Several isotopes (e.g., ¹H, ¹³C, ¹⁵N, ³¹P, ²⁷Al, ²⁹Si, etc.) carry a spin. This fundamental quantum-mechanical property can be described by an inherent spin angular moment of the atomic nucleus. In combination with its electric charge, the spin generates a magnetic moment which may interact with an external magnetic field or with other spins.

If a sample containing magnetically active nuclei of, for example, the above mentioned isotopes is inserted into the strong magnetic field of a superconducting magnet (e.g., at 9.4 T, approx. 200,000-fold stronger than earth's magnetic field) the spins will align and the energetic degeneracy of different spin states is lifted. This effect has been discovered in 1896 by Pieter Zeeman for the electron spin and has been awarded with the Nobel prize in Physics in 1902. Zeeman observed the splitting of optical absoprtion or emission lines in atomic vapors due to a magnetic field. In comparison to the Zeeman effect which relies on the relatively large spin moment of unpaired electrons, the nuclear Zeeman effect is about three order of magnitude smaller. Therefore, modern NMR spectroscopy requires very large magnetic fields at the edge of technical possibilities.

A nuclear spin inside a strong magnetic field which is also subject to an electromagnetic field oscillating at the nuclear Larmor frequency will show resonance. Thus, a nuclear spin transition is induced a an appropriate photon is absorbed. The resulting induction can be measured by the same radiofrequency pickup coil which has been used for pulse excitation, and the signal is detected and electronically stored. Because the nuclear spin of each atom inside a molecule senses a different magnetic environment (for example, due to local fields of other spins or shielding by electronic orbitals), a unique and individual resonance line will be measured in the NMR sepctrum.

At typical magnetic fields of 7–24 T, Larmor frequencies between 300 and 1000 MHz are obtained for protons (¹H nuclei of hydrogen). The chemical shift (i.e., the relative frequency shift due to electronic shielding) is only on the order of 10–20 ppm. FOr other nuclei the respective Larmor frequency is considerably smaller, however, the chemical shift range can take up much larger ranges (with spans of up to thounsands of ppm). This results in numerous application scenarios for NMR.

Based on the above-described basics, an NMR spectrometer contains the following components: (1) magnet, (2) NMR probe, (3) console, (4) work station.

1. The magnet generates the required magnetic field through the induction of a superconducting coil. Once cooled below its critical temperature and charged up to field, the current flows persistently and essentially without electrical resistance. This not only reduces the electrical power loss which makes operation with conventional magnets extremely cost expensive due to the electricity and cooling required; it also ensures an extremely stable magnetic field due to the purely direct current. This allows for spectral separation of NMR lines with an extremely high resolution in the ppb range and below. However, the magnet requires constant cooling with liquid Helium at a temperature of 4.2 K (–269 °C). In order to reduce cryogen losses, the helium cryostat is shielded by an insulating vacuum and an additional vessel cooled by liquid nitrogen. This increases the laboratory footprint, often resulting in the need for specialized spaces and multi-story clearance.
2. The NMR probe is holding the sample tube inside the sweet spot of the vertical magnet bore. More importantly, it contains the means to generate and pickup the radiofrequency fields crucial for NMR. For this, special inductive coils are utilized (e.g., saddle or solenoid). These are part of electromagnetic resonance circuits for increasing the conversion factor between magnetic field and electric signal. Furthermore, NMR probes often contain equipment for temperature variation or spinning of the sample and, particularly for analytical high-throughput applications, with automatic sample changers.
3. The console contains the electronic components for generation of the radiofrequency pulses and detection of the nuclear spin signals which are emitted or picked up, respectively, by the NMR coil. For this, frequency synthesizers, pulse or abitrary waveform generators as well as duplexers, preamplifiers and audio amplifiers are utilized in the homodyne method. Additionally, the console contains dedicated control components for the magnet (e.g., cryogen level sensors, shim coil systems for field homogeneity optimization, etc.) and for communication with the work station.
4. The work station acts as the interface between the spectroscopist and the spectrometer. This is typically a desktop PC operating a dedicated control and processing software in direct communication with the console via ethernet. Through the software, the operator may program special pulse sequences, vary experimental parameters or post-process obtained spectroscopic data for scientific analysis.

Interaction relvant to NMR are generally anisotropic, meaning the magnitude of the interactions depends on the orientation between the external magnetic field and the spin's environment (e.g., the molecule or crystal). Therefore, all NMR interaction are described by second rank tensors connecting two vector quantitites (e.g., one spin moment with the external field or two spin moments with each other).

Small molecules in solution may tumble fast enough due to Brownian motion that all orietations are effectivel averaged. Some interactions are consquently reduced to their isotropic mean value (e.g., chemical shift, J-coupling); this isotropic value is obtained by the trace of the interaction tensor. Other interactions are vanishing under fast tumbling since their respective interaction tensor is traceless.

In large macromolecules or polymers, highly viscous or vitreous solutions, or in (poly-)crystalline/amorphous solids molecular tumbling is significantly arrested. Therefore, anisotropic interactions are not averaged. In single crystalline samples this causes a shift of (often narrow) resonance lines under rotation of the macroscopic crystal within the magnetic field. By controlled angular adjustment (e.g., by a goniometer), the interaction tensors can thus be measured and even be correlated with the crystallographic axes.

In the case of polycriystalline or amorphpus samples all possible orientations are observed at the same time. This causes inhomogeneous broadening of the nuclear resonances and a so-called powder spectrum is obtained. The envelope of each resonance line describes the weighted spectral range which would be swept by rotation of a single crystal around all axes. However, overlap of resonances of different nuclei is often a problem due to the large inhomogeneous breadth; also, no direct correlation between interaction tensor orientation and crystallographic axes is possible. Nevertheless, information about magnitude and orientation of the tensor elements may be obtained by more sophisticated experiments and spectral simulation.

Another important advantage of solid-state NMR is the direct influence of dipolar spin-spin interactions on the obtained NMR spectrum. In contrast to solution NMR, these dipolar interactions cause a splitting of resonance lines which allows for the direct and model-free measurement of distances or angles between nuclei with an inherently high accuracy. Similarly, electrical field gradients at the position of a high-spin nucleus may be obtained by analysis of quadrupolar splitting schemes. From this, for example, information about electron pair distributions can be obtained or hydrogen bond formation can be confirmed.

However, this abundance of additional couplings not present in solution NMR comes with certain disadvanteges. Overlapping resonances from typically hundreds or even thousands of different nuclei within a sample system result in spectral crowding. Additionally, insufficient averaging of strong dipolar couplings may cause a rapid loss of spin decoherence which in turn results in significantly increased (homogeneous) line widths.

Anisotropic NMR interactions follow spherical harmonics. In this regard, a special role is taken by the second Legendre Polynomial P₂(x)=0.5·(3x²–1), where x is the Cosine of the angle with respect to the external magnetic field. This function describes the angular dependence of the most important di- and quadrupolar interactions as well as of the chemical shift anisotropy. Therefore, these interactions or anisotropies are vanishing if the relevant angle corresponds exactly to the magic angle of 54.736°. Cosequently, if, for example, the connecting vector between two coupled spins is oriented in this angle to the externam magnetic field the respective coupling has no influence on the solid-state NMR spectrum!

Particularly in polycrystalline or amorphous samples this unique orientation ist not possible since the individual crystallites or molecules are disordered. Therefore, the sample is rotated with high frequency around an axis oriented about the magic angle. If the rotational frequency is sufficient, all interactions orthogonal to this axis are avergaed and only interactions parallel to the magic angle potentially persist. As a consequence, all couplings and interactions following the 2nd Legendre polynomial are completely removed.

An analogous picture is obtained if the magic angle is considered as the space diagonal of a cube. Rotation about this diagonal projects all three orthogonal cubic axes onto each other which causes an effecting averaging of all interactions depending linearly (i.e., in first order) on the coordinates within the unit sphere.

For efficient averaging, rotational frequencies exceeding the magnitude of the anisotropic interactions are required.Therefore, efficient decoupling of the dipolar interaction between two directly bonded ¹³C nuclei with a distance of 154 pm is achieved with a rotational frequency several-fold larger than the coupling constant of 2 kHz. ¹H bonded directly to ¹³C has a coupling constant of 22 kHz; thus, heternonuclear decoupling by radio frequency pulses is used in addition to MAS. Two methylene hydrogens with a distance of 180 pm experience a similar coupling strength, however, due to the homonuclear nature of the interaction, this can only be suppressed by very fast MAS at frequencies up to the current technical limit of 110 kHz in commercial probes. For the controlled reintroduciton of the dipolar interaction (recoupling), dedicated pulse sequences may be applied. This allows for the measurement of distances and angles between nuclei.

If the MAS frequency is smaller than the magnitude of the anisotropy, its averaging is incomplete. Due to the evolution of the transverse magnetization under the modulated interaction the resulting dephasing will be periodically refocused and rotational echos will be observed after each complete MAS revolution during the free induction decay (FID). After Fourier Transform (FT), these rotational echos manifest as spinning side bands in equal distance corresponding to the MAS frequency. Since the integral area under all bands is conserved, increasing the MAS frequency does not only lead to better spectral separation of the center band (occuring at the isotropic frequency) from the side bads, but also to larger intensity due to a reduction of side bands' count and intensities.

For the implementation of MAS with frequencies up to 110 kHz modern stator-rotor systems are used. The stator is part of the NMR probe and is aligned at the magic angle inside the magnet bore. It consists of the housing, bearings, baffles and the drive unit. A radial air or nitrogen flow is injected into the space between bearing and rotor wall and holds the rotor contactless in place with minimal friction. At the same time a tangential jet drives the rotor’s turbine and spins it up to high MAS frequencies. The flow of the MAS gases is guided by baffles to the exhaust in order to prevent turbulences.

Most of the time, zirkonia (ZrO₂) is used as rotor material due to its hardness and ease of machining. Alternatively, a rotor made of a single crystal of synthetic sapphire (Al₂O₃) equally withstands the centrifugal forces during fast rotation. However, its brittleness makes it more prone to unplanned rapid loss of structural integrity (i.e., explosion) of the rotor. In contrast to zirconia, sapphire’s advantage lies in its transparency to electromagnetic radiation over a wide range of frequencies, making it possible to illuminate the sample inside the rotor with, for example, visible light or microwaves during MAS.

The rotor’s diameter directly limits the maximum MAS frequency. On the one hand, this belies on the dependence of the centrifugal force on the diameter; on the other hand, it has to be considered that the tangential velocity of the outside wall may approach the speed of sound of the gas medium used for bearing and drive. This limits the maximum rotational frequency particularly when sample cooling is achieved with cryogenic MAS gases. At the current time, typical rotor diameters vary between 7 mm (max. 7 kHz) and 0.7 mm (max. 110 kHz). Popular variants are 4 mm (15 kHz), 3.2 mm (25 kHz), 1.9 mm (40 kHz), and 1.3 mm (67 kHz), besides others.

The nuclear Zeeman effect is a very weak interaction. Thus, it effects also very small energy splittings of the spin states within technically achievable magnetic fields. This situation has several consequences. On the one hand, the long natural lifetimes of the excited spin states and the resulting long decoherence times cause extremely narrow homogeneous line widths. On the other hand, the small Boltzmann factor leads to extremely small polarization of the spin transition. As the population difference between the energetic ground and the excited state, this polarization directly limits the reachable NMR signal intensity of a system in thermal equilibrium. Together with the slow return of the spin system into equilibrium after a radio frequency pulse (relaxation), this results in large experimental time requirements for recording NMR spectra with sufficient signal-to-noise ratio.

The thermal spin polarization of unpaired electrons is about three orders of magnitude larger as compared to nuclear spins. Through dynamic nuclear polarization (DNP) this large polarization may be transferred from electrons to nuclei and the NMR sensitivity can be increased. For ¹H, the maximum DNP enhancement factor is about 660, for other nuclear spins this factor is larger according to their gyromagnetic ratio. However, losses due to relaxation and inefficiencies of transfer mechanisms result in typical enhancement factors of 20-200. Nevertheless, this factor effects an acceleration of NMR experiments by a factor of 400-40,000!

In solids there a several DNP mechanisms. Of those, the solid effect (SE) and cross effect (CE) are the two most relevant.

1. The solid effect has been the first known DNP mechanism to occur in electrically non-conductive (dielectric) solids. It is based on the direct excitation of zero and double quantum coherences of an electron and a nuclear spin which are nominally forbidden due to quantum mechanics. Therefore, strong microwave fields are required for their excitation and the efficiency of SE is consequently limited, particularly at high magnetic fields. Nevertheless, the SE is an important DNP mechanism due to its simplicity regarding the required minimal spin system.
2. The cross effect is very efficient and the most commonly evoked DNP mechanism. Here, uniquely tailored polarizing agents are utilized which contain two unpaired electrons (weakly coupled biradicals). If the EPR spectrum of one of the radical moieties is saturated by microwave irradiation, the polarization of the second electron may be transferred onto a nucleus due to magnetic coupling between the electron spins. This is particularly efficient during MAS.

The spin of unpaired electrons serves as the source of the large polarization which is transferred to nuclei during DNP. However, unpaired electrons in molecules are only chemically stable under certain conditions. These may be provided, for example, by sterical hindrance or mesomeric stabilization within persistent radicals, or by the intrinsically higher stability of the deeper lying open electronic shells of transitions metal ions or lanthanoids.

For SE; simple radicals such as trityl (triarylmethyl) or BDPA (1,3-bisdiphenylen-2-phenylallyl) are utilized. Alternatively, metal ion complexes of Gd³⁺ or Mn²⁺ may be used. For CE, nitroxides are tethered to dimers, most commonly on the basis of TEMPO [(2,2,6,6-tetramethylpiperidin-1-yl)oxyl], or a linked with trityl or BDPA to heterodimeric biradicals. Solubility in different environments is achieved by suitable derivatization.

Besides the central MAS-NMR spectrometer, DNP requires additional components: (1) the microwave source, (2) a DNP probe, and (3) a cryo-MAS heat exchanger.

1. A gyrotron is most often used as the microwave source for DNP. In this cyclotron electron beam MASER device, linearly accelerated electrons are injected into a magnetic field whereupon radiation with frequencies between approx. 100 and 1000 GHz may be generated at a power of up to ~100 W. The capability of generating that large microwave power at high frequencies is unique and has revolutionized MAS DNP during the 1990 decade. Once generated, the microwave beam is led to the DNP probe via an overmoded circular waveguide.
2. The DNP probe is based upon a conventional MAS NMR probe but features several necessary modifications. First, a waveguide allows the irradiation of the sample during MAS with microwaves and therefore the induction of a suitable DNP mechanism. Second, thermal insulation and the capability of rapid rotor eject and insert without removal of the probe is required because DNP is typically performed at cryogenic temperatures around –173°C (100 K). The sample cooling freezes molecular motion and thus guarantees a rigid spin network which allows the spreading of enhanced nuclear magnetization via spin diffusion; additionally, slowing of spin relaxation increases the DNP efficiency.
3. The cryo-MAS heat exchanger uses liquid nitrogen at a temperature of 196 °C (77 K) for cooling of gaseous nitrogen which in turn is used as MAS bearing and drive medium. This allows the lowering of the sample temperature down to –173 °C (100 K) and below.

Despite the existing quantum mechanical knowledge for the description of easy spin-systems, the distance prediction, based on polarization transfer via DNP is very difficult. This lays in the variety of different effects that can influence the DNP transfer. The hyperfine coupling between electron- and nuclei-spins enables the transfer of polarization, but at the same time, couplings to other spins can interfere with the main transfer. In bigger systems, this fact leads to a complex scheme of different interactions between electrons and nuclei.

The knowledge about the DNP transfer rate as a function of electron-nuclei-distance and other parameters such as rates of relaxation and spin diffusion can bring an advantage for specific applications. For example, specific electron spin labeling of molecules or complexes could allow efficient distance determination via DNP. Moreover, selective NMR detection of labeled components in a mixture would be possible.  

This protein plays an important role in the decomposition of other proteins while apoptosis. During the protolyses, one or several ubiquitin molecules are bound to the target molecule by a specific covalent binding on the C-terminus of a lysine. This complex is then recognized by the proteasome and degraded. Because of its important function in cell ubiquitin is a very good understood model system that still offers many possibilities for the investigation of proteolysis with DNP and was therefore well-suited for our research. In one of our studies, we selectively changed the amino acid sequence on three different positions for a specific attachment of spin-labels at these postions. These tags were based on the Gd³⁺ chelate-complexes and were covalently bound to the mutation sites in the protein. We moreover could show, that these metal ions could be used as a polarizing agent for site-specific hyperpolarization in the protein.

In our recent work, we look at the influence of the proton concentration in the sample to the direct DNP in Gd-DOTA-ubiquitin. Due to the deuteration of the protein while the recombinant expression a precise variation change of isotope rate between protons and deuterons is possible. Through this variation, the spin-diffusion, as well as the cross-relaxation rate of ¹³C and ¹⁵N, can be affected. This allows us a significant increase in direct DNP enhancement. Further, we could show, that direct DNP can in principle be used for distance measurements between the electron and nuclei. These results will be published soon.

Ribonucleoproteins are complexes consisting of proteins and RNA. One of the most prominent examples of a such system is the ribosome, composed of several RNAs and different protein units and plays an essential role in gene expression. Ribonucleoproteins mostly form very complex structures that take very specific, essential tasks in the cell. To understand the function of these complexes the knowledge of the structure plays a crucial role. The research in this field is highly relevant and has a huge significance in medical studies.

SnoRNAs are non-coding nucleic acids used in the modification of other functional RNA, RNA processing or genetic imprinting. In general, we can differentiate between two types of snoRNA: box H/ACA and box C/D. box H/ACA and box C/D snoRNAs form complexes with several proteins, which allow specific recognition of the target structures. The C/D snoRNA serves as a guide-RNA for the 2’-O-methylation of the ribose, whereas the H/ACA snoRNA is the guide-RNA for rRNA and snRNA during the pseudouridylation.

The box C/D is a complex of two snoRNAs that serve as a complex framework and several proteins (see Figure). The two snoRNAs have complementary base sequences and form base pairs at two different positions called C and D. The box C/D is used for the methylation of guide RNA, which is needed as protection against degradation or modification of catalytically functional RNA molecules. The malfunction of the box C/D, caused by for example mutation is associated with many diseases, such as diabetes or Prader Willie syndrome, which is caused by a gene mutation in chromosome 15.

In cooperation with university Hannover (Alexander Marchanka) our group investigates the possible application of DNP in such complexes. Here, especially the method of SCREAM-DNP seems to be promising.

Dynamic nuclear polarization (DNP) in biomolecular complexes can be very difficult. As DNP is typically performed at cryogenic temperatures, typically around 100 K, the investigated biomolecules have to be cryoprotected from degradation. This is mostly performed by adding glycerol to the sample, whereas it must be ensured that glycerol does not affect the correct folding or the function of the molecule. Moreover, the complexity of biomolecular NMR lays in the spectral signal overlapping as those contain similar structural building blocks such as amino acids in the case of proteins or nucleotides in the case of nucleic acids. Therefore our group focuses on DNP-methods that allow systematic investigation of specific sides in the biomolecule.

Ribonucleic acids (RNA) are not only essential for the translation of the genetic code into proteins, but they are more fulfill a multitude of tasks and functions in our cells. Despite the fact, that RNA constructed just by four building blocks, the nucleotides adenosine, guanosine, cytosine and uridine, the variety of RNA is enormous. In general, RNA can be distinguished in coding and non-coding RNA (ncRNA). While the coding messenger RNA (mRNA) is translated into the protein the ncRNAs perform different regulatory tasks in the cell. Some prominent examples for ncRNA are the ribosomal RNA (rRNA) and the transfer RNA (tRNA), which are needed for gene expression, the micro RNA (miRNA) that plays an important role in the chromatin folding, the small interfering RNA (si RNA) which is necessary for the histone modification, the small nuclear (snRNA) that is involved in the process of splicing in eukaryotic organisms or the so-called small nucleolar RNA (snoRNA), that is crucial for the processing and modification of other ribonucleic acids. The ncRNAs are often found in a complex with proteins, the so called ribonucleoproteins (RNPs). Another type of RNAs are the RNA-aptamers, which are mostly selected by the ability to bind a very specific ligand with high affinity. Those are interesting for applications in the analytical sciences or cancer research.

The TC-aptamer was originally found to investigate the interaction between RNA and the antibiotic tetracycline. But after several modifications, it could be used for the regulation of gene expression or splicing (Suess et al., Nucleic Acids Res. 2003, 31,1853). The antibiotic tetracycline contains three methyl-groups and is bound with a very high affinity by the RNA. As the RNA in its natural form does not contain any methyl groups the TC-aptamer was a perfect system to investigate SCREAM-DNP (see chapter SCREAM-DNP).  To do so tetracycline was labeled with NRM active carbon-nuclei (¹³C) and was used as a methyl-carrying spy molecule. If tetracycline was bound to the RNA characteristic spectral fingerprint of the RNA-aptamer occurred, which could be isolated from the unwanted background signal. By changing the experimental conditions and preventing binding between the RNA and the tetracycline, the specific signals do not occur.

This study was published in 2019 in the Journal Angewandten Chemie (Aladin et al., Angew. Chem. Int. Ed. 2019, 58, 4863), for which Victoria Aladin got the GDCh-Ernst-Award. This study demonstrates the first application of SCREAM-DNP for selective investigation in biomolecular complexes. Because of the very high specificity of this method the complexes could also be investigated beside other components in the sample, as the spectral components can be spectrally isolated from the unwanted background. 

The hammerhead-ribozyme (HHRz) is a structural component of the viral genome in plants. After the successful infection of the host cell, the HHRz catalyzes an enzymatic cleavage reaction, which allows the replication of viral RNA. For this cleavage a specific binding site has to be occupied with divalent ions such as Mg²⁺ or Mn²⁺.  For a long time, it was unknown if the coordination of the divalent ions entails significant structural change until Martick and Scott showed that the coordination of the divalent ion does not affect the crystal structure of the HHRz (Scott et al., Prog. Mol. Biol. Transl. Sci., 2013, 120, 1). Our group could confirm this hypothesis by using DNP enhanced MAS-NMR. As well in the presence as in the absence of  Mg²⁺ the 2D-correlation spectra (¹⁵N-¹³C-TEDOR) show the same tertial contacts between two RNA stem-loops by non-canonical reverse Hoogsteen-basepair. This strongly suggests, that also in solution the specific coordination of Mg²⁺ does not significantly affect the structural population. (Daube et al., Solid State Nucl. Magn. Reson. 2019, 101, 21).

Moreover, we could show that Mn²⁺ could be utilized as polarizing agent for performing site specific DNP. This is highly interesting as it shows that an exogenous polarizing agent, such as nitroxide radical, does not have to be added to the sample solution, as DNP is performed by using the bound metal ion inside the biomolecule as polarizing agent (Wenk et al., J. Biomol. NMR 2015, 63, 97).

NMR is a powerful method for structure determination, unfortunately, it suffers from an intrinsic low sensitivity due to low polarization. This fact leads to the problem that the signal intensity often is not sufficient enough to resolve the investigated structure. The method of dynamic nuclear polarization (DNP) allows inscreasing the sensitivity within several orders of magnitude. But even with DNP the structural investigation of biomolecules can be very challenging, especially when it comes to structural determination in the natural environment, as it is necessary to be able to differentiate between the biomolecule of interest and other cellular components.

To solve this problem our group is using a new method the so-called SCREAM-DNP (Specific Cross Relaxation Enhancement by Active Motions). In this method, we use methyl-carrying “spy-molecules” that can specifically polarize biomolecules if those are in close proximity to the molecule interest. This can be achieved if the methyl-carrying molecule is specifically bound by the protein- or RNA-molecule of interest.  We could show, that this method can be used for proof of binding between the “spy-molecule” and the biomolecule, moreover SCREAM-DNP can also serve as a spectral filter for components that do not interact with the spy-molecule.

In general, the polarization transfer from the electron spin to the nuclear spin can occur either through direct or indirect polarization transfer. In the case of direct polarization (Figure (A) blue arrows) the polarization is directly transferred from the electron to the nuclei of interest (in this example to the carbon nuclei). In the case of indirect polarization, the transfer happens with an intermediate step, where the polarization is first transferred to protons and only afterward to the nuclei of interest (Figure (A) red arrows). If the sample contains mobile methyl-groups, both polarization pathways can occur simultaneously. This effect is caused by the very fast reorientation dynamics of methyl-groups (Nuclear Overhauser Effect). In such a case, the direct pathway results in positive and indirect transfer in negative signals. This means that the resulting spectra contain negative as well as positive spectral intensities (Figure (B),DP, lilac). To separate the negative and positive compound of the spectra and isolate just the information caused by the methyl-groups, which would allow the investigation of the near surrounding of the methyl-group, another experiment has to be performed where the use of saturation pulses (Figure (A) τ_sat ) allows to suppressing the indirect pathway through protons. The resulting spectra contain only positive signals caused by direct polarization (Figure (B), DP_sat, blue). Mathematical subtraction of DP and DP_sat leads the spectral contribution from the indirect pathway caused due to the fast reorientation dynamics of methyl groups (Figure (B), ΔDP_sat ). This spectrum than contains information about the surrounding of the methyl-groups. Figure (C) shows schematic representation of the direct and indirect pathway on the example of glycine, which does not have any methyl-groups and alanine, which has exactly one methyl group and that shows significant SCREAM-activity.