![]() How to Label a Protein for Multidimensional NMR If your protein concentration is too low, you will need to collect data for longer. While these concentrations are not necessarily physiologically relevant, it serves to enhance your NMR signals and speed up data acquisition time. In multi-dimensional NMR experiments, which are less sensitive than 1D experiments, you want the concentration of your protein to be as high as possible without aggregation occurring.Īim for a sample concentration of ~0.3–1.0 mM. Sample Requirements for Multidimensional NMR A flowchart outlining the labeling and 2D 1H- 15N heteronuclear single-quantum coherence (HSQC) NMR workflow. Let’s start with a flowchart outlining the labeling and 2D 1H- 15N NMR workflow (Figure 1).įigure 1. Practical Aspects of Multidimensional NMR Not many biophysical techniques can offer such information about proteins in solution. Thus, multidimensional NMR provides a powerful platform to begin dissecting your protein’s structure, function, and dynamics at atomic resolution. See this practical website for an introduction to triple resonance experiments and resonance assignments. However, by following 1H- 13C- 15N magnetization pathways in your protein, individual resonances can be assigned to their nuclei of origin via multi-dimensional NMR. Typically, signals from 1D NMR can be bunched only into the following groups: Using 1D NMR, enormous signal overlap renders it impossible to determine which resonances arise from which nuclei. This is referred to as the “ 3D HNCA experiment.” Multidimensional NMR vs. And this simplifies your NMR data interpretation!Īn example of a triple resonance experiment includes 1H chemical shifts on the x-axis, backbone 15N chemical shifts on the y-axis, and 13C α chemical shifts on the z-axis. So, the construction of heteronuclear (i.e., 1H- 15N, 13C- 15N- 1H, etc.) 2D and 3D spectra (via double and triple resonance experiments, respectively) is possible. This enables the correlation of the chemical shifts of protons with chemical shifts of the 13C or 15N nuclei to which they are attached (although covalent attachment is not necessary). Well, instead of simply collecting NMR data on the ~1,000 1H nuclei (protons) in your ~100 residue protein, you can now include the ~150 15N and ~500 13C nuclei in a multidimensional spectrum to associate the position of each peak with two or more resonance frequencies, thereby vastly enhancing spectral resolution. Not satisfied? And wondering why you should bother labeling your protein with NMR-active ( 13C and/or 15N) isotopes? This results in cross-peaks that provide extra structural information about the sample and make the assignment of chemical shifts easier. Regarding point 2, this is because so-called magnetization transfer can take place between coupled nuclei. Regarding point 1, this is because the signals from the nuclei are spread over a 2D surface rather than a 1D chart. But in simple terms, the main benefits are: What Are the Benefits of Multidimensional NMR? Simple AnswerĪgain, the answers are quite high-level and rigorous. Plot them all together, and hey presto-a 2D NMR spectrum!Ĭheck out slide 26 of this resource for an example. This extra set of chemical shifts (also called frequencies) usually collected 13C or 15N nuclei in addition to the proton ( 1H) nuclei. So effectively, the plot is intensity vs. In a 2D NMR experiment, the spectrum obtained has an additional set of chemical shift values. In a 1D proton NMR experiment, the spectrum obtained is a plot of intensity vs. It’s a large and complicated subject, so I’ll keep the answer as simple as possible. In this third and final part, we discuss multidimensional NMR, touch on isotope labeling, and learn about a common 2D NMR experiment that can identify ligand binding sites. ![]() In part two, we learned about spin relaxation rates, such as T1 and T2, etc., and how they can be used to derive the oligomeric state of a sample protein. In part one, we explained some critical points when doing 1D proton NMR and learned how to quickly assess whether a sample protein is folded. Welcome to part three of “The Basics of NMR”, where we dive into multidimensional NMR.Īs a brief refresh, this three-part series describes how you can use NMR experiments to characterize your protein of interest. ![]()
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