Biomolecular NMR Assignment: Illustration Using ... - ACS Publications

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Biomolecular NMR Assignment: Illustration Using the Heme Signals in Horse Cytochrome c Ana V. Silva and Ricardo O. Louro* Instituto de Tecnologia Química e Biológica, António Xavier, Universidade Nova de Lisboa, Av. da República (EAN), 2780-157 Oeiras, Portugal S Supporting Information *

ABSTRACT: NMR spectroscopy is a powerful technique with applications in a myriad of areas of scientific research. In (bio)chemical research, NMR spectroscopy can provide unique insights on structure, dynamics, and reactivity of target molecules. These studies typically start with the assignment of the NMR signals to specific nuclei. Here, we describe an NMR assignment activity aimed at advanced undergraduate or graduate level students in either the biological or chemical sciences. Using a variety of NMR spectral data, students assigned the protons of the horse cytochrome c heme cofactor in a single 4 h session. It provides students with the opportunity to execute successfully a simulated research task and appreciate the complexity and challenges of an NMR assignment and the excitement of finding the solution. For instructors, it is designed to dispense the need to access NMR spectrometers, which facilitates its implementation. Ultimately, this increases the range of institutions and curricular units where it can be adopted. KEYWORDS: Upper-Division Undergraduate, Graduate Education/Research, Biochemistry, Biophysical Chemistry, Hands-On Learning/Manipulatives, Collaborative/Cooperative Learning, Bioinorganic Chemistry, NMR Spectroscopy

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INTRODUCTION

An in-depth and easy to grasp explanation of how raw NMR data are converted into relevant structural information is usually left out of chemistry and biochemistry curricula.10 An activity is described where students assign the heme signals of horse cytochrome c using a combination of multidimensional NMR experiments. Multidimensional NMR techniques such as COSY, TOCSY, and NOESY have the advantage of reducing spectral overlap by spreading the signals across the various dimensions. The COSY (homonuclear correlation spectroscopy) is the simplest two-dimensional proton NMR experiment and detects 1 H−1H scalar correlations.11,12 It generates a map with crosspeaks between pairs of protons that typically are no more than three chemical bonds apart4,13 (Figure 1). In TOCSY (total correlation spectroscopy), in addition to the cross-peaks present in a COSY spectrum, signals that originate from the interaction of all protons of a spin system are observed13 (Figure 1). In practical terms, a spin system is composed of the nuclei of hydrogens bound to consecutive heavy atoms such as carbons. A NOESY (nuclear Overhauser effect spectroscopy) experiment provides cross-peaks for pairs of protons at a short distance from one another (typically within 5 Å) even if they do not belong to the same spin system (Figure 1).13

NMR spectroscopy is a powerful physical technique with great impact on multiple areas of scientific research such as biology, biochemistry, chemistry, and material sciences.1−4 It can provide information on molecular structures at the atomic level and also probe the amplitude and rates of molecular dynamic phenomena in time scales that span more than 12 orders of magnitude, from picoseconds to days.5 The size of the molecule to be analyzed is no longer an unsurmountable limitation. With advances in the spectrometers’ magnetic field strength, the development of new pulse sequences,6 the use of selective labeling,7 and the combination with other techniques,8 it is possible to study large macromolecular complexes using NMR spectroscopy.9 NMR signal assignment relies on the principle that for a given molecule each atom occupies a unique chemical environment, thus having a unique chemical shift. The chemical shifts can be used to predict properties such as structure, redox state, and presence of metal ligands.2,10 In proteins with metallic cofactors, the assignment of the NMR signals associated with the active center is often enough to extract important insights on protein function and biochemical properties. This represents a major advantage since a full assignment of the polypeptide residues is not needed in order to gain valuable insight into the chemical function of the protein, in contrast to most NMR-based approaches for protein studies. Assigning only the redox center of the protein is much simpler and faster. © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: February 14, 2017 Revised: June 20, 2017

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DOI: 10.1021/acs.jchemed.7b00123 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Activity

follow-up to 3 h of theoretical introduction to the basic concepts of NMR spectroscopy. The students were divided in groups of 2−4, each with a computer. The spectra needed for this activity take several hours to collect and must be obtained by the instructor and provided to the students already processed.

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MATERIALS AND METHODS Description of spectral collection and processing is available as Supporting Information. Required Infrastructures

All spectra are provided as Supporting Information. This means that student access to a high-field NMR spectrometer is not necessary. Access to computers for student use in analyzing the spectra with Sparky v.3.115 is needed to implement this activity.

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RESULTS At the start of the activity, a brief theoretical overview was given by the instructor on the nature of each experiment. Students were also made aware of the common chemical shift values for the heme substituents (Table 1). Table 1. Typical Range of Chemical Shifts for the Heme Protons in Diamagnetic Hemes ca Figure 1. (a) Representation of a molecule that gives the spectrum represented in panel b. The dashed line represents a series of covalent bonds that make HD closer in space to HC despite being separated by numerous chemical bonds. (b) Scheme representation of cross-peaks observed in COSY (○), TOCSY (○ and □), and NOESY (○, □, and ☆) spectra. The dashed line represents the diagonal of the twodimensional spectra.

a

Group

Reference Shift Interval

Mesos Methyls Thioether methines Thioether methyls

9−10 ppm 2−4 ppm 5−7 ppm 1−3 ppm

Data from refs 14 and 18.

To facilitate the assignment, a paper print of a heme c structure was provided to the students (Supporting Information). This allowed them to predict the cross-peaks that they would observe in each NMR experiment as illustrated in Figure 2. After this introduction, students were given a print of the 1D proton spectrum of the protein in the reduced state (Supporting Information) and encouraged to assign the peaks in the spectrum to protons in the structure. Given the clear spectral region where the heme meso protons are found, this was the starting point. The protein was lyophilized twice in D2O before data collection to reduce the number of protein amino acid signals in the meso region. Next, the NOESY spectrum at 50 ms was provided to the students for use; this provided information on the cross-peaks in the typical spectral region of heme thioether methines. If no cross-peaks were found to putative thioether methines, the signal was tentatively assigned as a H15, H20 or protein amino acid. Otherwise, the meso was tentatively assigned as H5 or H10. Confirmation of the assignment of thioether methines was obtained using the TOCSY or COSY spectrum by looking for the cross-peak with the neighboring thioether methyl as illustrated in Figure 3 below the diagonal. Signals with cross-peaks in the 50 ms NOESY spectrum that did not match the expected region of heme methyls were tentatively assigned as protein amino acid signals and not explored further. Unequivocal identification of the signals was obtained from the 200 and 400 ms NOESY spectra that

This activity is adapted from the pioneering research work by Keller and Wuthrich14 and gives students a practical NMR assignment that does not rely on tabulated typical chemical shifts for amino acid protons. It aims to identify the signals of the heme cofactor of horse heart cytochrome c. Horse heart cytochrome c is a small (12 kDa), inexpensive, easily accessible, and nontoxic protein. It has two stable redox states: a diamagnetic iron 2+ state and a low-spin paramagnetic 3+ state. Furthermore, its convenient spectral properties15 arising from a single heme cofactor, ease of manipulation, and stability over a wide range of conditions16 make it a convenient target for teaching spectroscopic characterization of proteins containing metal cofactors. The pedagogic goal of this activity is to provide students with the experience of combining different kinds of homonuclear NMR experiments to perform the assignment of signals in biomolecules. It was designed as part of a curricular unit on biochemical and biophysical tools to explore the role of metals in biology. It was implemented three times in advanced summer schools focusing on methods to study metals in biology. These summer schools trained Ph.D. students and early stage postdocs with a broad range of background knowledge in biological and chemical sciences, but often with very little or no previous training in biomolecular NMR spectroscopy. This activity can also integrate into a cytochrome-c-based biochemistry course as has been proposed previously.17 The activity was designed to last 4 h, and it is the B



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cooperate to get the full assignment of the heme. As should be expected, students at first needed significant assistance with this data analysis strategy and with operating the software. However, as students gained proficiency with the use of Sparky and started to understand the procedure, they were interested and motivated, with the groups cooperating and competing to “solve the puzzle”. Using this approach, students typically could assign the heme proton signals within 3.5 h. Table 2 provides Table 2. Chemical Shift Table Resulting from the Assignment of the Reduced NOESY, COSY, and TOCSY Spectra

Figure 2. Diagram of heme c numbered according to the IUPAC-IUB nomenclature.19 The red lines show the heme protons involved in the short-range connectivities that are typically detected in a 50 ms NOESY spectrum; the blue lines indicate the long-range connectivities that can be observed in a 200 or 400 ms NOESY spectrum. Black lines indicate connectivities visible in TOCSY and COSY spectra. (Adapted from ref 18 with permission. Copyright 1992 Blackwell Publishing Limited.) Some correlations may not be observed in real experimental data due to the particular orientation of the thioether of the heme.

Group

Shift (ppm)

H5 H10 H15 H20 M21 M71 M121 M181 H31 H81 M32 M82

9.37 9.69 9.69 9.15 3.55 3.92 3.59 2.29 5.31 6.46 1.53 2.66

an answer key of the results that students should obtain; this table contains the averaged values reported by Sparky resulting from the assignment using the three types of spectra. In the final half hour, the students were provided with a print of the 1D proton spectrum of the partially oxidized protein (Supporting Information) and instructed to upload the NOESY spectrum obtained under the same conditions. This allowed them to appreciate the dramatic changes in spectral range and line width of the signals due to the presence of the unpaired electron. It also allowed them to appreciate that a NOESY experiment also detects chemical exchange in addition to dipolar interactions. The students were then invited to assign the signals of the heme methyls in the paramagnetic state using the exchange cross-peaks with the methyl peaks in the reduced state identified in the initial part of the activity (Table 3). This Table 3. Chemical Shift Table Resulting from the Assignment of the Methyls in the Oxidized NOESY Spectrum

Figure 3. Illustration of the assignment strategy applied to a meso of the H5 class. Above the diagonal, a 50 ms NOESY spectrum. Below the diagonal, a 60 ms TOCSY spectrum. Box A: Insert of 200 ms NOESY showing the cross-peak between H5 and M32. The edge of the heme showing the connectivities highlighted in the spectra is overlaid above the diagonal.

Group

Shift (ppm)

M21 M71 M121 M181

7.03 32.19 9.99 34.99

provided them with the opportunity to appreciate the fact that, for macromolecules, the NOESY experiment does not discriminate dipolar correlations from exchange correlations making the assignment of M21 and M121 uncertain without confirmatory data. This offered the opportunity to discuss the ROESY (rotating frame Overhauser effect spectroscopy) experiment20 and its advantages and weaknesses when compared to the NOESY. The most common difficulty encountered during this activity was the misassignment of peaks due to the presence of signals

allowed the observation of long-range connectivities between nuclei associated with each of the meso protons. As the students identified the various classes of protons, the instructor set up a table on the board so that they could compare their results with the results of other groups and C



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financial support through Projects PTDC/BBB-BQB/4178/ 2014 and Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020Programa Operacional Competitividade e Internacionalizaçaõ (POCI). This work was supported by COST action CM1305-ECOSTBIO. The NMR data were acquired at CERMAX which is a member of the Portuguese NMR network.

and cross-peaks resulting from the polypeptide chain. The table set up by the instructor with the assignment proposals of each group of students provided immediate feedback to the class by revealing if the groups were all reaching the same solution. Also, misassignment of cross-peaks was identified by large standard errors in the Sparky assignment table and solved by a more careful look at the shape of the peaks. Another challenge was the discrimination of the mesos H10 and H15 due to their very similar chemical shifts. To solve this, students had to refer to the 1D spectra and to the different pattern of cross-peaks in the various experiments to see which ones matched the pattern expected from Figure 2. In cases where 4 h are not available to complete this activity or where a cohort of students is struggling, the assignment of the heme methyl signals in the oxidized state can be omitted without compromising the pedagogical goal of the activity. The completion of the assignment during the class gives immediate confirmation that the pedagogical goal was achieved. Each group of students delivered a report containing their assignment results, a brief discussion of the structural, and dynamic information obtained from each experiment, and how that impacted the proton peak assignments.

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(1) Viegas, A.; Manso, J.; Nobrega, F. L.; Cabrita, E. J. Saturationtransfer difference (STD) NMR: A simple and fast method for ligand screening and characterization of protein binding. J. Chem. Educ. 2011, 88, 990−994. (2) Bertini, I.; McGreevy, K. S.; Parigi, G. NMR and Its Place in Mechanistic Systems Biology. In NMR of Biomolecules: Towards Mechanistic Systems Biology, 1st ed.; Bertini, I., McGreevy, K. S., Parigi, G., Eds.; Wiley-Blackwell: Weinheim, Germany, 2012; pp 3−6. (3) Barrett, P. J.; Chen, J.; Cho, M.-K. K.; Kim, J.-H. H.; Lu, Z.; Mathew, S.; Peng, D.; Song, Y.; Horn, W. D. V.; Zhuang, T.; Sönnichsen, F. D.; Sanders, C. R.; Van Horn, W. D.; Zhuang, T.; Sönnichsen, F. D.; Sanders, C. R.; et al. The quiet renaissance of protein nuclear magnetic resonance. Biochemistry 2013, 52, 1303− 1320. (4) Louro, R. O. Introduction to Biomolecular NMR and Metals. In Practical Approaches to Biological Inorganic Chemistry, 1st ed.; Crichton, R., Louro, R., Eds.; Elsevier: Amsterdam, The Netherlands, 2013; pp 77−107. (5) Ban, D.; Sabo, T.; Griesinger, C.; Lee, D. Measuring dynamic and kinetic information in the previously inaccessible supra- τc window of nanoseconds to microseconds by solution NMR spectroscopy. Molecules 2013, 18, 11904−11937. (6) Viegas, A.; Viennet, T.; Yu, T.-Y.; Schumann, F.; Bermel, W.; Wagner, G.; Etzkorn, M. UTOPIA NMR: activating unexploited magnetization using interleaved low-gamma detection. J. Biomol. NMR 2016, 64, 9−15. (7) Kerfah, R.; Plevin, M. J.; Sounier, R.; Gans, P.; Boisbouvier, J. Methyl-specific isotopic labeling: A molecular tool box for solution NMR studies of large proteins. Curr. Opin. Struct. Biol. 2015, 32, 113− 122. (8) Duss, O.; Yulikov, M.; Allain, F. H. T.; Jeschke, G. Combining NMR and EPR to determine structures of large RNAs and proteinRNA complexes in solution. Methods Enzymol. 2015, 558, 279−331. (9) Kay, L. E. New Views of Functionally Dynamic Proteins by Solution NMR Spectroscopy. J. Mol. Biol. 2016, 428 (2 Pt A), 323− 331. (10) Wright, N. T. Heteronuclear Multidimensional Protein NMR in a Teaching Laboratory. J. Chem. Educ. 2016, 93, 287−291. (11) Derome, A. E. Homonuclear Shift Correlation. In Modern NMR Techniques for Chemistry Research, 1st ed.; Baldwin, J. E., Ed.; Pergamon Press: Oxford, United Kingdom, 1987; pp 183−244. (12) Anderson-Wile, A. M. Introducing 2D NMR Spectroscopy to Second-Year Undergraduate Chemistry Majors Using a Building-Up Approach. J. Chem. Educ. 2016, 93, 699−703. (13) Ferella, L.; Rosato, A.; Turano, P. Determination of Protein Structure and Dynamics. In NMR of Biomolecules: Towards Mechanistic Systems Biology, 1st ed.; Bertini, I., McGreevy, K. S., Parigi, G., Eds.; Wiley-Blackwell: Weinheim, Germany, 2012; pp 53−94. (14) Keller, R. M.; Wuthrich, K. Assignment of the heme c resonances in the 360 MHz 1H NMR spectra of cytochrome c. Biochim. Biophys. Acta, Protein Struct. 1978, 533, 195−208. (15) Craig, D. B.; Nichols, E. R. Spectroscopic Measurement of the Redox Potential of Cytochrome c for the Undergraduate Biochemistry Laboratory. J. Chem. Educ. 2006, 83, 1325−1326. (16) Pettigrew, G. W.; Moore, G. R. Cytochromes c Biological Aspects, 1st ed.; Springer-Verlag: Berlin, 1987; pp 4−266. (17) Vincent, J. B.; Woski, S. A. Cytochrome c: A Biochemistry Laboratory Course. J. Chem. Educ. 2005, 82, 1211−1214.

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CONCLUSIONS This activity provides an inexpensive and versatile frame for training students interested in biomolecular structural determination. It can be held in multiple pedagogical contexts with respect to course level and subject of the overall studies plan. For example, this activity can be adapted to a curricular unit more centered on biomolecular NMR spectroscopy, and in that case, it becomes the follow-up of a previous practical class where the students set up their own 2D NMR experiments to collect the data. Alternatively, this activity can be implemented in the absence of access to advanced NMR instrumentation by making use of the Supporting Information. Thus, any instructor with a computer of modest performance can set up a version of this activity tailored to the specific needs of the course in which it would be integrated.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00123. 2D NMR spectra, 1D NMR spectra, list of pulse programs, Sparky step by step guide, and heme image (ZIP)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ana V. Silva: 0000-0002-1861-0339 Ricardo O. Louro: 0000-0002-2392-6450 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the members of the Inorganic Biochemistry and NMR Laboratory for critically reading the manuscript. Fundaçaõ para a Ciência e Tecnologia is acknowledged for D



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(18) Turner, D. L.; Salgueiro, C. A.; LeGall, J.; Xavier, A. V. Structural studies of Desulfovibrio vulgaris ferrocytochrome c3 by twodimensional NMR. Eur. J. Biochem. 1992, 210, 931−936. (19) Moss, G. P.; Smith, P. A. S.; Tavernier, D. Glossary of class names of organic compounds and reactivity intermediates based on structure (IUPAC Recommendations 1995). Pure Appl. Chem. 1995, 67, 1307−1375. (20) Bothner-By, A. A.; Stephens, R. L.; Lee, J.; Warren, C. D.; Jeanloz, R. W. Structure determination of a tetrasaccharide: transient nuclear Overhauser effects in the rotating frame. J. Am. Chem. Soc. 1984, 106 (3), 811−813.

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Biomolecular NMR Assignment: Illustration Using ... - ACS Publications

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