波谱学杂志  2014, Vol. 31 Issue (1): 61-70

Article Information

蒋先旺, 孙鹏, 肖楠, 蒋滨, 秦显国, 李从刚, 刘买利, 张许
JIANG Xian-wang, SUN Peng, XIAO Nan, JIANG Bin, QIN Xian-guo, LI Cong-gang, LIU Mai-li, ZHANG Xu
蛋白质芳香基团的1H-13C HSQC信号增强研究
Sensitivity Enhancement in 1H-13C HSQC Experiments on Aromatic Groups in Proteins
波谱学杂志, 2014, 31(1): 61-70
Chinese Journal of Magnetic Resonance, 2014, 31(1): 61-70

Article History

Received date: 2013-05-28
Revised date: 2013-06-05
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蒋先旺, 孙鹏, 肖楠, 蒋滨, 秦显国, 李从刚, 刘买利, 张许 .蛋白质芳香基团的1H-13C HSQC信号增强研究[J].波谱学杂志, 2014, 31(1): 61-70.
JIANG Xian-wang, SUN Peng, XIAO Nan, JIANG Bin, QIN Xian-guo, LI Cong-gang, LIU Mai-li, ZHANG Xu . Sensitivity Enhancement in 1H-13C HSQC Experiments on Aromatic Groups in Proteins[J]. Chinese Journal of Magnetic Resonance, 2014, 31(1): 61-70.
Sensitivity Enhancement in 1H-13C HSQC Experiments on Aromatic Groups in Proteins
JIANG Xian-wang1,2, SUN Peng1,2, XIAO Nan1,2, JIANG Bin1, QIN Xian-guo1, LI Cong-gang1, LIU Mai-li1, ZHANG Xu1     
1. State Kay Laboratory of Magnetic Resonance and Atomic and Molecular Physics (Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences), Wuhan 430071, China;
 2. University of Chinese Academy of Sciences, Beijing 100049, China
Received date: May 28 2013; Revised date: June 05 2013
Foundation item: National Natural Science Foundation of China (20875098, 21075132), National Major Basic Research Program of China (2009CB918603), and the Ministry of Science and Technology of China (2009IM030700)
Biography: JIANG Xian-wang(1984-), male, born in Hunan, specializing in NMR methodology and application;
* Corresponding author: LIU Mai-li, Tel: +86-27-87197305, E-mail: ml.liu@wipm.ac.cn; ZHANG Xu, Tel: +86-27 -87197056, E-mail: zhangxu@wipm.ac.cn
Abstract: Aromatic side chains are usually located in the hydrophobic core or ligand binding sites of proteins. They play important roles in protein functions such as structure stabilization and biological recognition through noncovalent interactions. Thus, aromatic side chains are considered to be a promising probe to determine the structure and dynamics of protein through NMR. However, they are not frequently studied in practice. One of the reasons is that some NMR signals of aromatic rings are rarely observed due to the severely line broadening induced by complex slow internal motion. In this study, CPMG-INEPT 1H-13C HSQC was employed to detect the aromatic resonances of protein GB1. It was found that some unobservable peaks in routine 1H-13C HSQC spectrum could be recovered in the corresponding CPMG-INEPT 1H-13C HSQC spectrum. This indicated that the CPMG-INEPT 1H-13C HSQC may be an effective alternation to routine 1H-13C HSQC for detecting the resonances of aromatic rings in proteins.
Key words: NMR     CPMG-INEPT     chemical exchange     sensitivity enhancement     aromatic side chain    
蛋白质芳香基团的1H-13C HSQC信号增强研究
蒋先旺1,2, 孙鹏1,2, 肖楠1,2, 蒋滨1, 秦显国1, 李从刚1, 刘买利1, 张许1     
1. 波谱与原子分子物理国家重点实验室（中国科学院武汉物理与数学研究所），湖北武汉 430071;
 2. 中国科学院大学，北京 100049
*通讯联系人: 刘买利，电话：027-87197305，E-mail: ml.liu@wipm.ac.cn; 张许，电话：027-87197056，E-mail: zhangxu@wipm.ac.cn
摘要: 含芳香环的氨基酸残基多处于蛋白质的疏水内核或者是蛋白质与配体的结合部位，这些残基间的非共价键相互作用对于蛋白质结构的稳定性和生物的特异性识别有重要意义.因此，在蛋白质的核磁共振研究中，芳香基团被认为是一个研究蛋白质结构动力学的潜在探针.但是由于芳香基团的1H-13C HSQC谱的灵敏度较低，目前，利用核磁共振对其进行研究乏善可陈.造成这一现象的主要原因可能在于芳香环体积较大，往往存在慢的翻转运动，导致其NMR信号被展宽.作者利用CPMG-INEPT HSQC序列可以有效地减小慢运动影响的特征，将其应用到了GB1蛋白质芳香环的1H-13C HSQC谱中，有效的增强了芳香基团HSQC谱信号的灵敏度，得到了部分在常规HSQC中无法观测的信号.
关键词： 核磁共振(NMR)     CPMG-INEPT     化学交换     信号增强     芳香环侧链    
Introduction

Aromatic side chains usually buried in hydrophobic core of protein or involved in protein-ligand interaction surface, can serve as a promising NMR probe to study protein structure and dynamics[1-19]. The 1H-1H Nuclear Overhauser Effect (NOE), that aromatic side chains involved in, usually provides long-range structural constraints in NMR based protein structure calculation. Therefore, the complete assignments of aromatic side chain resonances are beneficial for high-resolution structure determination of protein via solution NMR[11, 20-27].

Though aromatic side chain has received more and more attention, its application is still limited. This may be attributed to its large 1JC, C and 1JH, C scalar couplings, which hampering the spectral analyses and making the sequence specific assignment of aromatic side chains, cannot be achieved by 1H-1H J-correlated experiments like the aliphatic counterparts, as the scalar coupling network is interrupted by the quarternary carbon at the γ position[20, 24, 28]. Up to date, many methods have been developed to overcome the difficulty mentioned above. One large category of methods is to change the coherence or polarization transfer mode[29-40]. Another category is to develop a special selective labeling strategy[11, 21, 28, 41-45].

Another hindrance for NMR detection of aromatic resonance is line-broadening caused by chemical exchange associated with the slow complex flipping motion of the aromatic ring. When the flipping rate of aromatic ring is in the intermediate range, the chemical exchange induced by the conformation exchange will dramatically reduce the NMR signal intensity, making the signal undetectable. When the exchange rate is out of the intermediate range, the line width of NMR signal of aromatic rings can be narrowed down[3, 4, 16, 20, 46-51]. So optimizing the temperature is sometimes used for sensitivity enhancement of aromatic resonance, since the flipping rate of aromatic ring is temperature dependent[45, 46]. However, this approach cannot be applied generally, because the protein samples may become unstable due to the limited thermal stability and critical condition for protein for proper folding[45, 46, 52]. Besides, some NMR experiments have to be performed in a specific condition, because the biological process we are interested in is also affected by the sample condition[46, 52-60].

It was recognized that the dephasing of spin coherence by chemical exchange processes can be reduced by using proper pulse train[61-64]. Among those methods, XY16[65], one of the CPMG derived pulse train, has been found to be an effective method to regain the sensitivity loss caused by chemical exchange. It not only can be applied in direct acquisition time which named “divided evolution”[52], but also can be applied in indirect acquisition time. The apparent transverse relaxation time can be effectively inflated when the CPMG derived pulse train is applied with intervals shorter than the lifetime of the chemical exchange process[66]. XY16 pulse train has been applied in the magnetization transfer periods of HSQC, resulting in a known CPMG-INEPT method[67, 68], which has been successfully used to obtain 1H-15NHSQC of RNA[67] and protein[68] to reduce the sensitivity loss arisen from chemical exchange.

Since CPMG-INEPT is an effective way to recover the signals loss induced by chemical exchange[67, 68], it may be an effective solution to regain the signal loss of aromatic resonances. In the present work, we demonstrated that CPMG-INEPT 1H-13C HSQC can be used to obtain 1H-13C correlation of the aromatic resonance of protein GB1 with enhanced sensitivity and reducing possible signal loss associated with chemical exchange which is affected much by its complex flipping motion.

1 Materials and Methodology

All NMR experiments were performed with a sample of 0.7 mmol/L 13C, 15N-labeled GB1 solution (90% H2O/10% D2O, 50 mmol/L potassium phosphate buffer, pH=7.1), and carried out on a Bruker AVANCE Ⅲ 800 spectrometer equipped with a triple resonance cryoprobe, the corresponding 1H frequency is 800.20 MHz. 1H-13C routine HSQC and CPMG-INEPT HSQC spectra were recorded with 64×1024 complex data matrix corresponding to spectral widths of 6 038.6 and 10 683.8 Hz for F1 and F2 dimensions, respectively. 32 scans were collected per increment with interscan delay of 1.5 s, the coherence transfer time of INEPT and CPMG-INEPT was 3.03 ms corresponding to 1JH, C of 165 Hz. To clarify the influence of temperature on this exchange broadened signal loss, the two sets of experiments were all performed with a variant of temperature, which increased from 293 K to 308 K with a step of 5 K.

A set of multidimensional NMR experiments were also carried out to verify all the peaks detected in CPMG-INEPT 1H-13C HSQC, which include 2D (HB)CB(CGCD)HD experiment, 2D (HB)CB(CGCDCE)HE experiment[29], 3D 13C/15N-edited NOESY-HSQC experiment[69, 70] and (H)CCH-TOCSY experiment[71]. Both 2D (HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE experiments were recorded with complex data matrix of 50×1 024 corresponding to spectral widths of 3 018.1 and 12 820.5 Hz for F1 and F2 dimensions, respectively. The 3D 13C/15N-edited NOESY-HSQC experiment with NOE mixing time of 80 ms is recorded with 72×48×1024 complex data matrix and the spectral widths of 10 405.8, 12 077.3 and 10 416.7 Hz for F1, F2, and F3 dimensions, respectively. The (H)CCH-TOCSY is also recorded with 50×48×1 024 complex data matrix and the spectral widths of 12 077.3, 12 077.3 and 9 615.4 Hz for F1, F2, and F3 dimensions, respectively.

All spectra were processed and analyzed using the software package NMRPipe and peak heights were determined by parabolic interpolation[72]. For each HSQC experiment, the time domain data were multiplied with a square cosine function in the direct dimension and cosine function in the indirect dimension and zero-filled to 512×2 048 complex data matrix. The assignment of aromatic resonance was performed using the software package NMRView[73].

2 Results and Discussion

Fig. 1 shows the two sets of spectra of the aromatic ring resonance of protein GB1 acquired using CPMG-INEPT 1H-13C HSQC (bottom row) and routine 1H-13C HSQC (upper row) at different temperatures and plotted at the same contour level for comparison. It is clear that most of the cross peak intensitces are nearly the same for the two kinds of experiments. However, two extra peaks are observed in the CPMG-INEPT 1H-13C HSQC spectroscopy (peaks are assigned to residue 52F). The two new peaks are most likely to be affected by chemical exchange, since they are weaker than most of the other peaks in the same CPMG-INEPT HSQC spectroscopy. Nevertheless, these signals have been confirmed and assigned by (H)CCH-TOCSY and 3D NOESY-HSQC experiment. In addition, the new peak of 52F at 1H chemical shift of 7.13 can also be detected in routine HSQC by increasing the number of experiment scans from 32 up to 512.

Fig. 1 Traditional 1H-13C HSQC (upper part) and CPMG-INEPT 1H-13C HSQC (bottom) spectra of aromatic groups of GB1, in which the frequency offset of 13C was set to be at 120. The assignments of the resonances are indicated. The newly detected resonances are labeled in the CPMG-INEPT HSQC spectra (bottom)
图选项

The difference between CPMG-INEPT and routine HSQC spectra may be due to the fact that some aromatic rings experience slow complex flipping motions[3, 4, 16, 20, 46-51]. Considering CPMG-INEPT is an effective approach to reduce chemical exchange effect in ms time scale. The signal loss induced by chemical exchange during the magnetization transfer periods will be actively reduced when CPMG-INEPT has been applied. Since CPMG-INEPT affects little the signals without chemical exchange, it is suggested that CPMG-INEPT HSQC can be a better replace to routine HSQC experiment in detecting the resonances of aromatic rings of protein, and the signal intensity enhancement may help us to assign more aromatic resonances. On the other hand, except the two aromatic resonances of residue 52F, most of the signals from aromatic rings are present in both HSQC spectra, and their intensities do not change much with different detection methods, this may be due to their exchange rates at room temperature are not in the medium rang. This can be confirmed by the experiments at different temperatures which are also shown in Fig. 1. In all routine HSQC spectra (upper in Fig. 1), except the chemical shifts of the resonances changed by temperature, there are no significant changed in intensities for most peaks. The intensities of most peaks at 308 K changed no more than 15% that of 293 K, except the peak from residue 45Y at the 1H chemical shift of 6.2, whose intensity is doubled at 308 K. Nevertheless, the two new peaks from residue 52F are still missing in routine HSQC at temperatures between 293 K and 308 K. In summary, though some of the aromatic resonances in routine HSQC increased at higher temperature, most of them are insensitive to temperature, this indicates that temperature increase does work, but not efficient enough.

It is also interesting to find out that the tendency in the change of aromatic signals intensity with temperature is different. The new peaks from residue 52F at 1H chemical shift of 7.13 at 308 K are 30% strongerer than that at 298 K. While, the intensity of the other new peak from residue 52F at 1H chemical shift of 7.60 decreased apparently with the increase in temperature. This indicates that the internal motion of the aromatic residue is temperature related. This seems to be reasonable, because residue 52F is located in a hydrophobic core in the center of the molecule[74], and is crowded by other aromatic residues around (30F, 43W), therefore, the little space to the inernal motions may make the flip of the aromatic rings difficult.

In either spectrum, the signals from the same aromatic ring have different intensities. Except overlap, intensities of some resonances are many times higher than others, though they are from the same aromatic ring. For example, in residues 43W, resonance at 13C chemical shift of 116.5 is much stronger than that at 124.5. This indicates that the apparent relaxation rate of those signals may be different, except the relative small change for their J coupling. The same phenomena can also be found in aromatic ring resonances of residue 52F. Some aromatic ring resonances of residue 52F have stronger intensities and do not show obvious sensitive enhancement in the CPMG-INEPT 1H-13C HSQC, such as aromatic resonance from residue 52F at 13C chemical shift of 129.5. This may be due to the fact that chemical shift of some flipping states of the resonances do not have much difference. These phenomena have also been reported by Takeda M and co-workers[45].

Indeed, XY4 and XY8 pulse train[65] have also been applied in CPMG-INEPT pulse train to optimize the CPMG rate, and it was found that XY16 is the best choice and can give an unfluctuating signal enhancement, which is consistent with the work of Mulder et al. that XY16 is the best choice for CPMG-INEPT blocks in moderately slow exchange range[68]. Besides, the experiment has also been applied to detect the aromatic resonances of protein Ubiquitin. The same result has been found that aromatic CPMG-INEPT 1H-13C HSQC can help enhance the resonances which are too broad to be detected in routine aromatic 1H-13C HSQC (three extra peaks have been detected in CPMG-INEPT HSQC of Ubiquitin, data not shown). These observations suggest that the property of slow chemical exchange of aromatic ring at room temperature is normal in proteins[3, 4, 16, 20, 46-51]. Therefore, CPMG-INEPT 1H-13C HSQC method can be generally applied to aromatic resonances in NMR based protein study.

3 Conclusion

In summary, the XY16 incorporated 1H-13C CPMG-INEPT HSQC experiment is proved to be an effective method to avoid the signal loss of aromatic resonances in protein, which is due to the chemical exchange originated from the complex flipping motion of aromatic group. Besides, INEPT is a general part in most heteronuclear multidimensional experiment, CPMG-INEPT may also be applied to other multidimensional experiment that is related to aromatic resonances.

Acknowledgements: This research is supported by grants from National Natural Science Foundation of China (20875098 and 21075132), National Major Basic Research Program of China (2009CB918603), and the Ministry of Science and Technology of China (2009IM030700). We thank Dr. JIANG Ling for providing the 13C, 15N-labeled protein GB1, and Dr. LI Shen-hui for his valuable comments.
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Sensitivity Enhancement in 1H-13C HSQC Experiments on Aromatic Groups in Protein...
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