N]-HSQC spectrum of the native protein acquired using standard methods (Cavanagh et al., 1995)
  8. Dithiothreitol (DTT); > 99% purity (Gold Bio, catalog number: DTT50 )
  9. Deuterium oxide (D2O); ≥ 99.8% purity (Sigma-Aldrich, catalog number: 617385 )
  10. Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl); ≥ 99% purity (Fisher Scientific, catalog number: BP153 )
  11. Sodium chloride (NaCl); ≥ 99% purity (Fisher Scientific, catalog number: S271 )
  12. S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl (MTSL) methanesulfonothioate (Toronto Research Chemicals, catalog number: O875000 )
  13. Acetonitrile; ≥ 99.9% purity (Fisher Scientific, catalog number: A996 )
  14. Sodium phosphate monobasic (NaH2PO4); ≥ 98% purity (Fisher Scientific, catalog number: S369 )
  15. Sodium azide; ≥ 99% purity (Fisher Scientific, catalog number: S227I )
  16. Sodium ascorbate; ≥ 98% purity (Sigma-Aldrich, catalog number: A7631 )
  17. Labeling buffer (see Recipes)
  18. 200 mM MTSL stock solution (see Recipes)
  19. Example NMR buffer (see Recipes)
  20. 250 mM sodium ascorbate stock solution (see Recipes)

Note: These reagents were used to obtain the PRE data according to Sjodt et al., 2016. However, other brands of these materials may be used if necessary.


  1. Centrifuge (Beckman Coulter, model: Allegra X-14R )
  2. SX4750A swinging bucket rotor (Beckman Coulter, model: SX4750A ARIESTM Roter )
  3. MALDI-TOF mass spectrometer (Thermo Fisher Scientific, Applied BiosystemsTM, model: Voyager-DE-STR )
    Note: This product has been discontinued. Examples of other MALDI-TOF instruments include Shimadzu, model: AXIMA Assurance Linear MALDI-TOF ; Bruker, model: microflex LT/SH .
  4. NMR experiments system
    Note: NMR experiments used in this protocol are part of the Bruker standard pulse sequence library and were performed on Bruker Avance spectrometers equipped with triple-resonance cryogenic probes (Bruker Corporation).


  1. NMRPipe (Delaglio et al., 1995)
  2. Bruker TopSpinTM
  3. Sparky (Goddard and Kneller, 2008)
  4. Microsoft Excel
  5. XPLOR-NIH (Schwieters et al., 2003)


Figure 1A shows a flow chart of the steps that need to be performed in order to label a protein with MTSL and how PRE-NMR distance restraints can be measured. In the procedure, the MTSL probe is attached via a disulfide bond to a protein containing a single cysteine residue. NMR spectra are then acquired and interpreted to extract distance restraints. In order to interpret the NMR spectra, the backbone chemical shifts of the protein must first be obtained.

Figure 1. A flow chart of nitroxide labeling and representative PRE data. A. A flow chart describing the steps taken when site-specifically labeling proteins with nitroxide spin labels for PRE-derived structural studies. B. Representative [1H-15N]-HSQC spectrum of IsdHN2N3 (see Sjodt et al., 2016 for more information). Boxed regions indicate the representative sections of each PRE probe’s spectra shown in (C) (K499R1 and E559R1). C. Magnified regions showing the selective distance dependent line-broadening for the K499R1 (top) and E559R1 (bottom) probes. For each probe the diamagnetic and paramagnetic spectra are shown on the left and right panels, respectively. D. Representative PRE profile of the E559R1 probe data. Normalized intensity ratios (Iox/Ired) are shown as a function of residue number. The asterisk depicts the location of the probe. Errors of the ratio measurements are approximately 10-15% based on signal to noise of the NMR spectra, which can lead to values in excess of 1. Errors also can occur as a result of manipulating the sample (adding ascorbate to reduce the probe) and instrument instability. These errors are partially accounted for by adding ± 5 Å to the distance restraints that are obtained from the ratio data (Battiste and Wagner, 2000).

  1. Assign the backbone chemical shifts of the native protein
    1. Acquire a high quality 2D [1H-15N]-HSQC spectrum of the native protein to be used as a reference (i.e., a spectrum containing the appropriate number of resolved cross-peaks based on the amino acid sequence of the protein).
    2. Assign the backbone amide chemical shifts in the native protein using standard NMR procedures (for standard NMR experiments see Cavanagh et al., 1995).

  2. Generate a protein mutant with a single cysteine residue
    1. For these studies, the protein should contain a single cysteine residue. Check the protein’s primary sequence to determine if it contains native cysteine residues that could be inadvertently labeled by the nitroxide probe. If necessary, make conservative mutations to these cysteine residues to prevent non-specific labeling. If biochemical assays are available for the protein, they should be performed to ensure these mutations do not impair its function.
    2. Choose the desired location in the protein to which the nitroxide probe will be attached. This location needs to be at a residue whose sidechain is solvent-exposed. The residue should be located in a structured loop or within defined secondary structure. This is important because if the probe is located in a structurally disordered region of the protein, the increased flexibility of the polypeptide backbone can reduce the quantitative strength of the technique.
    3. Perform site-directed mutagenesis to incorporate a single cysteine residue at the desired location to which the nitroxide probe will be attached.
    4. Purify 15N-labeled protein containing the cysteine mutation. Use buffers that are supplemented with 2.5 mM DTT to prevent formation of inter-molecular disulfide bonds that can lead to protein aggregation (see Note 1). For a review on expression and purification of proteins see Gräslund et al. (2008).
    5. Prior to labeling the purified cysteine mutant with MTSL, verify that the cysteine mutation does not affect the structure of the protein. This can be accomplished by comparing the [1H-15N]-HSQC spectra of the mutant and native proteins. The chemical shift position of the cross-peaks in the spectra should be similar, with only localized changes occurring near the site of the mutation.

  3. Site-specific labeling the protein
    The aim of the following steps is to label the 1H-15N labeled purified cysteine mutant of the protein with MTSL. Care is taken to maintain the cysteine mutant under reducing conditions until it is modified with MTSL. The apparatus used to label the protein is shown in Figure 2, and consists of a conical tube and desalting column.

    Figure 2. Schematic of the apparatus used to label proteins with MTSL

    1. Buffer exchange the 15N-labeled cysteine mutant into labeling buffer that has been supplemented with fresh 2.5 mM DTT. The protein concentration should be at least 250-300 µM.
    2. Make a stock solution of MTSL by adding acetonitrile directly to the amber vial in which the MTSL was shipped. The final concentration of the stock solution should be 200 mM MTSL. Store the MTSL stock solution at -20 °C (see Notes 2 and 3).
    3. Immediately before starting the labeling reaction, dilute the protein with labeling buffer to a final concentration of 250-300 µM protein. The final volume of the diluted protein solution should be 1 ml. The protein should be diluted with labeling buffer that does not contain DTT. From this step forward, all buffers used in this procedure should not contain DTT.
    4. Equilibrate a Zeba spin desalting column with labeling buffer as per the manufacturer’s instructions.
    5. Construct the MTSL solution that will be used to label the protein. Pipette 2.5 ml of labeling buffer into a clean 15 ml conical tube. Add MTSL from the stock solution to the conical tube. The amount added should be sufficient to achieve a final concentration of MTSL that is 10x the molar amount of protein that will be labeled (e.g., if the final concentration of protein in the reaction will be 300 µM then the concentration of MTSL should be 3 mM). Make sure to shield all MTSL solutions from light by covering the tube with foil first.
    6. Construct the apparatus that will be used to label the protein (Figure 2). Place the equilibrated desalting column into the conical tube from step C5. The column will rest above the MTSL-containing solution and will leave room for the protein solution to flow through.
    7. Slowly pipette the protein onto the center of the column’s resin bed (Figure 2).
    8. Centrifuge as per the manufacturer’s instructions. The protein will flow through the column and into the labeling solution, while DTT present in the protein solution will remain on the column. The process is considered complete after the elution of all the protein solution into the MTSL solution at the bottom of the collection tube. This will produce a solution located at the bottom of the conical tube that has a total volume of ~3.5 ml. Discard the desalting column.
    9. Cover the conical tube containing the modification reaction with foil and let it incubate under agitation for 15 min at room temperature.
    10. After 15 min, add additional MTSL from the stock solution to the modification reaction. The amount of added MTSL should yield a final concentration that is 20x greater than the protein’s concentration in the conical tube.
    11. Place tube back onto a rotating device and let the protein:MTSL mixture incubate under agitation overnight at room temperature (~16 h).
    12. Buffer exchange the MTSL-labeled protein sample into the desired NMR buffer using a fresh Amicon Ultra-15 centrifugal filter. This will remove any excess (unligated) MTSL (see Notes 4-6). As mentioned above, the NMR buffer should not contain any DTT.
    13. Verify that the protein is labeled with MTSL by MALDI-TOF mass spectrometry. As compared to the unligated protein, the mass of the modified protein should increase by ~186 Da (the weight of the attached probe). Although MALDI-TOF is not quantitative, in the mass spectrum > 90% of the protein should be labeled with MTSL. If biochemical assays are available for the protein, they should be performed on labeled protein to verify that MTSL attachment does not impair its function.
    14. For NMR studies, the MTSL modified protein should have a final concentration of ~300 µM (see Note 7). The NMR buffer should contain 8-10% D2O, but be devoid of any DTT. Place the sample in a standard NMR tube and shield from light using foil.

  4. NMR data acquisition
    1. Use a standard 2D- [1H-15N]-HSQC pulse sequence to acquire a spectrum of the freshly MTSL-labeled protein. Typically, the 1H and 15N dimensions are defined by 2,048 and 256 complex points, respectively. At this time, the sample is in its paramagnetic state. Its spectrum should be well dispersed, indicating that the modified protein is folded and not aggregating.
    2. Make a stock solution of 250 mM sodium ascorbate by dissolving it in NMR buffer.
    3. After the [1H-15N]-HSQC spectrum has been acquired of the paramagnetic protein (step D1), remove the NMR tube from the magnet. Using a long pipette that fits into the NMR tube, add sodium ascorbate to a final concentration that is 5x greater than the concentration of the labeled protein. This step should be performed carefully, by pipetting the solution up and down slowly so as to mix the solution, while preventing any sample loss. Addition of sodium ascorbate will reduce MTSL’s unpaired electron to form the diamagnetic state. Let the sample reduce for a minimum of 3 h at room temperature (no agitation is needed).
    4. Acquire a [1H-15N]-HSQC spectrum of the reduced diamagnetic MTSL:protein sample using acquisition parameters that are identical to those used to acquire the paramagnetic spectrum described in step D1.

Data analysis

  1. Process and analyze the paramagnetic and diamagnetic NMR data
    1. Process the acquired NMR data using standard software (e.g., NMRPipe [Delaglio et al., 1995] or Bruker TopSpinTM software). Both the paramagnetic and diamagnetic datasets should be processed identically. Representative [1H-15N]-HSQC spectra of diamagnetic and paramagnetic samples are shown in Figures 1B and 1C.
    2. Analyze the NMR spectra using standard software (e.g., Sparky) (Goddard and Kneller, 2008). The diamagnetic spectrum should be analyzed first, as it is most closely related to the previously assigned spectrum of the native protein. Assign the cross-peaks in the diamagnetic spectrum using the known chemical shifts of the native protein. This can readily be accomplished by overlaying the [1H-15N]-HSQC spectra of the native and labeled proteins, and then transferring the chemical shift assignments. The cross peaks in the spectra should overlay well, with only localized differences occurring for cross peaks that originate from residues that are located near the attached probe. Considerable care must be taken to make sure that the assignments are correctly assigned.
    3. Measure the intensities of the cross peaks in the assigned spectrum of the diamagnetic protein. Care should be taken to make sure that the cross peaks that are analyzed are well resolved (see Note 8). When making the intensity measurement, make sure that it is made at the peak’s maximum. In our experience, measuring peak height is sufficient for extracting distance restraints. However, measuring cross peak volumes is also acceptable. It is critical to extensively check the sequence-specific chemical shift assignments of the diamagnetic spectrum. Generate a list of cross peak intensities using the analysis software. The list should contain the cross peak’s intensity and the identity of the residue.
    4. Label the cross peaks in the paramagnetic spectrum using the curated chemical shift list that was used to analyze the diamagnetic spectrum. Using the procedures outlined in Data analysis A3, measure the peak intensities of isolated cross peaks in the paramagnetic spectrum. If a cross-peak in the paramagnetic spectrum is significantly broadened such that its intensity is near the noise level (e.g., Figure 1C, residue S563 in the E559R1 paramagnetic panel), accurate measurement of its intensity is not possible. Therefore, these cross peaks should not be employed to quantitatively relate the NMR data to inter-atomic distances, and intensities from these cross peaks should not be analyzed further. However, the identity of residues exhibiting significant line broadening in the paramagnetic spectrum should be noted. This is because distance restraints to these amino acids can still be employed in the structure calculations as complete broadening indicates that they are within ~12 Å of the probe. Use the software to generate a list of cross peak intensities using the analysis software. The list should contain the cross peak’s intensity and the identity of the residue. A sample table depicting a list of cross peak intensities is shown in Table 1.

      Table 1. Sample list of peak intensities and ratiosa

      aNote: This data was adapted from the NMR study of IsdHN2N3 (Sjodt et al., 2016).

    5. Calculate the intensity ratios using standard analysis software (e.g., Microsoft Excel). Import both the diamagnetic and paramagnetic intensity lists (generated from Data analysis A3 and A4) into a single spreadsheet. For each residue, use the cross peak intensity data to calculate the ratio of its intensity in the paramagnetic (Iox) and diamagnetic (Ired) states. A representative plot of the intensity ratios versus protein sequence is shown in Figure 1D.

  2. Estimating distance restraints from the NMR data
    The aim of this section is to describe how to relate the cross-peak intensity ratio data calculated in the previous section to distance restraints. A detailed description describing the effects of paramagnetic relaxation on cross peak intensity, and its relationship to interatomic distance has been described in detail elsewhere (Battiste and Wagner, 2000; Clore, 2015). The reader is encouraged to consult these references to understand the underlying theory. Briefly, the effect can be described by the following equation:


    where, Iox and Ired are the measured intensities of the cross peaks for the paramagnetic and diamagnetic forms of the protein, respectively, t is the total evolution time of the transverse proton magnetization during the NMR experiment. The values of R correspond to the rate of the transverse relaxation of the amide proton, R2 is the intrinsic relaxation of the proton, R2sp, is the contribution to the relaxation caused by the paramagnetic probe. The value of R2sp is dependent upon r, the distance between the amide proton and the probe. This relationship is described using a modified version of the Solomon-Bloembergen as shown in Equation 2 (Solomon and Bloembergen, 1956; Battiste and Wagner, 2000)

    where, K is a constant (1.23 x 10-32 cm6 sec-2) that describes the spin properties of the MTSL spin label (Battiste and Wagner, 2000), ωh is the Larmor frequency of the proton spin, τC is the apparent PRE correlation time (Simon et al., 2010). As ωh is known, and values of τC can be estimated based on the molecular weight of the protein (Cavanagh et al., 1995), a calibration curve can be generated using Equations 1 and 2. The curve relates the intensity ratio for a given cross peak to the distance between the amide proton and the probe. For MTSL probes, Iox/Ired ratios less than 1 indicate that the probe and proton atom are within 13-25 Å, whereas Iox/Ired ratios equal to ~0 and ~1 indicate that the probe and proton are separated by less than ~12 Å, or more than ~30 Å, respectively. It is critical to note, that the calibration curves only provide an estimate of the interatomic distances as the probe exhibits significant flexibility. As a result, the distance restraints obtained from the intensity data are imprecise and structure calculation strategies represent the probe using multiple conformers (Iwahara et al., 2004; Clore, 2015).
    1. Construct an intensity to distance calibration curve (Iox/Ired versus r). This is done using Equations 1 and 2, and estimated values of τC and the average amide proton linewidth (R2) for structured residues in the protein. A representative calibration curve is shown in Figure 3 and employs values of 23 Hz and 16.4 ns for R2/π and τC, respectively.

       Figure 3. Calibration curve for the estimation of PRE-derived distances. A representative calibration curve used for the conversion of intensity ratios into distances for the NMR study of IsdHN2N3 (Sjodt et al., 2016). The curve was generated according to Equations 1 and 2 using an average linewidth (R2/π) and correlation time (τC) of 23 Hz and 16.4 ns, respectively.

    2. Convert the experimental intensity ratios into distance separations using the calibration curve generated in Data analysis B1.
    3. Construct a distance restraint table to be used in XPLOR-NIH calculations (Figure 4). The distance restraints should be made between the nitrogen atom of the MTSL ring and the affected amide proton using standard XPLOR-NIH restraint table format (Schwieters et al., 2003). It should be stressed that these distances boundaries are estimates and are not to be used as accurate distance measurements. Two types of distance restraints are employed in the calculations:
      1. If Iox/Ired is < 0.80, the restraint is called ‘attractive’ and is given the assigned distance from Data analysis B2 with an error of ± 5 Å (see Note 9).
      2. If Iox/Ired ≥ 0.80, the restraint is called ‘repulsive’ and is given a lower bound distance of 20 Å with no upper bound distance (Battiste and Wagner, 2000; Reckel et al., 2011; Gottstein et al., 2012).

        Figure 4. Example of XPLOR-NIH restraint table. An example of PRE-distance restraint input table in XPLOR-NIH format. This table was adapted from the NMR study of IsdHN2N3 (Sjodt et al., 2016). The ‘segid ALT’ portion identifies the MTSL molecule that was modeled onto the starting structure in three randomized orientations. (For more information on generating a restraint table, the reader is encouraged to refer to Schwieters et al., 2003; Iwahara et al., 2004; Sjodt et al., 2016).  


  1. Make fresh DTT solution before each use.
  2. MTSL will precipitate at high concentrations of acetonitrile, so keep the stock concentrations below 200 mM.
  3. MTSL is light- and air-sensitive.
  4. Make sure to extensively wash the protein in step C11. Excess unligated MTSL will lead to artifacts in the NMR experiments.
  5. The NMR buffer should be optimized for the protein of interest. For a review on buffers suitable for NMR see Kelly et al. (2002).
  6. The pH of the buffer should be chosen based on the stability of the protein as the MTSL radical is quite stable once ligated to the protein. However, at basic pH it is possible for some of the free MTSL groups to be hydrolyzed to thiols. These groups could react with the remaining free MTSL molecules and lead to formation of disulfide linked biradicals, which would decrease the labeling efficiency. Therefore, buffers that are above neutral values should be avoided. To account for this possibility, a 20-fold molar excess of MTSL is used during the labeling process to ensure efficient labeling of the protein.
  7. The protein concentration should be kept low to prevent non-specific inter-molecular PRE effects.
  8. It has been reported that the tail of a cross-peak that is completely broadened by PRE effects can reduce the height of a neighboring peak by up to 15%, giving rise to false positive broadening effects (Battiste and Wagner, 2000).
  9. The large error bounds placed on the attractive restraints are provided to account for the inherent flexibility of the MTSL probe.


  1. Labeling buffer
    50 mM Tris-HCl (pH 7.8)
    50 mM NaCl
  2. 200 mM MTSL stock solution
    Dissolve 10 mg MTSL into 18.92 μl acetonitrile
  3. Example NMR buffer
    20 mM NaH2PO4 (pH 6.0)
    50 mM NaCl
    0.01% NaN3
  4. 250 mM sodium ascorbate stock solution
    Dissolve 49.5 mg sodium ascorbate into deionized water to a final volume of 1 ml


This protocol was adapted from Sjodt et al. (2016). This work was supported by National Institutes of Health Grants AI52217 (to R.T.C.) and the National Institutes of Health Award F31GM101931 (to M.S.).


  1. Battiste, J. L. and Wagner, G. (2000). Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data. Biochemistry 39(18): 5355-5365.
  2. Bertoncini, C. W., Jung, Y. S., Fernandez, C. O., Hoyer, W., Griesinger, C., Jovin, T. M. and Zweckstetter, M. (2005). Release of long-range tertiary interactions potentiates aggregation of natively unstructured alpha-synuclein. Proc Natl Acad Sci U S A 102(5): 1430-1435.
  3. Cavanagh, J., Fairbrother, W. J., Palmer, A. G. and Skelton, N. J. (1995). Protein NMR Spectroscopy: Principles and Practice. Academic Press pp: 587
  4. Clore, G. M. (2015). Practical aspects of paramagnetic relaxation enhancement in biological macromolecules. Methods Enzymol 564: 485-497.
  5. Clore, G. M. and Iwahara, J. (2009). Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes. Chem Rev 109(9): 4108-4139.
  6. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J. and Bax, A. (1995). NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3): 277-293.
  7. Gräslund, S., Nordlund, P., Weigelt, J., Hallberg, B. M., Bray, J., Gileadi, O., Knapp, S., Oppermann, U., Arrowsmith, C., Hui, R., Ming, J., dhe-Paganon, S., Park, H. W., Savchenko, A., Yee, A., Edwards, A., Vincentelli, R., Cambillau, C., Kim, R., Kim, S. H., Rao, Z., Shi, Y., Terwilliger, T. C., Kim, C. Y., Hung, L. W., Waldo, G. S., Peleg, Y., Albeck, S., Unger, T., Dym, O., Prilusky, J., Sussman, J. L., Stevens, R. C., Lesley, S. A., Wilson, I. A., Joachimiak, A., Collart, F., Dementieva, I., Donnelly, M. I., Eschenfeldt, W. H., Kim, Y., Stols, L., Wu, R., Zhou, M., Burley, S. K., Emtage, J. S., Sauder, J. M., Thompson, D., Bain, K., Luz, J., Gheyi, T., Zhang, F., Atwell, S., Almo, S. C., Bonanno, J. B., Fiser, A., Swaminathan, S., Studier, F. W., Chance, M. R., Sali, A., Acton, T. B., Xiao, R., Zhao, L., Ma, L. C., Hunt, J. F., Tong, L., Cunningham, K., Inouye, M., Anderson, S., Janjua, H., Shastry, R., Ho, C. K., Wang, D., Wang, H., Jiang, M., Montelione, G. T., Stuart, D. I., Owens, R. J., Daenke, S., Schütz, A., Heinemann, U., Yokoyama, S., Büssow, K. and Gunsalus, K. C. (2008). Protein production and purification. Nat Methods 5(2): 135-146.
  8. Goddard, T. D. and Kneller, D. G. (2008). Sparky NMR analysis software.
  9. Gottstein, D., Reckel, S., Dotsch, V. and Guntert, P. (2012). Requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by NMR. Structure 20(6): 1019-1027.
  10. Iwahara, J., Schwieters, C. D. and Clore, G. M. (2004). Ensemble approach for NMR structure refinement against 1H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule. J Am Chem Soc 126(18): 5879-5896.
  11. Kelly, A. E., Ou, H. D., Withers, R. and Dotsch, V. (2002). Low-conductivity buffers for high-sensitivity NMR measurements. J Am Chem Soc 124(40): 12013-12019.
  12. Reckel, S., Gottstein, D., Stehle, J., Lohr, F., Verhoefen, M. K., Takeda, M., Silvers, R., Kainosho, M., Glaubitz, C., Wachtveitl, J., Bernhard, F., Schwalbe, H., Guntert, P. and Dotsch, V. (2011). Solution NMR structure of proteorhodopsin. Angew Chem Int Ed Engl 50(50): 11942-11946.
  13. Roosild, T. P., Greenwald, J., Vega, M., Castronovo, S., Riek, R. and Choe, S. (2005). NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science 307(5713): 1317-1321.
  14. Schwieters, C. D., Kuszewski, J. J., Tjandra, N. and Clore, G. M. (2003). The Xplor-NIH NMR molecular structure determination package. J Magn Reson 160(1): 65-73.
  15. Simon, B., Madl, T., Mackereth, C. D., Nilges, M. and Sattler, M. (2010). An efficient protocol for NMR-spectroscopy-based structure determination of protein complexes in solution. Angew Chem Int Ed Engl 49(11): 1967-1970.
  16. Sjodt, M., Macdonald, R., Spirig, T., Chan, A. H., Dickson, C. F., Fabian, M., Olson, J. S., Gell, D. A. and Clubb, R. T. (2016). The PRE-Derived NMR model of the 38.8-kDa Tri-Domain IsdH protein from staphylococcus aureus suggests that it adaptively recognizes human hemoglobin. J Mol Biol 428(6): 1107-1129.
  17. Solomon, I. and Bloembergen, N. (1956). Nuclear magnetic interactions in the HF molecule. J Chem Phys 25: 261.
  18. Tang, C., Schwieters, C. D. and Clore, G. M. (2007). Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR. Nature 449(7165): 1078-1082.
  19. Villareal, V. A., Spirig, T., Robson, S. A., Liu, M., Lei, B. and Clubb, R. T. (2011). Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus. J Am Chem Soc 133(36): 14176-14179.


顺磁自旋标记与生物分子的位点特异性连接导致距离依赖性线宽增加效应,可用于研究溶液中这些分子的结构和动力学。 该协议描述了如何将氮氧自由标记连接到蛋白质上,以及如何使用这些标记的样品收集和分析NMR数据。 我们还解释了如何导出顺磁性松弛增强核磁共振(PRE-NMR)研究的距离约束。

该协议描述了如何使用顺磁性松弛增强核磁共振(PRE-NMR)方法将氮氧自由标记附着到蛋白质上以及如何使用修饰的蛋白质来导出距离约束。顺磁自旋标记对蛋白质的位点特异性附着增强了附近细胞核的横向松弛率,导致了可用于导出距离约束的线宽变化效应(Battiste和Wagner,2000; Iwahara等人。 ,2004; Clore和Iwahara,2009)。已经使用PRE衍生的距离限制来表征各种分子的结构,包括膜蛋白(Roosild等人,2005),显示域间动力学的多结构域蛋白(Sjodt 单链蛋白(Battiste和Wagner,2000),蛋白质-DNA复合物(Clore和Iwahara,2009),瞬时蛋白质 - 蛋白质相互作用(Tang等人, ,2007; Villareal等人,2011)和本质上无序的蛋白质(Bertoncini等人,2005)。通常使用两种方法来获得用顺磁探针标记的蛋白质来获得PRE衍生的距离约束。第一种方法通过确定Γ2(由岩华和克洛)详细描述,定量测量探针对附近蛋白质核的横向松弛率的影响(Iwahara等, 2004; Clore and Iwahara,2009)。第二种方法定量较少,但实际上更容易实现。它最初由Battiste和Wagner使用,并通过比较标记蛋白的抗磁性和顺磁性光谱中的交叉峰强度比来测量探针的作用(Battiste和Wagner,2000)。硝基氧自旋标记MTSL经常用作蛋白质的PRE-NMR研究中的顺磁探针,因为它容易通过二硫键连接到半胱氨酸残基,并且也相对较小,便宜且可商购。该协议的目的是描述如何将MTSL氮氧自由标记附着在蛋白质上,以及如何按照Battiste和Wagner(2000)所述的方法得出PRE距离约束。

关键字:顺磁弛豫增强, 大蛋白质NMR, 核磁共振光谱法, 硝基氧自旋标记, 距离约束


  1. 琥珀色小瓶
  2. 脱盐旋转柱,例如Zeba< sup>旋转脱盐柱,7k MWCO,2ml(Thermo Fisher Scientific,Thermo Scientific& TM,目录号:89889)
  3. 15 ml锥形试管
  4. 铝箔
  5. 离心过滤器,例如Amicon Ultra-15离心过滤器(EMD Millipore,目录号:UFC900308)
  6. 标准5毫米NMR管(SP Industries,目录号:535-PP-7)
  7. 使用标准方法获得的天然蛋白质的2D [ 1 H- 15 N] -HSQC光谱(Cavanagh等人,1995) />
  8. 二硫苏糖醇(DTT); > 99%纯度(Gold Bio,目录号:DTT50)
  9. 氧化氘(D 2 O); ≥99.8%纯度(Sigma-Aldrich,目录号:617385)
  10. 三(羟甲基)氨基甲烷盐酸盐(Tris-HCl); ≥99%的纯度(Fisher Scientific,目录号:BP153)
  11. 氯化钠(NaCl); ≥99%的纯度(Fisher Scientific,目录号:S271)
  12. S-(1-氧基-2,2,5,5-四甲基-2,5-二氢-1H-吡咯-3-基)甲基(MTSL)甲磺酸酯(多伦多研究化学公司,目录号:O875000)
  13. 乙腈; ≥99.9%纯度(Fisher Scientific,目录号:A996)
  14. 磷酸二氢钠(NaH 2 PO 4); ≥98%纯度(Fisher Scientific,目录号:S369)
  15. 叠氮化钠≥99%纯度(Fisher Scientific,目录号:S227I)
  16. 抗坏血酸钠≥98%纯度(Sigma-Aldrich,目录号:A7631)
  17. 标签缓冲液(见配方)
  18. 200mM MTSL储备溶液(参见食谱)
  19. 实施例NMR缓冲液(参见食谱)
  20. 250mM抗坏血酸钠储备溶液(参见食谱)



  1. 离心机(Beckman Coulter,型号:Allegra X-14R)
  2. SX4750A摆轮转子(Beckman Coulter,型号:SX4750A ARIES TM Roter)
  3. MALDI-TOF质谱仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:Voyager-DE-STR)
    注意:本产品已停产。其他MALDI-TOF仪器的实例包括Shimadzu,型号:AXIMA Assurance Linear MALDI-TOF; Bruker,型号:microlex LT/SH。
  4. NMR实验系统
    注意:本方案中使用的NMR实验是Bruker标准脉冲序列文库的一部分,并在装备有三重共振低温探针(Bruker Corporation)的Bruker Avance光谱仪上进行。


  1. NMRPipe(Delaglio等人,1995)
  2. Bruker TopSpin TM
  3. Sparky(戈达德和Kneller,2008)
  4. Microsoft Excel
  5. XPLOR-NIH(Schwieters等人,2003)



图1.硝基氧物标记和代表性PRE数据的流程图。A.一个流程图,描述当用特异性标记蛋白质的硝酸氧自由标记用于PRE衍生的结构研究时所采取的步骤。 B.代表IsdH N2N3的代表性[参见Sjodt等人]。 ,2016获取更多信息)。盒状区域表示(C)(K499R1和E559R1)所示的每个PRE探针的光谱的代表性部分。 C.显示K499R1(上)和E559R1(底部)探针的选择性距离相关线宽度的放大区域。对于每个探针,反磁性和顺磁性光谱分别显示在左侧和右侧。 D. E559R1探针数据的代表性PRE概况。归一化强度比(I ox/I>红色)显示为残基数的函数。星号描绘了探头的位置。基于NMR光谱的信噪比,比率测量的误差约为10-15%,这可能导致超过1的值。由于操作样品(添加抗坏血酸剂以减少探针),也可能发生错误。和仪器不稳定。通过从比率数据获得的距离限制(Battiste和Wagner,2000)添加±5埃,部分地考虑了这些误差。

  1. 分配天然蛋白质的骨架化学位移
    1. 获得待用作参考的天然蛋白质的高质量2D [ 1 H- 15 N] -HSQC光谱(基于蛋白质的氨基酸序列的包含适当数目的解析交叉峰的光谱)
    2. 使用标准NMR方法分配天然蛋白质中的主链酰胺化学位移(对于标准NMR实验参见Cavanagh等人,1995)。

  2. 用单个半胱氨酸残基生成蛋白质突变体
    1. 对于这些研究,蛋白质应含有单个半胱氨酸残基。检查蛋白质的一级序列,以确定其是否含有天然半胱氨酸残基,这些残基可能被氮氧自由基探针无意地标记。如有必要,对这些半胱氨酸残基进行保守突变以防止非特异性标记。如果生物化学测定可用于蛋白质,则应进行它们以确保这些突变不会损害其功能。
    2. 选择要连接氮氧自由基探针的蛋白质的所需位置。该位置需要在侧链溶剂暴露的残留物处。残留物应位于结构化的环或定义的二级结构内。这是重要的,因为如果探针位于蛋白质的结构上无序的区域,则多肽骨架的增加的灵活性可以降低该技术的定量强度。
    3. 进行定点诱变,以在氮氧自由基探针将附着的所需位置引入单个半胱氨酸残基。
    4. 纯化含有半胱氨酸突变的N-标记蛋白。使用补充有2.5 mM DTT的缓冲液,以防止会导致蛋白质聚集的分子间二硫键的形成(见注1)。关于蛋白质的表达和纯化的综述,参见Gräslund等人。 (2008)
    5. 在用MTSL标记纯化的半胱氨酸突变体之前,验证半胱氨酸突变不影响蛋白质的结构。这可以通过比较突变体和天然蛋白质的[ 1 H- 15 N] -HSQC光谱来实现。光谱中交叉峰的化学位移应该相似,仅在突变位点附近发生局部变化。

  3. 场地特异性标记蛋白质
    以下步骤的目的是用MTSL标记蛋白质的标记纯化的半胱氨酸突变体的 1 。注意在还原条件下保持半胱氨酸突变体,直到用MTSL修饰。用于标记蛋白质的装置如图2所示,由锥形管和脱盐柱组成。

    图2.用于使用MTSL 标记蛋白质的设备的示意图

    1. 缓冲液将 15 N-标记的半胱氨酸突变体交换到补充有新鲜2.5mM DTT的标记缓冲液中。蛋白质浓度应至少为250-300μM
    2. 通过将乙腈直接加入到MTSL运输的琥珀小瓶中来制备MTSL的储备溶液。储备溶液的最终浓度应为200mM MTSL。将MTSL储存液储存在-20°C(见注2和3)。
    3. 在开始标记反应之前,用标记缓冲液稀释蛋白质至终浓度为250-300μM蛋白质。稀释的蛋白质溶液的最终体积应为1ml。蛋白质应用不含DTT的标记缓冲液进行稀释。从此步骤中,此过程中使用的所有缓冲区不应包含DTT。
    4. 按照制造商的说明书,将Zeba旋转脱盐柱与标记缓冲液平衡。
    5. 构建将用于标记蛋白质的MTSL溶液。将2.5ml标记缓冲液吸入干净的15 ml锥形管中。将MTSL从储备溶液中加入到锥形管中。添加的量应足以达到MTSL的最终浓度,其为将被标记的蛋白质的摩尔量的10倍(例如,如果反应中的蛋白质的最终浓度将为300μM,则MTSL的浓度应为3mM)。

    6. 构建将用于标记蛋白质的仪器(图2)。将平衡的脱盐塔从步骤C5放入锥形管中。该柱将位于含MTSL的溶液之上,并且将留下蛋白质溶液流过的空间。
    7. 慢慢地将蛋白质移液到柱的树脂床的中心(图2)
    8. 离心机根据制造商的说明。蛋白质将流过柱并进入标记溶液,而存在于蛋白质溶液中的DTT将保留在柱上。在将所有蛋白质溶液洗脱到收集管底部的MTSL溶液中之后,该过程被认为是完全的。这将产生位于锥形管底部的溶液,其总体积为〜3.5ml。丢弃脱盐柱。
    9. 用箔包住含有改性反应的锥形管,让其在室温下搅拌孵育15分钟
    10. 15分钟后,从储备溶液中加入额外的MTSL至改性反应。添加的MTSL的量应该产生比锥形管中蛋白质浓度大20倍的最终浓度。
    11. 将管放回旋转装置,让蛋白质:MTSL混合物在室温下搅拌过夜(〜16h)。
    12. 使用新鲜的Amicon Ultra-15离心过滤器将MTSL标记的蛋白质样品交换到所需的NMR缓冲液中。这将清除任何超额(未经承诺的)MTSL(见注释4-6)。如上所述,NMR缓冲液不应含有任何DTT。
    13. 通过MALDI-TOF质谱法验证蛋白质是否被MTSL标记。与未分离的蛋白质相比,改性蛋白质的质量应增加〜186Da(附着的探针的重量)。尽管MALDI-TOF不是定量的,但在质谱中蛋白质的90%应用MTSL标记。如果生物化学测定可用于蛋白质,则应对标记的蛋白质进行测试,以验证MTSL附着物不会损害其功能。
    14. 对于NMR研究,MTSL修饰的蛋白质应具有〜300μM的终浓度(参见附注7)。 NMR缓冲液应含有8-10%的D 2 O,但不含任何DTT。将样品置于标准NMR管中,并使用箔屏蔽光。

  4. NMR数据采集
    1. 使用标准的2D- [ 1 H- 15 N] -HSQC脉冲序列以获得新近MTSL标记的蛋白质的光谱。通常, 1 H和 15维度分别由2,048个和256个复点定义。此时,样品处于顺磁状态。其光谱应良好分散,表明改性蛋白质折叠而不聚集。
    2. 通过将其溶于NMR缓冲液中制备250mM抗坏血酸钠的储备溶液
    3. 在获得顺磁性蛋白质(步骤D1)的[ 1 H- 15 N] -HSQC光谱后,从磁铁中取出NMR管。使用适合于NMR管的长移液管,加入抗坏血酸钠至比标记蛋白质浓度大5倍的终浓度。该步骤应仔细进行,通过缓慢上下移液来混合溶液,同时防止任何样品损失。添加抗坏血酸钠会降低MTSL的不成对电子,形成反磁性状态。让样品在室温下减少至少3小时(不需要搅拌)。
    4. 使用与用于获得所记载的顺磁光谱相同的采集参数获得还原反应的MTSL:蛋白质样品的[ 1 H- 15 N] -HSQC光谱步骤D1。


  1. 处理和分析顺磁和反磁性NMR数据
    1. 使用标准软件(例如,NMRPipe [Delaglio等人,1995]或Bruker TopSpin TM软件)处理获得的NMR数据。顺磁性和反磁性数据集都应该进行相同的处理。反磁性和顺磁性样品的代表性如图1B和1C所示。
    2. 使用标准软件(例如,Sparky)分析NMR谱(Goddard和Kneller,2008)。应首先分析抗磁谱,因为它与以前分配的天然蛋白质谱最密切相关。使用天然蛋白质的已知化学位移分配抗磁谱中的交叉峰。这可以通过覆盖天然和标记的蛋白质的[ 1 H 15 N] -HSQC光谱,然后转移化学位移分配来实现。光谱中的交叉峰应该覆盖得很好,只有位于附近探针附近的残基的交叉峰发生局部差异。必须非常小心确保分配正确分配。
    3. 测量抗磁性蛋白质指定光谱中交叉峰的强度。应注意确保分析的交叉峰值得到很好的解决(参见附注8)。进行强度测量时,请确保在峰值最大值。根据我们的经验,测量峰值高度足以提取距离约束。然而,测量交叉峰值体积也是可以接受的。广泛检查抗磁谱的序列特异性化学位移是非常重要的。使用分析软件生成交叉峰强度列表。该列表应包含交叉峰的强度和残差的身份。
    4. 使用用于分析抗磁谱的策略化学位移列表标记顺磁谱中的交叉峰。使用数据分析A3中概述的程序,测量顺磁光谱中孤立交叉峰的峰值强度。如果顺磁谱中的交叉峰显着变宽,使得其强度接近噪声水平(例如,图1C,E559R1顺磁性面板中的残基S563),其强度的精确测量为不可能。因此,这些交叉峰不应用于将NMR数据与原子间距离定量相关,不应进一步分析来自这些交叉峰的强度。然而,应注意在顺磁光谱中表现出明显的线展宽的残基的身份。这是因为在结构计算中仍然可以使用这些氨基酸的距离约束,因为完全扩展表明它们在探针的〜12Å内。使用软件使用分析软件生成交叉峰强度列表。该列表应包含交叉峰的强度和残留物的身份。描述交叉峰强度列表的示例表如表1所示

      a 注意:该数据是从IsdH N2N3的NMR研究(Sjodt等,2016)改编的。
    5. 使用标准分析软件(例如,,Microsoft Excel)计算强度比。将反磁性和顺磁性强度列表(从数据分析A3和A4生成)导入单个电子表格。对于每个残留物,使用交叉峰强度数据来计算其在顺磁(I ox)和反磁性(I Red )状态下的强度比。强度比对蛋白质序列的代表性图示于图1D中
  2. 从NMR数据估算距离约束
    本节的目的是描述如何将前一节中计算的交叉峰值强度比数据与距离约束相关联。描述顺磁弛豫对交叉峰强度的影响及其与原子间距离的关系的详细描述已在其他地方详细描述(Battiste和Wagner,2000; Clore,2015)。鼓励读者参考这些参考资料,了解基础理论。简而言之,效果可以通过以下等式来描述:

    其中,分别为蛋白质的顺磁性和反磁性形式的交叉峰的测量强度,t分别为横向的总进化时间NMR实验期间的质子磁化。 R的值对应于酰胺质子的横向弛豫速率,R 2是质子的固有弛豫,R 2> sup> sp ,是由顺磁探针引起的松弛的贡献。 R 2 sp 的值取决于r,酰胺质子和探针之间的距离。这个关系是用等式2所示的Solomon-Bloembergen的修改版本来描述的(Solomon and Bloembergen,1956; Battiste和Wagner,2000)

    其中,K 是描述旋转的常数(1.23×10 -32 cm 6 sec -2 ) MTSL自旋标签的性质(Battiste和Wagner,2000),ωh是质子自旋的拉莫尔频率,τC是表观PRE相关时间(Simon < em>等人,2010)。已知ωh,可以基于蛋白质的分子量来估计τC的值(Cavanagh等,1995) ),可以使用等式1和2生成校准曲线。该曲线将给定交叉峰的强度比与酰胺质子和探针之间的距离相关。对于MTSL探针,小于1的低氧/低分子比例表明探针和质子原子在13-25范围内,而I /I red 等于〜0和〜1表示探针和质子分别分开小于〜或大于〜30。值得注意的是,校准曲线仅提供原子间距离的估计,因为探头具有显着的灵活性。因此,从强度数据获得的距离约束是不精确的,结构计算策略表示使用多个构象异构体的探针(Iwahara等人,2004; Clore,2015)。
    1. 构建距离校准曲线(r)的强度,以及r)。这是通过使用等式1和2,以及蛋白质中结构残基的τsub值和平均酰胺质子线宽(π/R sub2)的估计值来完成的。代表性的校准曲线如图3所示,分别对π/R 2和τC分别使用23Hz和16.4ns的值。

      图3.用于估计PRE衍生距离的校准曲线。用于将强度比转换为IsdH N2N3的NMR研究的距离的代表性校准曲线 (Sjodt等人,2016)。根据等式1和2,分别使用23Hz和16.4ns的平均线宽(π/R 2 2)和相关时间(τsub)C C来生成该曲线。

    2. 使用数据分析B1中生成的校准曲线将实验强度比转换为距离分离。
    3. 构建XPLOR-NIH计算中使用的距离限制表(图4)。应使用标准XPLOR-NIH约束表格式,在MTSL环的氮原子和受影响的酰胺质子之间进行距离约束(Schwieters等人,2003)。应该强调,这些距离边界是估计值,不能用作准确的距离测量。计算中采用两种类型的距离约束:
      1. 如果Iox/Ired为0.80,约束被称为"吸引力",并给出距离数据分析B2的指定距离,误差为±5Å(见注9)。
      2. 如果Iox/Ired≥0.80,约束被称为"排斥性",并给予20Å的下限距离,无上限距离(Battiste和Wagner,2000; Reckel等人,2011; Gottstein等人,2012)。

        图4. XPLOR-NIH约束表的示例。 XPLOR-NIH格式的PRE距离约束输入表的示例。该表由IsdH N2N3(Sjodt等人,2016)的NMR研究改编而成。 "segid ALT"部分识别在三个随机取向中建模到起始结构上的MTSL分子。 (有关生成约束表的更多信息,请鼓励读者参考Schwieters等人,2003; Iwahara等人,2004; Sjodt等人al 。,2016)。  


  1. 在每次使用前,制作新的DTT溶液。
  2. MTSL将在高浓度的乙腈中沉淀,因此保持原料浓度低于200mM。
  3. MTSL是光和空气敏感的。
  4. 确保在步骤C11中广泛洗涤蛋白质。过多的未经承认的MTSL将导致核磁共振实验中的人造物。
  5. 应该对所关注的蛋白质优化NMR缓冲液。关于适用于NMR的缓冲液的综述,参见Kelly等人。 (2002)。
  6. 缓冲液的pH应该基于蛋白质的稳定性来选择,因为一旦连接到蛋白质,MTSL基团是相当稳定的。然而,在碱性pH下,一些游离的MTSL基团可能被水解成硫醇。这些基团可以与剩余的游离MTSL分子发生反应并导致形成二硫键连接的双基,这会降低标记效率。因此,应避免超过中性值的缓冲液。为了解决这种可能性,在标记过程中使用20倍摩尔过量的MTSL以确保蛋白质的有效标记。
  7. 蛋白质浓度应保持较低,以防止非特异性分子间PRE效应
  8. 据报道,通过PRE效应完全扩展的交叉峰的尾部可以将相邻峰值的高度降低高达15%,导致假的积极扩大效应(Battiste和Wagner,2000)。 />
  9. 提供了放置在有吸引力的约束上的大误差范围,以说明MTSL探针的内在灵活性


  1. 标签缓冲区
    50mM Tris-HCl(pH7.8)
    50 mM NaCl
  2. 200毫升MTSL储备溶液
    将10mg MTSL溶解于18.92μl乙腈中
  3. 实施例NMR缓冲液
    20mM NaH 2 PO 4(pH 6.0)
    50 mM NaCl
    0.01%NaN 3
  4. 250mM抗坏血酸钠储备溶液


该协议由Sjodt等人改编。 (2016)。这项工作得到了国家卫生研究院AI52217(至R.T.C.)和美国国家卫生研究院奖F31GM101931(至美国)的支持。


  1. Battiste,JL和Wagner,G.(2000)。  利用现场定向自旋标记和高分辨率异核核磁共振,对具有有限核过程效应数据的大蛋白进行全局折叠测定。生物化学39(18):5355-5365。
  2. Bertoncini,CW,Jung,YS,Fernandez,CO,Hoyer,W.,Griesinger,C.,Jovin,TM和Zweckstetter,M。(2005)。长距离三级相互作用的释放增强了天然非结构化α-突触核蛋白的聚集。 美国国家科学院院士102(5):1430-1435。
  3. Cavanagh,J.,Fairbrother,WJ,Palmer,AG和Skelton,NJ(1995)。< a class ="ke-insertfile"href ="https://books.google.com.tw/books?hl= zh-CN&lr =&id = 85rYGWiBJ1kC&oi = fnd&pg = PP1&dq =蛋白质+ NMR +光谱:+原理+和+实践&ots = L4nwFr72uy&sig = 53_rQLZr3vtzZlD08eMJcJhZ-XY&redir_esc = y#v = onepage&q =蛋白质%20NMR%20光谱%3A%20原则%20和%20实践& = false"target ="_ blank">蛋白质核磁共振光谱:原理与实践 学术出版社 pp:587
  4. Clore,GM(2015)。顺磁松弛的实践方面增强生物大分子。方法Enzymol 564:485-497。
  5. Clore,GM and Iwahara,J。(2009)。  用于表征生物大分子及其复合物的瞬时低种群状态的顺磁性松弛增强的理论,实践和应用。 109(9):4108-4139。 />
  6. Delaglio,F.,Grzesiek,S.,Vuister,GW,Zhu,G.,Pfeifer,J。和Bax,A。(1995)。 NMRPipe:基于UNIX管道的多维光谱处理系统。 J Biomol NMR 6(3) :277-293。
  7. Gräslund,S.,Nordlund,P.,Weigelt,J.,Hallberg,BM,Bray,J.,Gileadi,O.,Knapp,S.,Oppermann,U.,Arrowsmith,C.,Hui,R.,Ming ,J.,dhe-Paganon,S.,Park,HW,Savchenko,A.,Yee,A.,Edwards,A.,Vincentelli,R.,Cambillau,C.,Kim,R.,Kim,SH,Rao ,Z.,Shi,Y.,Terwilliger,TC,Kim,CY,Hung,LW,Waldo,GS,Peleg,Y.,Albeck,S.,Unger,T.,Dym,O.,Prilusky, Sussman,JL,Stevens,RC,Lesley,SA,Wilson,IA,Joachimiak,A.,Collart,F.,Dementieva,I.,Donnelly,MI,Eschenfeldt,WH,Kim,Y.,Stols,L.,Wu ,R.,Zhou,M.,Burley,SK,Emtage,JS,Sauder,JM,Thompson,D.,Bain,K.,Luz,J.,Gheyi,T.,Zhang,F.,Atwell, ,Almo,SC,Bonanno,JB,Fiser,A.,Swaminathan,S.,Studier,FW,Chance,MR,Sali,A.,Acton,TB,Xiao,R.,Zhao,L.,Ma, Hunt,JF,Tong,L.,Cunningham,K.,Inouye,M.,Anderson,S.,Janjua,H.,Shastry,R.,Ho,CK,Wang,D.,Wang,H., M.,Montelione,GT,Stuart,DI, Owens,RJ,Daenke,S.,Schütz,A.,Heinemann,U.,Yokoyama,S.,Büssow,K.and Gunsalus,KC(2008)。< a class ="ke-insertfile"href = https://www.ncbi.nlm.nih.gov/pubmed/18235434"target ="_ blank">蛋白质生产和纯化。方法 5(2):135-146 。
  8. Goddard,T.D。和Kneller,D.G。(2008)。 Sparky NMR分析软件
  9. Gottstein,D.,Reckel,S.,Dotsch,V。和Guntert,P。(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22560730"target ="_ blank">通过NMR测定膜蛋白质结构的顺磁性松弛增强数据的要求。结构 20(6):1019-1027。
  10. Iwahara,J.,Schwieters,CD和Clore,GM(2004)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15125681"target = "_blank">用于核磁共振结构细化的组合方法,从与大分子连接的柔性顺磁性基团产生的 1 H顺磁性松弛增强数据。 126(18):5879-5896。
  11. Kelly,AE,Ou,HD,Withers,R.and Dotsch,V。(2002)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12358548"target ="_ blank">用于高灵敏度NMR测量的低电导率缓冲液.JA Am Chem Soc。124(40):12013-12019。
  12. Reckel,S.,Gottstein,D.,Stehle,J.,Lohr,F.,Verhoefen,MK,Takeda,M.,Silvers,R.,Kainosho,M.,Glaubitz,C.,Wachtveitl,J.,Bernhard ,F.,Schwalbe,H.,Guntert,P。和Dotsch,V.(2011)。蛋白质视紫红质的解决方案NMR结构。 Angew Chem Int Ed Engl 50(50):11942-11946。
  13. Roosild,TP,Greenwald,J.,Vega,M.,Castronovo,S.,Riek,R。和Choe,S。(2005)。 Mistic的核磁共振结构,用于膜蛋白表达的膜整合蛋白。 科学 307(5713 ):1317-1321。
  14. Schwieters,CD,Kuszewski,JJ,Tjandra,N.和Clore,GM(2003)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12565051"target ="_ blank"> Xplor-NIH NMR分子结构测定软件包。 Magneto磁盘160(1):65-73。
  15. Simon,B.,Madl,T.,Mackereth,CD,Nilges,M。和Sattler,M。(2010)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm .nih.gov/pubmed/20148424"target ="_ blank">一种用于溶液中蛋白质复合物的基于NMR光谱的结构测定的有效方案 Angew Chem Int Ed Engl 11):1967-1970。
  16. Sjodt,M.,Macdonald,R.,Spirig,T.,Chan,AH,Dickson,CF,Fabian,M.,Olson,JS,Gell,DA and Clubb,RT(2016)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25687963"target ="_ blank">来自金黄色葡萄球菌的38.8-kDa三结构域IsdH蛋白的PRE衍生NMR模型表明它自适应地识别人血红蛋白。 J Mol Biol 428(6):1107-1129。
  17. 所罗门,I.和Bloembergen,N.(1956)。< a class ="ke-insertfile"href ="http://scitation.aip.org/content/aip/journal/jcp/25/2/10.1063 /1.1742867"target ="_ blank"> HF分子中的核磁相互作用 J. Chem Phys 25:261.
  18. Tang,C.,Schwieters,CD and Clore,GM(2007)。  通过顺磁性核磁共振观察到的载脂蛋白麦芽糖结合蛋白的开 - 关转换。自然 449(7165):1078-1082。
  19. Villareal,VA,Spirig,T.,Robson,SA,Liu,M.,Lei,B.and Clubb,RT(2011)。  瞬时弱蛋白 - 蛋白复合物将血红素转移到金黄色葡萄球菌的细胞壁上。 J. Am Chem Soc 133 (36):14176-14179。
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引用:Sjodt, M. and Clubb, R. T. (2017). Nitroxide Labeling of Proteins and the Determination of Paramagnetic Relaxation Derived Distance Restraints for NMR Studies. Bio-protocol 7(7): e2207. DOI: 10.21769/BioProtoc.2207.