1600.e1 Cell 166 , September 8, 2016 ©2016 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2016.08.061
SnapShot: Biomolecular NMR
David Wemmer
Department of Chemistry, University of California, Berkeley, Berkeley CA, 94720, USA
Nuclear magnetic resonance (NMR) spectroscopy uses the intrinsic magnetic moments of nuclei that arise from quantum mechanical spin and couple to an applied static
magnetic field. In modern spectrometers, NMR data are collected by using short rf pulses near the resonance frequency to induce a coherence between the energy levels. This
coherent state has a magnetic moment that induces an oscillating voltage that is detected as a function of time after the pulse. Fourier transform (FT) of this signal gives the
amplitudes and frequencies of spins in the sample. With a sample beginning at equilibrium, the FT of the response to a single pulse produces the absorption spectrum.
Multidimensional NMR was a key development in extending NMR to large, complicated molecules. In such experiments, there are multiple rf pulses and multiple variable
time periods in the experiment. Data are collected by systematically collecting the transient response generated for many combinations of the variable time periods. For
example, in an experiment with three time periods, designated t 1 , t 2 , and t 3 , where frequencies need to be determined, the signal as a function of real time t 3 will be collected
separately for each of a set of values of t 1 and t 2 that are systematically arrayed (e.g., nDt 1 and mDt 2 , where n and m are integers and the D values are a fixed increment in time).
The multidimensional spectra are generated by carrying out successive Fourier transforms over each time variable. The cross peaks (different frequencies in different dimen-
sions) are described as n-tuples of frequencies, reflecting the spin that carried the magnetization in each dimension. These cross peaks can only occur when the correlation
mechanism is active for them, for example that they are coupled or that they are near in space—the specific mechanisms can be different in different dimensions and are
determined by the pulse sequence. The correlation information is the key both to identifying resonances, associating them with specific spins, and also for doing structural or
dynamic analysis. There are thousands of variations of multidimensional experiments using different combinations of couplings and proximity to generate correlations. The
increased resolution and sensitivity of multidimensional NMR are critical for biomolecular applications, particularly for large molecules.
Isotope labeling has become a critical aspect of most biomolecular NMR experiments. Enrichment of^13 C and^15 N to near 100% allows use of the many couplings between
them, and also with^1 H, to generate correlations. For work in very large proteins, it is advantageous to replace most of the^1 H with^2 H (that has a much smaller magnetic
moment) to reduce the linewidth of resonances. With selective isotope labeling, and special pulse sequences designed to produce sharp signals, it has been possible to probe
very large macromolecules (up to the megaDalton range in favorable cases). Although full structure determination is not possible at such molecular weights, localization of
binding interactions, probing of conformational changes, and determination of dynamics can be done.
Basic Concepts
Nuclear Magnetic Resonance = NMR
Many nuclei have magnetic moments that couple to an applied magnetic field H0. NMR spectroscopy uses radio frequency “light” to drive transitions between spin down
and spin up states. Because the energy gap between the states is small, the populations of down and up states are nearly equal and the observed signal is proportional to the
population difference. Higher fields provide stronger signals, with the sensitivity scaling roughly as the square of the applied field.
Spin 1/2 Nuclei
Spin 1/2 nuclei have sharp NMR spectra. In available magnets, the frequencies of spins are in the radio-frequency (rf) range (now up to 1,000 MHz for protons, approaching
the microwave range) Nuclei most useful for probing molecules include (with natural abundance):
Nucleus:^1 H (99.9%)^13 C (1.1%)^15 N (0.3%)^19 F (100%)^31 P (100%)
Frequency (14.1 Tesla field): 600 MHz 150 MHz 60 MHz 560 MHz 240 MHz
Chemical Shift
Movement of electrons around a nucleus in an applied magnetic field generate a response field in proportion. This is called a chemical shift and reflects the local electronic
environment. Chemical shifts are reported in parts per million of the applied field (ppm). Because the shift is linearly proportional to the field, but the width of individual reso-
nances is less dependent on field, the resolution increases with field, making it desirable to work at the highest available field. Unfortunately, very high field magnets are much
more expensive.
Spin-Spin Couplings
Nuclei also feel magnetic fields from nearby nuclei. Couplings are transmitted by bonding electrons and decrease in size with increasing numbers of bonds. Couplings are
independent of applied field and are reported as frequencies in hertz (Hz). If molecules are partially ordered, residual direct dipolar couplings (RDCs) are present and add to
the isotopic couplings.
Fourier Transform
The FT is a mathematical process that analyzes a signal occurring as a function of time to determine the amplitude and frequency of each component of the signal. For
NMR, signals are usually cosine or sine functions that decay exponentially, the FT gives a Lorentzian peak at the frequency of trig function, with a width inversely proportional
to the decay rate (rapid decay = broad line).
Relaxation
Pulses perturb populations of the up and down spin states and also create coherences, so the spins are not at equilibrium. Each spin feels magnetic fields arising from
other nearby spins, and from chemical shifts, that fluctuate as the molecule rotates randomly in solution. The fluctuating magnetic fields induce transitions of the spins, allow-
ing them to return to equilibrium. The time constant for populations to return to equilibrium is called T 1 and is affected most by fluctuations at the spin’s resonance frequency.
The time constant for coherences to dissipate is called T 2 and is affected by fast fluctuations but even more so by slow ones. 2D experiments can be used to measure the T 1
and T 2 values for spins in each residue of a biomolecule and hence site specifically probe motions.
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