Real-time NMR spectroscopy in the study of biomolecular kinetics and dynamics

The review describes the application of NMR spectroscopy to study kinetics of folding, refolding and aggregation of proteins, RNA and DNA. Time-resolved NMR experiments can be conducted in a reversible or an irreversible manner. In particular irreversible folding experiments pose large requirements on (i) the signal-to-noise due to the time limitations and (ii) on synchronizing the refolding steps. Thus, this contribution discusses the application of methods for signal-to-noise increases including dynamic nuclear polarization, hyperpolarization and photo-CIDNP for the study of time-resolved NMR 15 studies. Further, methods are reviewed ranging from pressureand temperature-jump, light induction and rapid mixing to induce rapidly non-equilibrium conditions required to initiate folding.


Introduction
In 1993, the journal Current Opinion in Structural Biology published a special edition on protein-nucleic acid interactions edited by D. Moras and S. Philipps and on protein folding edited by C. Dobson. The edition featured an editorial article by 20 Phillips and Moras (1993) on protein-nucleic acid interactions, and reviews on transcription factor structure and DNA binding by Wolberger (1993), zinc-finger proteins by Berg (1993), DNA repair enzymes by Morikawa (1993), restriction endonucleases and modification methylases by Anderson (1993), DNA-and RNA-dependent DNA polymerases by Steitz (1993), aminoacyl-tRNA synthetases by Cusack (1993), work on ribosomes by Yonath and Franceschi (1993) and contributions by Robert (Rob) Kaptein on "protein-nucleic acid interaction by NMR" (Kaptein, 1993). This first part on 25 protein-nucleic acids complexes was accompanied by a second part, introduced in the editorial article by Chris Dobson (1993), on protein folding with contributions by Dyson and Wright (1993) on peptide conformation and protein folding, on denatured states of proteins by Shortle (1993), on principles of protein stability by Fersht and Serrano (1993), H/D exchange experiments by Baldwin (1993), molecular simulation of peptide and protein folding by Brooks (1993), on protein folding by Dill (1993), accessory protein in protein folding by Jaenicke (1993), and antibody-antigen interaction by Wilson and 30  Biomacromolecules can be unfolded in many different ways (Fürtig et al., 2007a;Roder et al., 2004). Proteins can be chemically denatured using high concentrations (6-8 M) of guanidinium chloride (GdnCl) (Logan et al., 1994;Zeeb and Balbach, 2004) or urea (Egan et al., 1993;Neri et al., 1992;Schwalbe et al., 1997), but also organic solvents including TFE or DMSO (Buck, 1998;Buck et al., 1995Buck et al., , 1993Nishimura et al., 2005). The (re-)folding of chemically denatured proteins 130 can then be initiated with a rapid dilution into native buffer conditions (Balbach et al., 1995) or vice versa for unfolding of native proteins (Kiefhaber et al., 1995). Alternatively, a rapid pH-change can be used to re-or de-nature proteins (Balbach et al., 1996;Corazza et al., 2010;Dobson and Hore, 1998a;Schanda et al., 2007;Zeeb and Balbach, 2004). The rapid mixing design introduced by Mok et al. (Mok et al., 2003b) has also found widespread application for studies on folding of nucleic acids (see below). 135

Pressure jump
Pressure is a physical state parameter that can influence the conformation of biomolecules. High pressure can denature proteins. This process is usually reversible and upon release of pressure the protein folds back to its native state. The required pressure can be adjusted by use of chaotropic agents to lower the overall stability, or by introducing specific mutations to introduce internal cavities in the folded structure (Bouvignies et al., 2011;Mulder et al., 2001). Static high 140 pressure NMR spectroscopy is a long established method to assess the thermodynamic profile of proteins (Balbach et al., 2019), and allows detailed thermodynamic characterization of the energy landscape (Akasaka et al., 2013;Roche et al., 2019). There are further reviews about equilibrium high-pressure measurements as reviewed in (Caro and Wand, 2018;Nguyen and Roche, 2017;Roche et al., 2017). Here, we focus on more recent developments regarding the rapid change of pressure inside the spectrometer to study kinetics of biomolecules, especially protein folding kinetics. High pressure NMR measurements require a special NMR tube (made from either quartz, sapphire or zirconia) that can withstand high pressures up to a few thousand bar. The most often used ones are made from zirconium oxide, as they are commercially available and are specified up to a maximum 3000 bar (Daedalus Innovations LLC). The pressure is usually realized by use of an external hydrostatic pressure pump connected via pressure withstanding tubing to the sample inside the spectrometer. Usually, mineral oils are used to transmit the pressure providing phase separation from the typical water 150 samples. The up to now most advanced setup was developed by (Charlier et al., 2018a) and the schematic is show in Figure   2. The system uses a high pressure and an atmospheric pressure reservoir, connected through a hydraulic valve to the sample.
By opening the valve from the high pressure reservoir, the pressure is rapidly equilibrated in the sample to the high pressure, while the pressure rapidly drops in the sample by changing to the low pressure reservoir. The oil reservoir is kept in a close system under nitrogen atmosphere to avoid oxidation. 155 Figure 2: Schematic representation of the rapid pressure-jump NMR apparatus, developed by (Charlier et al., 2018a). The apparatus is mounted onto a frame with pneumatic lift (light gray) to adjust the height to any spectrometer. The pressure apparatus is a closed system under N 2 atmosphere and uses a high-pressure and atmospheric pressure reservoir to increase or to decrease the pressure, correspondingly inside the sample stored in a Zirconia tube inside the spectrometer. Reprinted with 160 permission from (Charlier et al., 2018a).
The major advantage of pressure jump compared to other methods to study protein folding and energy landscape lies in the reversibility of the induced conformational transition. In combination with the shortest time (1-5 ms) requirement to change between folding and unfolding conditions this method allows complex NMR experiment designs to study in details the folding pathways, mechanism and even the structure of intermediates. 165

Light induction
Folding can be initiated photochemically by irradiation within the NMR spectrometer. Laser irradiation of biomolecules within the NMR-spectrometers has been introduced by Kaptein in photo-CIDNP NMR (Kaptein et al., 1978), before first https: //doi.org/10.5194/mr-2021-16 Discussions Open Access Preprint. Discussion started: 10 February 2021 c Author(s) 2021. CC BY 4.0 License. real-time folding applications have been conducted. In folding applications, high-power laser irradiation (up to 8-10 W primary output) is coupled to the NMR-spectrometer by a quartz fibre ending in the NMR-tube within the spectrometer. 170 Figure 3 shows the setup of two lasers coupled to an NMR spectrometer as it is used at BMRZ in Frankfurt. To achieve reasonable irradiation times (typically between 0.2-4s) depending on the folding rate to be observed, not only the applied power is important but also the homogeneous light illumination within the sample. Different methods that advance the setup presented by Berliner (Scheffler et al., 1985), such as using a cone shaped quartz tip (Kühn and Schwalbe, 2000) or stepwise tapered (Kuprov and Hore, 2004) or sandblasted quartz fibre ends (Feldmeier et al., 2013) have been introduced to achieve 175 this. Most importantly, the approach relies on the presence of a chromophore within the NMR sample. This can either be a biomolecule carrying a photosensitive group such as the yellow protein (Derix et al., 2003), or folding can be initiated by release of cofactors from photo-labile chelators (Kühn and Schwalbe, 2000), from caged ligands (Buck et al., 2007) or from photo-labile precursors that cage biomolecular conformation (Wenter et al., 2005). 185

Temperature jump
Next to pressure, temperature is the second thermodynamic state parameter. It is coupled to the free enthalpy of a conformational equilibrium. Thus, the change of temperature was one of the first methods to initiate changes in biomolecular systems and ultrafast T-jump experiments have been introduced by M. Eigen and awarded with the Nobel prize in Chemistry in 1967. The first application of temperature-jump in combination with NMR spectroscopy was the study of proline cis-trans 190 isomerization in oligopeptides as an alternative to the jump in pH(D) by Wüthrich (Grathwohl and Wüthrich, 1981). Additionally, temperature changes can initiate refolding of biomolecules exhibiting temperature-dependent conformations (Reining et al., 2013;Rinnenthal et al., 2010), denature proteins at high temperature, or refold cold-denatured proteins. Later, different technical setups were developed to speed up the temperature change to study dynamic changes with faster 200 reaction rates. A list of the different techniques to achieve a temperature jump is given in Table 1. In this table, important parameters are described for each technique. Out of all the different T-jump techniques, microwave (MW) and radiofrequency (RF) heating proved to be the most suitable for biomolecular NMR. In both cases, inductive and dielectric heating effects take place, the latter is the major factor and couples well to lossy samples (salt-containing aqueous samples). RF heating allows easy coupling to the spectrometer, due to built-in RF generators and amplifier system. It can reach relatively 205 fast heating with 20 K/sec, although slower than MW setups, but offers a more homogeneous heating profile which is required for high resolution NMR spectroscopy. The latest RF heating setup, shown in Fig. 4, described in the literature (Rinnenthal et al., 2015) uses a built-in RF coil to 210 initiate the jump with an additional optimized gas-heating to stabilize final temperature. The range of the temperature jump can be adjusted by the number of heating RF pulses applied, and for longer measurements, the gas-heating provides stability at the final temperature. The suitability of this setup to study folding mechanism of proteins has been demonstrated on the cold-denatured barstar, where the temperature jump initiated the complete reversible refolding of the protein.

General pulse scheme for RT-NMR 215
In the context of real-time NMR, a number of aspects within pulse sequences has to be conceptualized. Firstly, the timing of NMR excitation, synchronous triggering of folding, and the correlation of NMR coherences or polarizations have to be designed (shown in Fig. 5). Secondly, the best excitation pulses and detection schemes have to be applied. The pulse sequences used to measure time-resolved NMR experiments depend on the trigger and on the system under study. These can be divided into two major groups: non-reversible ( Fig. 5a) or reversible systems (Fig. 5b-c). In both cases, before initiating 220 the kinetic experiment, reference spectra are recorded. For non-reversible systems, the basic scheme is a simple trigger after which a series of 1D-NMR spectra is recorded, allowing the best time resolution. While two-dimensional experiments can provide higher chemical shift resolution, they can only be utilized for slow kinetic measurements. Depending on the timescale of the observed kinetics, 15 N/ 13 C-1 H correlation spectra can be measured. Modifications to these experiments can HMQC techniques (Favier and Brutscher, 2011; with longitudinal relaxation optimization (Farjon et al., 2009), Hadamard frequency encoding  or ultrafast approaches (Gal et al., 2007) or in case of NOESY experiments Looped-PROjected SpectroscopY (L-PROSY) (Novakovic et al., 2020a(Novakovic et al., , 2018. Furthermore, several of the pulse sequences can be combined with non-uniform sampling (NUS) (Gołowicz et al., 2020) to further reduce the final measurement time up to a few seconds. 230 For reversible systems, two dimensional experiments can be recorded with the same time resolution as 1D-NMR experiments. To achieve this, only one increment of the indirect dimension is recorded in every kinetic experiment and time incrementation in the indirect dimension is achieved after each new folding trigger. Repeating the experiments and measuring the required indirect dimension points and finally concatenating together the corresponding time points, kinetic measurements with high temporal and spectral resolution can be achieved (Alderson et al., 2017;Harper et al., 2004;Kremer 235 et al., 2011;Naito et al., 1990).  It is noteworthy that reversible systems in combination with magnetization transfer between starting state one (at the start) and state two (new equilibrium) allow recording of a so called state-correlated (SC) spectrum. In SC-spectrum, the pulse sequence starts with magnetization transfer to other nuclei before the actual physical parameter (light, pressure or temperature) is changed. The final detection takes place already in the new equilibrium state. The limiting factor for such 245 application is the speed of the physical parameter change, as it has to be faster than the T 1 relaxation time. Additionally, double jump experiments can also be utilized to observe alternative folding pathways if rapid changes between state one and state two in both directions are possible (Charlier et al., 2018c;Kremer et al., 2011;Pintér and Schwalbe, 2020).

Signal enhancement
It is beyond the scope of this article to cover the physical principles of the plethora of signal-to-noise enhancement 250 approaches in NMR spectroscopy. Time-resolved experiments, however, provide very stringent requirements on signal-tonoise as the kinetics of structural transitions in biomolecular folding define the time window that is available. In the following, we will discuss the coupling of time-resolved experiments to dynamic nuclear polarization (DNP) in solid-state NMR (Becerra et al., 1993;Corzilius, 2020), to hyperpolarization (Ardenkjaer-Larsen et al., 2003;Ragavan et al., 2011) and to photo-CIDNP experiments in liquid-state NMR. 255

DNP
Dynamic nuclear polarization describes a set of mechanisms by which nuclear non-Boltzmann magnetization is created (for a recent review see (Corzilius, 2020)). So far, it works best for solid-state NMR and especially polarisation transfer via the cross-effect using biradicals as polarisers has progressed from the proof-of-concept stage to numerous applications.
Enhancements of up to 150-fold have been reported on complex systems such as GPCRs (Joedicke et al., 2018) or ribosome 260 nascent chain complexes (Schulte et al., 2020). This significant boost in sensitivity can be highly beneficial for real-time NMR applications. However, a sufficiently long electron relaxation for an efficient polarisation transfer to the nuclei is required, which has to be obtained under cryogenic conditions. Therefore, DNP-enhanced solid-state NMR experiments are usually performed around 100 K or even below making them less compatible with real-time studies but offering great opportunities for cryotrapping of intermediate protein states. First attempts, initially without the help of DNP, date back to 265 1994 (Ramilo et al., 1994). Recently, remarkable progress has been achieved by combining rapid mixing/freeze quenching with DNP by which a time-resolution in the ms-range could be achieved (Jeon et al., 2019). Another possibility is cryotrapping of light-induced photoreceptor intermediates under DNP conditions, which is briefly addressed below.

Dissolution-DNP -Hyperpolarization
The application of DNP enhancement schemes employing direct microwave irradiation and use of polarizing agents is 270 limited in liquid state NMR. Due to the large dielectric losses in water, the sample volume is usually limited to nanolitre range. However, polarization of the solvent in solid-state followed by rapid heating and injection e.g. of polarized solvent can be utilized for signal enhancement in real-time NMR measurements of biomolecules. It has been first demonstrated by Ardenkjaer-Larsen (Ardenkjaer-Larsen et al., 2003) and used for example to enhance the sensitivity of 13 C detected in vivo metabolic MRI (Kurhanewicz et al., 2019) and later developed by the Frydman group for biomolecules. 275 The solvent water, in a pellet form, is hyperpolarized at low temperature in a hyperpolarizer. The pellet is heated to liquid state by flushing with hot solvent. The commercially available Hypersense (Oxford Instruments Plc) instrument uses 3-4 ml of hot water while the Frydman group has developed an elegant method to reduce the dissolution factor that also extracts the radical agent, using hot non-polar solvents (Harris et al., 2011). The latter has the advantage of removing the polarizing This method has been demonstrated on the intrinsically disordered protein p27 Kip1 , which refolds upon interaction with Cdk2/cyclin (Ragavan et al., 2017). Direct polarization of p27 Kip1 in deuterated form was used to increase T 1 relaxation time was.
The group of Hilty was the first to demonstrate the applicability of dissolution-DNP to folding experiments (Chen et al., 285 2013). In this application, the ribosomal protein L23 was hyperpolarized and subsequently injected to folding buffer of higher pH and folding is monitored by 13 C 1D spectra.
In a more recent application, Novakovic et al. used dissolution-DNP to monitor the real-time refolding of the RNA aptamer domain of guanine-sensing riboswitch (GSW) upon ligand binding (Novakovic et al., 2020b). Different to proteins, the exchange of solvent water with imino sites in RNAs is sufficiently fast not only in unstructured but also in structured regions 290 of the RNA, and thus, all RNA sites can benefit from increase signal-to-noise due to exchange transfer. In this paper, the GSW specific ligand (hypoxanthine) was injected parallel with the hyperpolarized solvent water to the RNA sample. Coinjection of hyperpolarized water and refolding initiating ligand inducd refolding of the aptamer, observable with an almost 300-fold imino signal enhancement. The obtained signal enhancement is sufficient to even record series of 2D-NMR spectrum using selective HMQC pulse sequence. 295

Photo-CIDNP
Beside microwave-driven DNP, Chemically Induced Dynamic Nuclear Polarization (CIDNP) is a method that can selectively enhance the signal-to-noise of NMR signals. Especially photo-CIDNP offers the possibility to probe the solvent accessibility of amino acids in proteins and peptides. During protein folding and refolding, the solvent accessible area of a protein is changing due to hydrophobic collapse that rearranges the conformation of solvent exposed amino acids. This 300 probing of differential accessibility makes photo-CIDNP a powerful tool for the (real-time) investigation of protein folding (Hore and Broadhurst, 1993;Kuhn, 2013;Morozova and Ivanov, 2019).
The positive or negative signal enhancement is based on a chemical reaction between a laser-induced excited photosensitizer and a CIDNP-active aromatic group. The radical pair mechanism explains the photo-CINDP effect with an excited photosensitizer that is present in the triplet state, after intersystem crossing, accepting an electron or hydrogen from a donor 305 molecule. A radical ion pair is formed and the singlet recombination probability of this pair is influenced by the hyperfine coupling constants of the present magnetic field (Hore and Broadhurst, 1993). The hyperfine coupling leads to differences in the population of the nuclear spin energy levels and therefore emissive or absorptive NMR signals, predictable by Kaptein's rule (Kaptein, 1971). The setting of a basic photo-CIDNP pulse sequence is quite uncomplicated. After an optional presaturation, the sample is illuminated for short time controlled by a mechanical shutter followed by the desired 310 experimental pulse sequence. Alternating light (with laser irradiation) and dark (without irradiation) spectra are recorded and subtracted to obtain the difference spectra with the enhanced signals (Hore and Broadhurst, 1993). Several amino acids exhibit polarization by photo-CIDNP in solution, these are tryptophan, tyrosine, histidine, and also methionine, glycine and methylcysteine, although to different extent (Stob and Kaptein, 1989;Morozova et al., 2005;Morozova and Yurkovskaya, 2008;Morozova et al., 2016). Embedded in a protein or peptide, Trp, Tyr, His and Met show signal enhancement if they are 315 accessible to a photosensitizer (Kaptein et al., 1978;Hore and Broadhurst, 1993). For the photo-CIDNP reaction different dyes as photosensitizer can be used, the most common ones are substituted flavins, 2,2-dipyridyl (DP), 4-carboxybenzophenone (4-CBP), and 3,3',4,4'-tetracarboxybenzophenone (TCBP) (Morozova and Ivanov, 2019).
The first presented photo-CIDNP studies on proteins were published by Kaptein et al. (1978) on bovine pancreatic trypsin inhibitor (BPTI). The investigation of BPTI by photo-CIDNP showed that the enhanced signals of tyrosine residues were in 320 line with ones exposed on the surface in the crystal structure. In the following years, the time-resolved investigation of the kinetics of folding or refolding proteins with photo-CIDNP should become more and more important. This is also due to the fact that time-resolved photo-CIDNP studies have a better time resolution than other NMR techniques, because of the laserinduced generation of nuclear polarization. The repetition rate is determined on electron relaxation, not nuclear relaxation rates. (Day et al., 2009;Kuhn, 2013). Also the small chemical shift resolution in non-native or not folded states of proteins 325 can be overcome due to the fact that only solvent accessible amino acids are photo-CIDNP sensitive and the investigation of unfolded or partially folded structures at a residue-specific level is possible (Schlörb et al., 2006). During folding, several conformational states including random coil, molten globule states as well as folding intermediates, non-native states, partially folded states and native states can be characterized in a residue specific manner (Kuhn, 2013).
Beside amino acids, also DNA and RNA mononucleotides are CINDP-active including guanosine, adenosine and thymidine 330 Pouwels et al., 1994). With a self-complementary tetramer it was shown that photo-CIDNP can only be detected in single stranded regions, when the nucleobase is accessible to the solvent and the photosensitizer (McCord et al., 1984a). photo-CIDNP-studies on tRNA were the first investigations of such kind on larger nucleic acids. Temperaturedependent photo-CIDNP experiments showed changes consistent to the melting of tertiary and secondary structures (McCord et al., 1984b). Therefore, photo-CIDNP can also be a powerful tool for the characterization of the accessibility of 335 nucleobases in RNA or DNA that play key roles in RNA-protein binding sites.

Overview of light and rapid mixing applications
Folding induction using rapid mixing approaches coupled to NMR is universally applicable and the installation of the rapid mixing apparatus is not costly. Experimental challenges, including deterioration of NMR spectra, remain, however. The coupling of laser irradiation to trigger biomacromoleular folding reactions is conceptually more elegant but puts more 340 stringent requirements to the systems under study. Advantages are obvious: the dead time of folding induction is no longer determined by built-up of NMR homogeneity and absence of flow-related susceptibility inhomogeneities across the sample, but homogeneous illumination that depends on photophysical properties, including concentration-dependent extinction coefficients. Triggering times can thus be shortened as signal-to-noise is increased. A number of biomacromolecules carry an Other systems can be modified by photo-active non-natural groups or, alternatively cofactors or ligands can be masked by a photolabile group. These applications are discussed in chapter 4.2 along with applications of rapid mixing as the two methods complement each other on some of the systems investigated.

Photo-active yellow protein
The photo-active yellow protein (PYP) from the Ectothiorhodospira halophile shows negative phototactic response towards intense blue light (Sprenger et al., 1993). Light excitation of PYP induces a photocycle during which PYP undergoes structural and dynamic changes. The photocycle is defined by three states, the ground state pG, the red-shifted intermediate pR and the long-lived blue-shifted intermediate pB. The photocylce is coupled to the formation of a covalently bound thiol 355 ester-linked chromophoric group, the p-coumarin acid, which undergoes cis-trans isomerization upon light excitation (Hoff et al., 1994). Early on, Kaptein realized that due to the small size of the protein (14-kDa) and the chromophore, PYP is an excellent model system for the investigation of the processes occurring during photoreception in solution at atomic resolution. Kaptein's group thus investigated the blue-shifted intermediate pB of the photocycle of PYP Rubinstenn et al., 1999Rubinstenn et al., , 1998 (Rubinstenn et al., 1999) experiment as a further development of the previously introduced 1 H NMR SCOTCH experiment (Kemmink et al., 1986b). With this, the long-lived intermediate pB populated on the 365 photocycle could be generated by light and its resonances could be correlated to the pG state as an early example of a statecorrelated 2D NMR experiment. In the SCOTCH experiment, the 15 N chemical shift of pG is correlated with 1 H chemical shift of the 15 N attached proton of pB ( Fig. 6 B,C). The experiments revealed that pB is structurally disordered in solution populating an ensemble of conformers in exchange on a millisecond timescale. This finding was in contrast to previous crystal structures that showed a single structure for state pB with just minor changes around the chromophore (Genick et al., 370 1997). Further investigations of PYP in solution with hydrogen-deuterium exchange, pH studies, a mutant lacking negative charge and an N-terminally truncated variant revealed more detailed information about the protein and its photo active cofactor (Bernard et al., 2005;Craven et al., 2000;Derix et al., 2003).

BLUF domain
Proteins containing a BLUF (sensors of blue-light using flavin adenine dinucleotide) domain are another representative for proteins reacting to light. The BLUF domain is a FAD-binding domain and was found in various proteins, mainly present in 380 proteobacteria, cyanobacteria and a few eukaryotic organism (Gomelsky and Klug, 2002). In comparison to PYP, the chromophore in BLUF domains is not covalently bound (Wu and Gardner, 2009). The proteins detect blue light using their chromophore, followed by a reversible red shift and formation of a photo-activated conformation, the signalling state that decays spontaneously to the ground state if the system returns to a dark environment (Zirak et al., 2006). Illumination induces different structural and functional outputs as regulation of catalytic activity of enzymes and second messengers, 385 photophobic responses and expression control of photosynthetic genes (Gomelsky and Klug, 2002). Beside other methods as time-resolved fluorescence or absorption spectroscopy, NMR coupled with light is a powerful tool for the investigation of the photoreaction mechanisms in BLUF domains after light irradiation at atomic resolution. The best characterized BLUF photoreactions analysed by NMR are the ones of AppA (Gauden et al., 2007;Grinstead et al., 2006aGrinstead et al., , 2006b, and BlrB (Jung et al., 2005;Wu et al., 2008) (from Rhodobacter sphaeroides), BlrP1 (from Klebsiella pneumonia) (Wu and Gardner, 2009) 390 and YcgF (from Escherichia coli) (Schroeder et al., 2008). Kaptein and his co-workers studied the AppA BLUF domain and presented a solution structure as well as evidence for structural changes in the light-induced state, e.g. for surface residues and the flipping of a glutamine side chains followed by the formation of a hydrogens bond (Grinstead et al., 2006b(Grinstead et al., , 2006a. By mutation of aromatic amino acids that are in short distance to the FAD cofactor, they were able to gain more information about the electron-transfer pathways in BLUF domains (Gauden et al., 2007). With NMR under light and dark conditions, 395 Gardner et al. observed structural changes in BlrB for amino acids near the flavin-binding pocket but also more than 1.5 nanometer apart. This finding indicates that the light-induced signal is propagated from the flavin through the protein resulting in the initiation of the regulatory function (Wu et al., 2008). Together with the Essen group in Marburg, Schwalbe and coworkers investigated YcgF from E. coli (reconstituted with FMN and FAD) and in comparison to HSQC spectra of AppA and BlrB, much stronger chemical shift perturbations were observed upon light excitation (Fig. 7A) (Schroeder et al., 400 2008). Furthermore, they recorded kinetics for the dark state recovery of YcgF by proton NMR spectroscopy in a temperature-dependent manner. Additionally, 31 P kinetic measurements were performed to investigate the kinetic behavior of the chromophore signals upon illumination (Fig. 7B-C). The results combined with UV/Vis spectroscopy show a heterogeneous distribution of half-live times for the light to dark conversion, suggesting hysteresis effects.  -Jurkauskas et al., 2008;Ni et al., 2018). From a technical point of view, the NMRspectroscopic time-resolved investigation of the photocycle of the eukaryotic, visual bovine rhodopsin, the mammalian visual dim-light G-protein coupled photoreceptor, represents one of the most challenging biophysical studies to characterize key intermediates and kinetics of its photocycle as its photocycle is irreversible, highly light sensitive and the spectra of 415 functional rhodopsin of extremely low signal-to-noise. Such functional rhodopsin can only be prepared using eukaryotic expression systems (Reeves et al., 2002).
Opsin, the apo-protein, is covalently attached to the chromophore 11-cis-retinal through Schiff base formation. Upon photon absorption, the retinal undergoes a E/Z isomerization of the cis-configured double bond to all-trans-retinal. Thus, isomerization induces conformational transitions and population of several high-energy photocycle intermediates, whose 420 decay leads to the formation of the meta II signal state. The aim of our time-resolved studies was the characterization of the decay kinetics of this meta-II state resolved on individual amino acid reporter signals. For this, we could assign the 1 H, 15 N tryptophan side chain indole resonances using selectively 15 N-labelled tryptophan expressed in HEK293 cells  and we also pursed the attachment of fluorinated reported groups to cysteine thiol groups to utilize 19 F-NMR to follow these conformational changes in a time-resolved manner (Loewen et al., 2001). After in-situ illumination of the dark state of 425 rhodopsin, we could detect the NMR signals in the meta-II state in 2D correlation spectra and could analyse the decay kinetics of meta-II. These kinetics are bifurcated: next to the known formation of opsin, we could show the meta-III state to be populated (Fig. 8). This meta-III-state is not signalling active but considered as storage system (Stehle et al., 2014).

435
This finding is important, as continued activation of the photocycle of rhodopsin leads to the accumulation of all-trans-retinal in the rod outer segments (ROS). For retinal homeostasis, deactivation processes are required to delay the release of retinal.
Bovine visual arrestin (Arr(Tr)) has been previously proposed to play a key role in the deactivation process and in fact, timeresolved NMR together with optical spectroscopy conducted by the group of Wachtveitl could show that formation of the rhodopsin-arrestin complex markedly influences partitioning in the decay kinetics of rhodopsin. Binding of Arr(Tr) leads to 440 an increase in the population of the meta III state that is simultaneously formed with meta II from meta I (Chatterjee et al., 2015). We further studied the retinal-disease-relevant G90D bovine rhodopsin mutant by time-resolved liquid-state and DNP-enhanced solid-state NMR with the group of Glaubitz as well as by advanced optical spectroscopy with the group of Wachtveitl (Kubatova et al., 2020). The G90D mutation is one of numerous mutations that impair the visual cycle of the mammalian dim-light photoreceptor rhodopsin; it is a constitutively active mutant form that causes CSNB disease. Different 445 to previous crystallographic reports, we could detect two long-lived dark states, both of which contain the retinal in 11-cis configuration. By studying the photocycle with DNP-enhanced solid-state NMR, we could detect the dark state, the bathorhodopsin and the meta-II state and could show that all these states retain their conformational heterogeneity. This conformational heterogeneity is linked to a substantially altered photocycle as shown by optical spectroscopy. photoreceptors such as rhodopsins offers insight to link their 3D structures with their photochemical properties. Typical readout parameters are isotropic and anisotropic chemical shifts, homo-and heteronuclear dipole couplings or torsion angles by which finest alterations within the chromophores during the photocycle could be detected (Becker-Baldus et al., 2015;Carravetta et al., 2004;Concistrè et al., 2008). The use of DNP in these systems was demonstrated for bacteriorhodopsin (Bajaj et al., 2009) and the discovery of many new rhodopsins inspired a series of new experiments covering the marine 455 photoreceptor proteorhodopsin (Mehler et al., 2017), the light-gated ion channel channelrhodpsin-2 1 or the light-driven Napump KR2 2 (Jakdetchai et al., 2021). In order to trap a desired photointermediate for DNP solid-state NMR analysis, an optimized MAS-NMR setup is needed allowing simultaneous sample illumination by light with the desired wavelength as well as microwave irradiation (Fig. 9a). Furthermore, a suitable cryotrapping protocol has to be established, which depends   We investigated the Ca 2+ -dependent transition from the unfolded to the folded state of BLA in the presence of urea at neutral pH using photochemical triggering by releasing Ca 2+ from the photolabile chelator DM-Nitrophen Schwalbe, 2000, 2000;Schlepckow et al., 2008) using laser irradiation. We could show that folding under these conditions proceeds via parallel folding pathways (Schlepckow et al., 2008). Coupling of light-induced folding and photo-CIDNP using two lasers 495 coupled into one quarzfiber allowed us to characterize a folding intermediate with a non-native environment that is populated already after 200 ms and has disappears again after 1.5s (Wirmer et al., 2001) see Figure 10.  Balbach et al. (1995) showed as early as 1995 that a folding intermediate of refolding from the GdnCl-unfolded state of lactalbumin by rapid dilution resembles the molten globule of the protein by comparison of kinetic and static 1D spectra. In 505 the second study (Balbach et al., 1996), they investigated the cooperative nature of folding from the molten globule state to the native state by raising the pH during mixing in the absence of denaturant. They extracted kinetic rates on a per residue basis from one HSQC-spectrum recorded during folding by simulating the observed line shapes. Schanda et al. directly measured folding rates of this folding process by implementing fluid turbulence-adapted SOFAST-HMQC measurements, which allowed them to record HMQC spectra every 10.9 s during the folding process . They observed 510 uniform mono-exponential folding rates throughout the molecule confirming the presence of a single transition state.

(a) An experimental setup is required which allows simultaneous light and microwave irradiation under MAS-NMR conditions at low temperatures. (b) The pentameric proton pump proteorhodopsin undergoes a photocycle with a number of distinct intermediate states, which can be trapped for DNP solid-sate NMR (Mehler et al., 2017). (c) Upon retinal isomerisation and Schiff base deprotonation, two distinct M-states form as shown here by a NC-TEDOR spectrum. (d) In the M-state, the tautomeric and rotameric state of H75, which forms a functionally important triad with proton acceptor D97 and W34 across the
Other proteins that were investigated using folding initiation by mixing and detection using 2D and 3D NMR spectroscopy include S54G/P55N ribonuclease T 1 (Haupt et al., 2011) and  2 -microglobulin (B2M) (Franco et al., 2017b;Rennella et al.,  The rapid mixing technology used for the investigation of the proteins mentioned above can be combined with photo-520 CIDNP: refolding of hen egg white lysozyme (HEWL) (Dobson and Hore, 1998b;Hore et al., 1997), the histidinecontaining phosphocarrier protein HPr (Canet et al., 2003), and ribonuclease A (Day et al., 2009) have been studied.
The concept of photochemical triggering of biochemical processes by light is also appealing to solid-state NMR. So far, only few examples have been reported in which reactions catalysed by membrane proteins have been followed by real-time MAS NMR spectroscopy (Kaur et al., 2016;Ullrich et al., 2011). Such studies are challenging since the nature of MAS-NMR 525 makes the samples sealed within a MAS rotor inaccessible during the experiment preventing titration of reagents. The reaction can therefore only be triggered for example by a T-jump on a sample mixture stored in a pre-cooled MAS rotor.
However, the feasibility to release substrates protected by photolabile groups directly during the MAS NMR experiment followed in situ illumination has been recently demonstrated for the first time (de Mos et al., 2020): The E. coli lipid regulator diacylglycerolkinase phosphorylates its lipid substrate diacylglycerol under ATP consumption. It was possible to 530 demonstrate that both ATP as well as the lipid substrate protected by an NPE-group can be uncaged directly under MAS-NMR triggering DGK's enzymatic activity, which could be followed by 31 P detection (Fig. 11).  While the previous examples for protein folding use cofactors or internal chromophores for the initiation of folding, site 540 specific photoprotection of amino acids has not been used in time resolved NMR, yet. A breakthrough for the site-specific labeling of proteins for NMR was the incorporation of unnatural amino acids in vivo. By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair unnatural amino acids can be integrated in proteins in response to a TAG amber frame shift codon . These side-specific incorporated unnatural amino acids are attractive for the investigation of ligand binding or protein folding in vitro and in vivo (Jones et al., 2009). Especially for NMR, isotope 545 labeled ( 19 F, 13 C, 15 N (Cellitti et al., 2008;Hammill et al., 2007;Jackson et al., 2007) photo-caged, spin-labeled and metal chelating (Lee et al., 2009;Otting, 2008;Xie et al., 2007) unnatural amino acids are of high interest. Here, we will focus on the photo-caged amino acids, represented e.g. by o-nritobenzyl (o-NB) caged tyrosine (Deiters et al., 2006), cysteine (Wu et al., 2004), lysine (Chen et al., 2009), the 4,5-dimethoxy-2-nitrobenzyl caged serine (DMNB) (Lemke et al., 2007) (Cellitti et al., 2008). After cleavage of the photo-cage via UV light binding was reestablished demonstrating that site-specific labeling via photo cages can be achieved without 555 modifying the protein sequence, but with the possibility to inhibit and regenerate the function of the natural amino acid after cleavage. Another example for inactivation of function is the use of an o-NB caged cysteine at the active site of a proapoptotic cysteine protease caspase-3. After cleavage the natural amino acid is obtained and 40% of its activity is restored (Wu et al., 2004). Photo-caged amino acids can also be used to allow for selective covalent modifications in proteins after cleavage of the cage. With site specific incorporation of a photo-caged selenocysteine and following uncaging, it is possible 560 to site-specific modify these due to their higher reactivity in comparison to competing cysteine residues (Welegedara et al., 2018).

RNA
In time resolved NMR studies characterising the folding or refolding of RNAs, two main strategies for the utilization of photo-caged compounds can be employed. Either the RNA itself or a folding-inducing ligand can be modified (Fürtig et al., 565 2007a). Folding-inducing ligands include high affinity, low molecular-weight ligands, e.g. in riboswitch folding, or divalent ions, in particular Mg 2+ . If the RNA itself is modified, several strategies can be applied with regard to choice and positioning of the photolabile functional group. One approach is to modify the nucleobases in order to sterically and chemically prevent the formation of mutual exclusive base pairs within the different conformations whose interconversion shall be studied (Höbartner et al., 2004). The second approach is to place the photo-cage at a functional group within the backbone of the 570 RNA. This can be for example the 2'-OH group that is important in the establishment of stabilising interactions as well as being the mediator of RNA-catalysed reactions (Manoharan et al., 2009). For the minimal hammerhead ribozyme, caging of the active 2'OHgroup at the active site in combination with position selective 13 C-labelling revealed a concerted motion of both nucleotides of the catalytic centre during the catalysed cleavage reaction (Fürtig et al., , 2008. Application of the approach to cage the nucleobase led to the characterisation of refolding events in various bistable RNAs 575 ( Figure 12) (Wenter et al., 2006(Wenter et al., , 2005, to the formulation of generalized folding rules that correlate the number of base pairs to refolding rates (Fürtig et al., 2007c) and to the delineation of transition state conformations in RNA refolding reactions (Fürtig et al., 2020(Fürtig et al., , 2010. When applied to RNAs for which refolding is intimately linked to biological function, the exact folding pathways during transcription involving the metastable states could be determined (Helmling et al., 2018). The introduction of photo-caged protecting groups normally requires the production of the RNA by chemical solid phase 580 synthesis (Brieke et al., 2012;Mayer and Heckel, 2006) rendering the simultaneous incorporation of isotope labelled nucleotides laborious and expensive (Quant et al., 1994). However, new chemo-enzymatic techniques that are able to combine chemically modified and in vitro transcribed, isotope labelled strands within a single RNA resolve these difficulties (Keyhani et al., 2018), and will enable light-triggered folding studies of more sizeable and complex RNAs in the future.
Likewise, tremendous advances are also made in the chemistry of photo-protecting groups utilized to cage RNAs. Whereas 585 early studies mainly focus on the 1-(2-nitrophenyl)ethyl that needs to be cleaved with UV light and has a limited steric demand (Ellis-Davies and Kaplan, 1988), new concepts using photo-caging groups with either more red-shifted absorption or higher destabilizing potency emerge (Ruble et al., 2015;Seyfried et al., 2018). Right from the beginning, the methodology of caging interacting ligands could be utilized in the study of more complex RNA systems at the size limit of liquid state NMR. First studies on the aptamer domain of the guanine-sensing riboswitch where the ligand hypoxanthine was caged 590 revealed a two-state folding trajectory (Buck et al., 2007). This an important molecular feature that enables fast discrimination of cognate over near-cognate ligands and enables the kinetic control of transcription termination within the two-domain full-length riboswitch (Steinert et al., 2017). In this application, resolving the folding dynamics at the level of individual nucleotides was only possible by application of nucleotide type selective isotope labelling in conjunction with xfiltered and X-nuclei-edited real-time NMR experiments. 595 Caging of divalent ions is challenging for RNA as they are often needed for proper folding of tertiary interactions but as also the affinities stay in the micro-to millimolar range. However, for the Diels−Alder ribozyme the difference in reactivity for different mutants could be traced down to the differences in local dynamics around the catalytic pocket (Manoharan et al., 2009). In this case, besides mixing also release of the divalent ions from a photo-caged chelator was possible (Fürtig et al., 2007b). More recently we also investigated folding of RNA using the rapid mixing methodology. Here, folding can be induced e.g. by rapid mixing of metal-ions, as has been exemplified for ribozymes by addition of Ca 2+ (Manoharan et al., 2009) and structural changes in RNA riboswitches by adding their specific ligand (Reining et al., 2013). All these applications share the 610 detection of the kinetics on the imino-resonances in 1D spectra.

DNA and RNA non-canonical structures -Time-resolved NMR studies of DNA and RNA G-quadruplexes and DNA i-motifs folding and refolding
Non-canonical DNA structures including G-quadruplexes (G4) and i-motifs typically coexist in several heterogeneously folded conformations. This pronounced structural polymorphism and the associated inherent dynamic character make these 615 structural motifs prime examples for time-resolved NMR studies. The pH-induced folding of a DNA i-motif revealed that the folding follows kinetic partitioning, with re-equilibration processes subsequent to the initial folding (Lannes et al., 2015;Lieblein et al., 2012). Similar findings have been made for a telomeric DNA G4 that coexists in two conformations with different folding topologies. For G4 DNA, folding can be induced by rapid injection of a K + -buffer solution, since monovalent cations are essential for G4 formation. G4 folding follows complex folding pathways, which involve long-lived 620 intermediate states that persist for several hours and the re-equilibration proceeds over days at room temperature (Bessi et al., 2015).
Recently, we investigated the folding and refolding kinetics of an 18-mer DNA (G4) forming oligonucleotide sequence from the human cMYC proto-oncogene promoter (Fig. 13). This G4 coexists in two conformations that are distinguished by a register shift of one G-rich strand-segment along the stacked tetrads. To study the conformational dynamics in this system, 625 we used a combination of K + -induced folding with an approach, where we photochemically trapped a single conformation https: //doi.org/10.5194/mr-2021-16 Discussions Open Access Preprint. Discussion started: 10 February 2021 c Author(s) 2021. CC BY 4.0 License. and induced refolding with in situ laser irradiation. By site-specific incorporation of photocages, we could block the base pair interactions for distinct nucleotides. This strategy allowed us to separate the two conformations and study the refolding mechanism in detail. The proposed kinetic model, based on kinetic and thermodynamic experimental data, reveals that after initial folding the two conformations can directly refold into each other. The proposed transition state requires only a 630 minimal degree of unfolding. The slow refolding kinetics (0.9 h -1 ) are caused by a relatively high activation energy that is needed for an initial opening of the base paired tetrads. Further, we showed that folding kinetics induced by rapid-mixing deviate by several orders of magnitude from light induced folding. This finding highlights that the altered energy landscapes under different non-equilibrium conditions have a severe impact on the folding dynamics. Photolabile protecting groups here are an optimal tool to investigate native, unmodified (after photocleavage) oligonucleotides dynamics under constant 635 experimental (pressure, temperature, buffer composition) and physiological conditions (Grün et al., 2020).

Temperature jump
There have been several different techniques for T-jump experiments in combination with real-time NMR spectroscopy, but so far applications on biomolecular systems have been limited. In most cases, RNase A was used as a test system to either follow its heat denaturation kinetics (Akasaka et al., 1991;Naito et al., 1990) or in case of flow system (Yamasaki et al.,650 2013) the refolding from its heat denatured state was followed. In all cases simple two-state kinetics were observed. The most recent application of rf-heating in combination with cold denatured barstar allowed detailed characterisation of different folding pathways. A stable intermediate was observed on the slow folding pathway (Fig. 14), where the rate limiting step is the trans-cis isomerization of Tyr47-Pro48 amide bond. Additionally, the reversibility of the system and the 660 slow cis-trans isomerization allowed the measurement of double-jump experiment to study alternative folding pathway. The equilibrium folded barstar was denatured by short cooling (2.5 min) time, keeping the Tyr47-Pro48 residue in cis conformation. From this non-equilibrium denatured state, a state-correlated spectrum was used. Although reaction rates could not be determined, evidence for an ensemble of intermediates was observed.

Pressure jump 665
Pressure-jump experiments have undergone rapid development in the last 10 years, thanks to the technical advancement.
First Kremer et al (Kremer et al., 2011) showed in 2011 that pressure jump of around 800 bar can be achieved in both direction to de-or renature the protein. They used a model protein histidine-containing phosphocarrierprotein (HPr) to design and demonstrate new pulse sequences to study protein folding. The pressure change is an order of magnitude faster than the longitudinal relaxation time of proteins therefore it can be directly incorporated into the pulse sequence. It is a 670 similar approach as the SCOTCH experiment designed by Rubinstenn et al. (Rubinstenn et al., 1999) for light sensitive proteins. Their approach was either at the start of the pulse sequence (pressure perturbation transient state spectroscopy PPTSS) or during the pulse sequence (pressure perturbation state correlation spectroscopy PPSCS) to change the pressure.
With these experiments they could demonstrate how pressure can be introduced as a new dimension into the pulse sequence and measure the k UN and k NU of HPr. 675 Roche et al. focused on the staphylococcal nuclease (SNase) with relatively long relaxation time to the new equilibrium state (even up to 24 hours), allowing even manual pressure perturbation (Roche et al., 2013). They investigated the effect of the introduction of different cavities by mutations and their effect on the folding kinetics. They observed drastic changes both in terms of stability and folding pathways. The I92A mutant showed structurally heterogeneous ensemble at the folding barrier with multiple folding pathways, while the WT SNase and the hyperstable D+PHS mutants have a well defined transient state 680 and folding pathway.
The next major developments come from Charlier et al. (Alderson et al., 2017;Charlier et al., 2018aCharlier et al., , 2018bCharlier et al., , 2018c in a series of publications. Their new P-jump system, as discussed in chapter 2.3 allows in both directions very fast pressure change. Their pulse sequence approaches are similar to what Kremer et al has shown already but developed even more complex ways to incorporate into the pulse sequence and to study the folding mechanism of the pressure sensitive Ubiquitin. 685 First the combination of H/D exchange with pressure jump revealed biexponential refolding kinetics attributed to an off pathway oligomeric intermediate. Next the use of P-jump in combination of 3D NMR spectroscopy allowed the chemical shift and 15 N transverse relaxation analyses of the still unfolded protein but already at low pressure. This revealed a very short lived intermediate, which is different from the hydrogen exchange revealed one. They proposed a folding mechanism of ubiquitin where two parallel but similarly efficient folding pathways take place: direct folding with no intermediate and 690 folding via a short lived intermediate state. Finally, they could prove by incorporating double pressure jump into the pulse sequence and measuring the chemical shifts that this short lived intermediate closely resembles the folded state with differences as they write in "the C-terminal strand, 5, and its preceding loop, strand 1, and the C-terminal residues of strand 3, with 5 being sandwiched between 1 and 3 in the natively folded state".

"Slow" kinetics 695
A number of processes are slow enough to be investigated by time-resolved NMR without the need of any device to initiate folding. One example is the formation of very large macromolecular assemblies. The group of Boisbouvier studied for example the self-assembly pathway of the 0.5 MDa proteolytic machinery TET2 (Macek et al., 2017), the group of Schwarzer investigated histone modification by time-resolved NMR-spectroscopy (Liokatis et al., 2016).
The investigation of protein modification and in particular phosphorylation has been put forward by Selenko in 2010 and 700 been used since than in a couple of applications (Kosten et al., 2014, p. 129;Landrieu et al., 2006;Liokatis et al., 2010;Mylona et al., 2016). Such studies are even possible in cellular environment by time-resolved in-cell NMR (Theillet et al., 2013). Other applications of time-resolved in-cell NMR are the proteolytic alpha-synuclein processing (Limatola et al., 2018), the methylation of lysines in cell, the investigation of ligand binding in cellular environment (Luchinat et al., 2020a(Luchinat et al., , 2020b) and the modulation of bound GTP levels of RAS (Zhao et al., 2020). Time-resolved NMR experiments are now also 705 pursued to characterize metabolic flux in patient-derived primary cells (Alshamleh et al., 2020;Reed et al., 2019).
The formation of amyloids is another highly relevant slow process. While fast tumbling monomers and flexible tails are amenable to liquid state NMR spectroscopy, residues in intermediate exchange present in oligomers or residues in fibrils cannot be observed. Therefore, the aggregation is monitored as monomer loss kinetics following the signal decrease of the resonances present at the beginning of the experiment. 710 The misfolding of the prion protein into fibrils is observed in neurogenerative prion diseases. While the prion protein is a mainly -helical globular protein with an unfolded tail in its native state, -sheet structures are enriched in the polymeric fibrillar forms. We investigated the kinetics of fibril formation from the unfolded state on a per residue basis of human prion protein (Kumar et al., 2010) and murine prion protein . Comparison of HSQC spectra directly dissolving the human protein (90-230) and after 4 and 7 days reveals that signals are lost fast for the core of the fibril 715 (145-223), while N-terminal signals decreases slower and signals close to the C-terminal (224-230) change their chemical shift indicating a structural change in the latter region within the fibril. A more detailed view of the fibril formation was obtained on the murine prion protein where signal loss rates could be obtained from multiple HSQC measurements on a per residue basis. Here we found that residues in close proximity to the disulfide bridge (C179-C214) broaden first which we attribute to initial molecular contacts in oligomer formation, while in a second stage of the aggregation fibrils are formed. 720 Another disease attributed to protein misfolding is the Alzheimer's disease, in which A peptides are forming fibrils. The aggregation of A1-40 and A1-42 has been investigated by a number of groups (Bellomo et al., 2018;Pauwels et al., 2012;Roche et al., 2016) by monitoring the loss of the monomers. Using the decay of the methylgroup region in a proton 1D as reporter of aggregation, Luchinat and coworkers (Bellomo et al., 2018)  certain oligomer size. These fibrils grow (by addition and release of monomers) and undergo fibril fragmentation resulting in smaller fibrils that in turn grow further.
Switching gears completely: another impressive example for the application of liquid NMR spectroscopy for the investigation of "slow" kinetics is the study of tRNA maturation. Barraud et al. (2019) were able to investigate the enzymatic 730 modification of tRNA Phe in yeast cell-extract over 26 h using a series of HSQC spectra. Figure 15 shows HSQC spectra of the maturation after 12-14 h after addition of the tRNA Phe to yeast extract showing that modifications are inserted in a specific order along a defined route. This application nicely demonstrates the power of NMR to investigate complex mechanisms on the per site resolution.

Conclusions 740
In this review, we have discussed the application of time-resolved NMR studies to study biomacromolecular folding, refolding, modification and aggregation. These studies utilize the power of NMR spectroscopy to determine kinetics of structural transitions together with site-resolution. Different to other structural techniques, NMR does not only provide snapshots of folding trajectories, but provides positive evidence for the transition of two or several conformational states. It can determine the associated kinetic rates with rates as fast as 5000 s -1 in a significant temperature range, allowing to classify 745 structural transitions to follow Arrhenius or non-Arrhenius behaviour. Particularly interesting are biomolecular systems whose folding trajectory is subject to kinetic partitioning. Starting from a single state, folding pathways diverge, multiple folding pathways are populated, with kinetically or thermodynamically driven conformational states. Together with mutational studies, NMR is key in delineating transitions state characteristics and to detected lowly populated states and prime examples have been reported for proteins (Korzhnev et al., 2004) and for DNA (Kimsey et al., 2018) and their 750 complexes (Afek et al., 2020). Future applications, thanks to unstoppable developments to increase signal-to-noise and resolution in NMR, will devise more sophisticated experiments to characterize transient conformations that often represent the key states carrying the biomolecular function.

Acknowledgements
We wish to acknowledge numerous collaborations with scientists that contributed ideas to the work present. In particular, we 755 wish to express our gratitude to R. Kaptein and R. Boelens for their help in early days of time-resolved NMR spectroscopy in the groups of the authors. Funding is acknowledged from Deutsche Forschungsgemeinschaft (CRC902, GRK1986, Normalverfahren), European commission (ITN, iNEXT, iNEXT-discovery), German Cancer research center (DKTK), and the state of Hesse (BMRZ).