Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology

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Aug 2014



Reconstitution of integral membrane proteins (IMP) in aqueous solutions of detergent micelles has been extensively used in structural biology, using either X-ray crystallography or NMR in solution. Further progress could be achieved by establishing a rational basis for the selection of detergent and buffer conditions, since the stringent bottleneck that slows down the structural biology of IMPs is the preparation of diffracting crystals or concentrated solutions of stable isotope labeled IMPs. Here, we describe procedures to monitor the quality of aqueous solutions of [2H, 15N]-labeled IMPs reconstituted in detergent micelles. This approach has been developed for studies of β-barrel IMPs, where it was successfully applied for numerous NMR structure determinations, and it has also been adapted for use with α-helical IMPs, in particular GPCRs, in guiding crystallization trials and optimizing samples for NMR studies (Horst et al., 2013). 2D [15N, 1H]-correlation maps are used as “fingerprints” to assess the foldedness of the IMP in solution. For promising samples, these “inexpensive” data are then supplemented with measurements of the translational and rotational diffusion coefficients, which give information on the shape and size of the IMP/detergent mixed micelles. Using microcoil equipment for these NMR experiments enables data collection with only micrograms of protein and detergent. This makes serial screens of variable solution conditions viable, enabling the optimization of parameters such as the detergent concentration, sample temperature, pH and the composition of the buffer.

Keywords: Micro-scale NMR (微型核磁共振), Structural Biology (结构生物学), Integral Membrane Proteins (完整的膜蛋白), Transverse relaxation optimized spectroscopy (TROSY) (横向弛豫优化光谱仪(TROSY)), NMR sample optimization (NMR样品优化)

Materials and Reagents

Studies of IMPs

  1. Phosphocholine-detergents (Avanti Polar Lipids)
  2. Tris Base (Thermo Fisher Scientific, catalog number: BP1521 )
  3. HCl (Thermo Fisher Scientific, catalog number: A144212 )    
  4. Urea (Thermo Fisher Scientific, catalog number: BP169212 )
  5. Ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, catalog number: BP1201 )
  6. L-Arginine (L-Arg) (Sigma-Aldrich, catalog number: A5131100G )
  7. Phosphate buffer (Sigma-Aldrich, catalog number: P7994 )
  8. Sodium Azide (NaN3) (Sigma-Aldrich, catalog number: S803225G )
  9. NaCl (Thermo Fisher Scientific, catalog number: BP358212 )
  10. Stock solutions of unfolded protein (see Recipes)
  11. Refolding buffer (see Recipes)
  12. NMR buffer (see Recipes)


NMR data collection

  1. NMR experiment set-ups used in this protocol are either part of the Bruker standard pulse sequence library or are described in the Appendix. These experiments were implemented on a Bruker DRX spectrometer equipped with microprobes (Bruker Corporation,model:1 mm TXI, 1.7 mm TXI )
  2. The following experiments were used to monitor the quality of aqueous solutions of [2H, 15N]-labeled IMPs: 2D [15N, 1H]-TROSY experiments (Pervushin et al., 1997), 1H-TRO-STE (Horst et al., 2011) and TRACT (Lee et al., 2006). 


  1. NMR sample preparation
    1. Add 100 µl of freshly prepared stock solution of unfolded protein to 600 μl refolding buffer and stir overnight at 4 °C.
    2.  Exchange with NMR buffer by repeated dilution/concentration cycles. ‘‘Upconcentration’’ of the detergent during these dilution/concentration cycles was taken into account when adjusting the detergent concentration in the NMR sample. For details on how to adjust these cycles, see Stanczak et al. (2009). For example, 10 mM 30-Fos in the NMR buffer results in approximately 160 mM 30-Fos in the NMR sample (Stanczak et al., 2012).
    3. Added 5 μl of D2O and 1 μl of a 100 mM solution of 4, 4-dimethyl-4-silapentane-1-sulfonic acid (DSS) as an internal reference for the 1H chemical shifts to 45 μl of the protein solution.
  2. Evaluation of different sample conditions based on 2D [15N, 1H]-TROSY experiments. The following criteria were used (see Zhang et al., 2008; Stanczak et al., 2012 for examples):
    1. Completeness of NMR observation of the IMP by comparison of the number of observed backbone 15N–1H correlation peaks with the number expected from the amino acid sequence.
    2. High average peak intensity and uniform distribution of peak intensities.
    3. Analysis of the peak line shapes to support the interpretation of the peak intensity measurements.
  3. Evaluation of the hydrodynamic radius of IMP/detergent mixed micelles, using 1H-TRO-STE (Horst et al., 2011) and TRACT (Lee et al., 2006) experiments.
    1. Determine the translational diffusion coefficient, Dt, using the 1H-TRO-STE experiment.
    2. Determine the rotational diffusion coefficient, Dr, using the TRACT experiment.
    3. Calculate the hydrodynamic radius, Rh, using the formula Rh = (3Dt/4Dr)1/2.
    4. Optimize sample conditions towards small Rh values, which favors the recording of high-quality NMR data.

Data analysis

Processing and analysis of NMR datasets:
Process all NMR experiments with the Bruker standard software Topspin. For all experiments, the data matrices are multiplied with an exponential window function in the 1H-dimension, and for 2D [15N, 1H]-TROSY a 75°-shifted sine bell window (De Marco and Wüthrich, 1976) is applied in the 15N-dimension The 2D [15N, 1H]-TROSY data sets were analyzed using the XEASY module (Bartels et al., 1995) of the CARA release 1.5.5 (www.cara.nmr.ch). The 1H-TRO-STE datasets are analyzed using the Bruker T1/T2-software package, as described in the Topspin DOSY application manual, chapter 3.2. The TROSY and anti-TROSY components of the TRACT data set are first separated using the Bruker standard AU program split, and then individually integrated using the Bruker integration module. The integrals were fitted to a mono-exponential decay, using the program XMGRACE (http://plasma-gate.weizmann.ac.il).


Optimization of NMR acquisition parameters for the 2D [15N, 1H]-TROSY, 1H-TRO-STE and TRACT experiments:

  1. For all experiments
    1. Determine the lengths of high power radio-frequency pulses as described in the user manual for the spectrometer. Typical pulse lengths are 8 μs and 35 μs for 1H and 15N, respectively.  
    2. Set the carrier frequency to the water line by minimizing the residual water signal in the Bruker standard pulse sequence zggppr.
    3. Optimize the water flip-back pulses sp2 and sp3, using the experiment shown in Listing 1 of the Appendix to interactively minimize the residual water signal in the topspin gs acquisition mode.
  2. 2D [15N, 1H]-TROSY and TRACT experiments (listings 2 and 4 of the Appendix)
    1. Adjust the WATERGATE soft pulse sp1, using the Bruker standard pulse sequence zggpwg by minimizing the residual water signal interactively in the topspin gs acquisition mode.
    2. Adjust the [15N, 1H]-INEPT transfer delay, d2, to maximize the signal from the protein amide moieties in the first increment of the TRACT experiment. Typical values for d2 are between 2.0 and 2.5 ms.
    3. Adjust the number of complex points and the maximum evolution time, t1max, for the 15N-dimension in the 2D [15N, 1H]-TROSY experiment. Typical values for IMP’s reconstituted in detergent micelles are 100 points and 35 ms, respectively. Use the same t1max value for the TRACT experiment.
  3. 1H-TRO-STE experiment (listing 3 in the Appendix)
    1. Calibrate the pulsed-field gradient strengths with the residual 1H signal of 99.9% D2O, using the Bruker standard pulse sequence ledgp2s1d ( Dt = (1.902 ± 0.09) × 10‒9 m2s‒1 for HDO at 25 °C).  
    2. Optimize the [15N,1H]-CRINEPT transfer delay, d2, by maximizing the signal from the protein amide moieties in the first increment of the spectrum.
    3. Optimize the diffusion delay and the gradient duration, d20 and p30, respectively, using the standard procedures described in the Topspin DOSY application manual, Chapter 2.1.


  1. Stock solutions of unfolded protein
    [2H, 15N]-labeled protein (12 mg/ml) in 20 mM Tris-HCl at pH 8.0 and 6 M urea
  2. Refolding buffer
    20 mM Tris-HCl at pH 7.5
    5 mM EDTA
    600 mM L-Arg
    45 mM detergent
  3. NMR buffer
    5 mM phosphate buffer at pH 6.8
    10 mM NaCl, 0.3 % (v/w) NaN3
    10 mM detergent


This work was adapted from previously published studies on the E. coli outer membrane protein X (OmpX) (Stanczak et al., 2009), and was used as a platform for the structure determination of E. coli OmpW (Horst et al., 2014). The procedures described in this protocol were also used to characterize E. coli OmpA in lipid bilayer nanodiscs and detergent micelles (Susac et al., 2014). This work was supported by the Roadmap initiative grant P50 GM073197 for technology development. Kurt Wüthrich is the Cecil H. and Ida M. Green Professor of Structural Biology at the Scripps Research Institute.


  1. Bartels, C., Xia, T. H., Billeter, M., Guntert, P. and Wuthrich, K. (1995). The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J Biomol NMR 6(1): 1-10.
  2. De Marco, A. and Wüthrich, K. (1976). Digital filtering with a sinusoidal window function: an alternative technique for resolution enhancement in FT NMR. J Magn Reson 24(2): 201-204.
  3. Horst, R., Horwich, A. L. and Wuthrich, K. (2011). Translational diffusion of macromolecular assemblies measured using transverse-relaxation-optimized pulsed field gradient NMR. J Am Chem Soc 133(41): 16354-16357.
  4. Horst, R., Stanczak, P. and Wuthrich, K. (2014). NMR polypeptide backbone conformation of the E. coli outer membrane protein W. Structure 22(8): 1204-1209.
  5. Horst, R., Stanczak, P., Stevens, R. C. and Wuthrich, K. (2013). beta2-Adrenergic receptor solutions for structural biology analyzed with microscale NMR diffusion measurements. Angew Chem Int Ed Engl 52(1): 331-335.
  6. Lee, D., Hilty, C., Wider, G. and Wüthrich, K. (2006). Effective rotational correlation times of proteins from NMR relaxation interference. J Magn Reson 178(1): 72-76.
  7. Pervushin, K., Riek, R., Wider, G. and Wuthrich, K. (1997). Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci U S A 94(23): 12366-12371.
  8. Stanczak, P., Horst, R., Serrano, P. and Wuthrich, K. (2009). NMR characterization of membrane protein-detergent micelle solutions by use of microcoil equipment. J Am Chem Soc 131(51): 18450-18456.
  9. Stanczak, P., Zhang, Q., Horst, R., Serrano, P. and Wuthrich, K. (2012). Micro-coil NMR to monitor optimization of the reconstitution conditions for the integral membrane protein OmpW in detergent micelles. J Biomol NMR 54(2): 129-133.
  10. Susac, L., Horst, R. and Wuthrich, K. (2014). Solution-NMR characterization of outer-membrane protein A from E. coli in lipid bilayer nanodiscs and detergent micelles. Chembiochem 15(7): 995-1000.
  11. Zhang, Q., Horst, R., Geralt, M., Ma, X., Hong, W. X., Finn, M. G., Stevens, R. C. and Wuthrich, K. (2008). Microscale NMR screening of new detergents for membrane protein structural biology. J Am Chem Soc 130(23): 7357-7363.


在洗涤剂胶束的水溶液中的内在膜蛋白(IMP)的重建已经广泛地用于结构生物学中,在溶液中使用X-射线晶体学或NMR。通过建立用于选择洗涤剂和缓冲液条件的合理基础可以实现进一步的进展,因为减缓IMP的结构生物学的严格瓶颈是制备衍射晶体或稳定同位素标记的IMP的浓缩溶液。这里,我们描述了监测在洗涤剂胶束中重构的[2 H],15 N] - 标记的IMP的水溶液的质量的程序。该方法已经开发用于β-桶IMP的研究,其中其成功地应用于许多NMR结构测定,并且其也适用于使用α-螺旋IMP,特别是GPCR,用于指导结晶试验和优化样品NMR研究(Horst等人,2013)。使用相关图作为"指纹"以评估解在溶液中的IMP的折叠度。对于有希望的样品,然后用平移和旋转扩散系数的测量补充这些"廉价的"数据,其给出关于IMP /洗涤剂混合胶束的形状和尺寸的信息。使用微线圈设备进行这些NMR实验能够仅收集微克蛋白质和洗涤剂来收集数据。这使得可变溶液条件的连续筛选可行,使得能够优化参数,例如洗涤剂浓度,样品温度,pH和缓冲液的组成。

关键字:微型核磁共振, 结构生物学, 完整的膜蛋白, 横向弛豫优化光谱仪(TROSY), NMR样品优化



  1. 磷酸胆碱清洁剂(Avanti Polar Lipids)
  2. Tris Base(Thermo Fisher Scientific,目录号:BP1521)
  3. HCl(Thermo Fisher Scientific,目录号:A144212)   
  4. 尿素(Thermo Fisher Scientific,目录号:BP169212)
  5. 乙二胺四乙酸(EDTA)(Thermo Fisher Scientific,目录号:BP1201)
  6. L-精氨酸(L-Arg)(Sigma-Aldrich,目录号:A5131100G)
  7. 磷酸盐缓冲液(Sigma-Aldrich,目录号:P7994)
  8. 叠氮化钠(NaN 3)(Sigma-Aldrich,目录号:S803225G)
  9. NaCl(Thermo Fisher Scientific,目录号:BP358212)
  10. 展开蛋白的储备溶液(参见配方)
  11. 重折叠缓冲区(参见配方)
  12. NMR缓冲液(参见配方)



  1. 在该方案中使用的NMR实验装置或者是Bruker标准脉冲序列文库的一部分,或者在附录。 这些实验在装有微探针的Bruker DRX光谱仪(Bruker Corporation,型号: 1 mm TXI,1.7 mm TXI
  2. 使用以下实验来监测[2 H],15 N] - 标记的IMP:2D [15 C] 15 N的水溶液的质量, 1 H-TRO-STE(Horst等人,1997),H-TRO-STE(Horst et al。 ,2011)和TRACT(Lee ,2006)。


  1. NMR样品制备
    1. 加入100微升新鲜制备的解折叠蛋白储备溶液600微升重折叠缓冲液,并在4℃下搅拌过夜。
    2.  通过重复稀释/浓缩循环与NMR缓冲液交换。 在这些稀释/浓缩过程中洗涤剂的"浓度"   在调节洗涤剂时考虑循环 NMR样品中的浓度。 有关如何调整这些的详细信息 循环,参见Stanczak等人(2009)。 例如,在NMR中的10mM 30-Fos   缓冲液在NMR样品中产生约160mM 30-Fos (Stanczak等人,2012)。
    3. 加入5μl的D 2 O和1μl的100mM 的4,4-二甲基-4-硅戊烷-1-磺酸(DSS)的溶液 1 H化学物质的内部参考转移到45μl的蛋白质 解。
  2. 基于2D [1 H] -TROSY实验的不同样品条件的评价。 使用以下标准(例如,参见Zhang等人,2008; Stanczak等人,2012年):
    1. 通过比较的IMP的NMR观察的完整性 观察到的主链的数目与数字的相关峰 预期从氨基酸序列。
    2. 高平均峰强度和峰强度分布均匀。
    3. 分析峰线形状以支持峰强度测量的解释。
  3. 使用H-TRO-STE(Horst等人,2011)和TRACT(Lee等人)对IMP /去污剂混合胶束的流体动力学半径进行评估 。,2006)。
    1. 使用 1 H-TRO-STE实验确定平移扩散系数 D 。
    2. 使用TRACT实验确定旋转扩散系数 D r
    3. 使用公式 R 计算流体动力学半径 R h em> t /4 r 1/2
    4. 将样本条件优化为小的R h 值,这有利于记录高质量NMR数据。


使用Bruker标准软件Topspin处理所有NMR实验。对于所有实验,数据矩阵乘以在H维中的指数窗函数,并且对于2D [ 15 N, 1 H] -TROSY在75°的正弦窗口(De Marco和Wüthrich,1976)应用于N维的2D [ 15 N,www.cara.nmr.ch )。如Topspin DOSY应用手册,第3.2章中所述,使用Bruker T1/T2软件包分析 1 H-TRO-STE数据集。首先使用Bruker标准AU程序 split 分离TRACT数据集的TROSY和anti-TROSY组件,然后使用Bruker积分模块单独积分。使用程序XMGRACE( http://plasma-gate)将积分拟合为单指数衰减。 Weizmann.ac.il )。


优化用于2D [1 H] 15 H,1 H] -TROSY,1 H-TRO-STE和TRACT实验的NMR采集参数:

  1. 对于所有实验
    1. 确定高功率射频脉冲的长度 如光谱仪用户手册中所述。 典型脉冲长度   对于 1 H和 15 N,分别为8μs和35μs。  
    2. 设置 载波频率通过最小化残留水到水线 Bruker标准脉冲序列 zggppr 中的信号。
    3. 优化 水翻转脉冲 sp2 和 sp3 ,使用 附录的清单1,以交互方式最小化残留水 信号在上采集 采集模式。
  2. 2D [ 15 N, 1 H] -TROSY和TRACT实验(附录的列表2和4)
    1. 使用Bruker标准调整WATERGATE软脉冲 sp1 脉冲序列 zggpwg 通过最小化残留水信号 以上层交互方式 gs 获取模式
    2. Bruker标准脉冲序列 zggppr 中的信号。
    3. 优化 水翻转脉冲 sp2 和 sp3 ,使用 附录的清单1,以交互方式最小化残留水 信号在上采集 采集模式。
  3. 2D [ 15 N, 1 H] -TROSY和TRACT实验(附录的列表2和4)
    1. 使用Bruker标准调整WATERGATE软脉冲 sp1 脉冲序列 zggpwg 通过最小化残留水信号 以上层交互方式 gs 获取模式
    2. ...
    3. Optimize the [15N,1H]-CRINEPT transfer delay, d2, by maximizing the signal from the protein amide moieties in the first increment of the spectrum.
    4. Optimize the diffusion delay and the gradient duration, d20 and p30, respectively, using the standard procedures described in the Topspin DOSY application manual, Chapter 2.1.


  1. 未折叠蛋白的储备溶液
    在pH 8.0的20mM Tris-HCl和6M尿素中的[2 H],15 N]标记蛋白(12mg/ml)
  2. 重折叠缓冲区
    20mM Tris-HCl,pH7.5 5 mM EDTA
    600 mM L-Arg
    45 mM洗涤剂
  3. NMR缓冲液
    5mM磷酸盐缓冲液,pH 6.8 10mM NaCl,0.3%(v/w)NaN 3
    10 mM洗涤剂


这项工作改编自以前发表的关于E的研究。大肠杆菌外膜蛋白X(OmpX)(Stanczak等人,2009),并用作E的结构测定的平台。大肠杆菌 OmpW(Horst等人,2014)。该方案中描述的程序也用于表征E。在脂质双层纳米圆盘和洗涤剂胶束中的大肠杆菌OmpA(Susac等人,2014)。这项工作得到了路线图计划赠款P50 GM073197技术开发的支持。 KurtWüthrich是Scripps研究所的Cecil H.和Ida M. Green结构生物学教授。


  1. Bartels,C.,Xia,T.H.,Billeter,M.,Guntert,P。和Wuthrich,K。(1995)。 计算机支持的生物大分子NMR光谱分析程序XEASY。 J Biomol NMR 6(1):1-10。
  2. De Marco,A。和Wüthrich,K。(1976)。 使用正弦窗函数的数字滤波:FT NMR中分辨率增强的替代技术。 J Magn Reson 24(2):201-204。
  3. Horst,R.,Horwich,A.L。和Wuthrich,K。(2011)。 使用横向弛豫优化脉冲场梯度NMR测量的大分子组装的平移扩散。 J Am Chem Soc 133(41):16354-16357。
  4. Horst,R.,Stanczak,P。和Wuthrich,K。(2014)。 E多肽的NMR多肽骨架构象。大肠杆菌外膜蛋白W. 结构 22(8):1204-1209。
  5. Horst,R.,Stanczak,P.,Stevens,R.C.和Wuthrich,K。(2013)。 beta2-肾上腺素能受体溶液用于结构生物学分析的微量NMR扩散测量。 Angew Chem Int Ed Engl 52(1):331-335
  6. Lee,D.,Hilty,C.,Wider,G.andWüthrich,K。(2006)。 来自NMR弛豫干扰的蛋白质的有效旋转相关时间。 J Magn Reson 178(1):72-76。
  7. Pervushin,K.,Riek,R.,Wider,G.and Wuthrich,K。(1997)。 通过偶极 - 偶极耦合和化学位移各向异性的相互消除的衰减的T2弛豫表示NMR结构的途径的非常大的生物大分子在溶液中。 Proc Natl Acad Sci USA 94(23):12366-12371。
  8. Stanczak,P.,Horst,R.,Serrano,P.and Wuthrich,K。(2009)。 使用微型线圈设备对膜蛋白 - 洗涤剂胶束溶液的NMR表征。 J Am Chem Soc 131(51):18450-18456。
  9. Stanczak,P.,Zhang,Q.,Horst,R.,Serrano,P.and Wuthrich,K。(2012)。 微线圈NMR以监测洗涤剂胶束中内在膜蛋白OmpW的重构条件的优化。 < J Biomol NMR> 54(2):129-133。
  10. Susac,L.,Horst,R。和Wuthrich,K。(2014)。 来自E的外膜蛋白A的溶液-NMR表征。大肠杆菌在脂质双层纳米圆盘和洗涤剂胶束中。 Chembiochem 15(7):995-1000。
  11. Zhang,Q.,Horst,R.,Geralt,M.,Ma,X.,Hong,W.X.,Finn,M.G.,Stevens,R.C.and Wuthrich,K.(2008)。 微尺度NMR筛选膜蛋白结构生物学的新型去污剂。 Chem Soc 130(23):7357-7363。
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引用:Horst, R. and Wüthrich, K. (2015). Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology. Bio-protocol 5(14): e1539. DOI: 10.21769/BioProtoc.1539.