Spin-state-selective methods in solution- and solid-state biomolecular 13C NMR

Progress in Nuclear Magnetic Resonance Spectroscopy 84–85 (2015) 1–13

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Progress in Nuclear Magnetic Resonance Spectroscopy journal homepage: www.elsevier.com/locate/pnmrs

Spin-state-selective methods in solution- and solid-state biomolecular C NMR q

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Isabella C. Felli ⇑, Roberta Pierattelli ⇑ Magnetic Resonance Center (CERM) and Department of Chemistry ‘‘Ugo Schiff’’, University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 14 October 2014 Accepted 26 October 2014 Available online 1 November 2014

Spin-state-selective methods to achieve homonuclear decoupling in the direct acquisition dimension of 13 C detected NMR experiments have been one of the key contributors to converting 13C detected NMR experiments into really useful tools for studying biomolecules. We discuss here in detail the various methods that have been proposed, summarize the large array of new experiments that have been developed and present applications to different kinds of proteins in different aggregation states. Ó 2014 Elsevier B.V. All rights reserved.

Keywords: Biomolecules Homodecoupling Scalar coupling IDP Paramagnetic molecules

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 C direct detection and the problem of homonuclear decoupling . . . . . . . . . . . . . Spin-state-selective methods for homonuclear decoupling . . . . . . . . . . . . . . . . . . . Implementation in multidimensional NMR experiments . . . . . . . . . . . . . . . . . . . . What if more than one homonuclear coupling is to be suppressed? . . . . . . . . . . . Spin-state-selective methods to measure couplings . . . . . . . . . . . . . . . . . . . . . . . . Implementation in solution and solid-state multidimensional NMR experiments Selected examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Direct 13C detection now provides a useful tool for biomolecular NMR applications both in solution and in solid state NMR [1]. Multidimensional NMR experiments can be acquired on many proteins of interest and provide information about nuclear chemical shifts, scalar and residual dipolar couplings and relaxation rates. All these observables can be used to study the structural

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Edited by Beat H. Meier and Gareth A. Morris.

⇑ Corresponding authors. Tel.: +39 0554574242/4265; fax: +39 0554574923. E-mail addresses: (R. Pierattelli).

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(I.C.

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http://dx.doi.org/10.1016/j.pnmrs.2014.10.001 0079-6565/Ó 2014 Elsevier B.V. All rights reserved.

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1 2 3 5 6 8 9 10 12 12 12

and dynamic properties of proteins, their interactions and their post-translational modifications. The intrinsically lower sensitivity of 13C with respect to 1H has been independently optimized by the impressive improvements in instrumental sensitivity that we have recently experienced [2–4] enabling us to perform experiments that we could only dream of until a few years ago. The continuous efforts to further improve sensitivity through a variety of different strategies are obviously going to have a strong impact on broadening the applicability of 13C direct detection NMR to a wider range of cases. The peculiar properties of 13C can thus be exploited to achieve information that is useful in general but that becomes particularly interesting when 1H detection NMR finds

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limitations either because proton lines are too broad or are not well dispersed. Focusing on 13C detection however also poses some challenges. The large one-bond homonuclear couplings between directlybound 13C spins cause large signal splittings in the direct acquisition dimension, in contrast to 1H detection where obviously one bond couplings are not present and only smaller two and three bonds couplings are observed with a much lower impact on spectral resolution and sensitivity. Therefore a key aspect to convert 13C direct detection NMR experiments into a useful tool for biomolecular applications consists in solving the problem of homonuclear decoupling in the direct acquisition dimension. This represents a novel aspect also with respect to applications of 13C direct detection to small molecules, which in general are based on samples at natural abundance. In the case of biomolecules, isotopic enrichment is mandatory to enable us to acquire multidimensional NMR experiments with sufficient resolution, and is of course very useful for 13C-detected NMR experiments provided the problem of the large homonuclear coupling in the direct acquisition dimension can be solved. As an example of the huge impact of this on the quality of the spectra and on the amount of atomic resolution information that can be achieved, Fig. 1 shows two different kinds of 13C-detected spectra acquired on different proteins in different aggregation states with and without homonuclear decoupling in the direct acquisition dimension. The removal of signal splitting is crucial to simplifying

Fig. 1. Two examples of 2D NMR spectra that only differ for the application of homonuclear decoupling in the direct 13C acquisition dimension. Top: 2D double-CP (H)NCO spectra acquired in the solid state on microcrystalline 13C, 15N labeled dimeric Cu(II)Zn(II) superoxide dismutase (SOD) without (left) and with (right) application of S3E C0 –Ca homonuclear decoupling in the direct 13C acquisition dimension. Spectra were acquired at 850 MHz using a 3.2 mm triple resonance probe at 20 kHz MAS [43]. Bottom: 2D CON spectra acquired in solution on 13C, 15N labeled human a-synuclein without (left) and with (right) application of IPAP C0 –Ca homonuclear decoupling in the direct 13C acquisition dimension. Spectra were acquired at 700 MHz, using a triple resonance cryoprobe optimized for 13C detection [73].

solution NMR spectra, but is also very relevant for increasing signal resolution in solid state NMR. The main focus of this review is thus to discuss the various strategies for obtaining highly resolved spectra in both states. Among these the most widely applied approach is based on exploiting spin-state-selective methods in various ways, giving a variety of different solutions. Therefore the basic principles and their implementation for homonuclear decoupling of the two 13C backbone nuclear spins in proteins (C0 and Ca) are extensively discussed here from simple implementation in the 1D mode, through implementation in different kinds of 2D experiments, to their use in a wide variety of more complex multidimensional experiments for solution and solid state applications. Finally, as an example, their application to selected kinds of proteins will also be presented.

2. 13C direct detection and the problem of homonuclear decoupling In principle all 13C spins can be detected without taking care of homonuclear decoupling, as initially proposed for biomolecular applications [2,5–10]. However, as already pointed out (Fig. 1), the complex multiplet structures of 13C signals in uniformly labeled proteins often contribute to a dramatic reduction in resolution and sensitivity of the spectra, to the extent that 13C-detected experiments turn out to be useful only for the study of small proteins with well dispersed signals. The presence of the large onebond carbon–carbon scalar couplings constitutes a peculiarity of 13 C spectroscopy of biomolecules, as no analogous homonuclear couplings occur for 1H and 15N in proteins. The presence of these couplings is very useful for coherence transfer and it is actively exploited in a variety of experiments. On the other hand, they do complicate the spectra in the direct 13C dimension. Several 1D 13C NMR spectra, acquired on different proteins in different aggregation states, are shown in Fig. 2. The different linewidths and signals dispersion clearly indicate that different experimental methods may be needed for different kinds of proteins, depending on their size, their structural and dynamic properties, their aggregation state, etc. On the other hand, common aspects are clearly evident, as it is well known in biomolecular NMR. The chemical shift range observed in 13C 1D spectra (about 200 ppm) is larger than that observed for protons (about 10 ppm); also, when comparing these numbers in absolute frequency units, the frequency range in Hz over which 13C spins resonate is larger by a factor of about 5 compared to that for 1H spins. This also holds if we compare 13C and 1H spins of similar chemical nature, for example aliphatic spins, that resonate in a region of about 70 ppm and 6 ppm respectively, which still implies a larger frequency range of a factor of about 3 for 13C resonances compared to 1H ones. Therefore, it is worthwhile to exploit as much as possible this property of 13C spins, to increase the dispersion of the observed resonances and more generally of cross peaks in multidimensional NMR spectra. The characteristic chemical shift ranges over which the different kinds of 13C spins (C0 , Cali, Ca, Caro) resonate also constitute a general property of 13C protein NMR. 13C band-selective pulses can be used to manipulate them selectively. In particular, C0 can be easily manipulated separately from Ca or from aliphatic spins (and vice versa); with some compromises for specific types of amino acids, one can also manipulate Ca separately from Cb; finally, aromatic spins can also be manipulated as a group. On the other hand it is not easy to find general ways to distinguish different kinds of aliphatic (or aromatic) side chain spins one from another through band-selective pulses. Coupling topologies for backbone 13C spins (C0 or Ca) do not depend on the kind of amino acid (except for glycines), but this

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Fig. 2. 13C 1D NMR spectra of various proteins in different aggregation states. From top to bottom: human a-synuclein in solution, ubiquitin in solution, dimeric Cu(II)Zn(II)SOD in solution and microcrystalline dimeric Cu(II)Zn(II)SOD under MAS. All samples were uniformly 13C, 15N labeled; the Cu(II)Zn(II)SOD sample was also 2H labeled, back-exchanged in H2O. The 13C 1D NMR spectra in solution were acquired at 700 MHz with similar parameters; the 13C spectrum in the solid state was acquired on microcrystalline Cu(II)Zn(II)SOD at 850 MHz, using a 1.3 mm triple resonance probe at 60 kHz MAS.

does not hold moving away from the backbone. C0 signals are split by the Ca–C0 coupling (54 Hz), while Ca signals (except for glycines) are further split by the Ca–Cb coupling (35 Hz). Therefore, most of the experiments recently developed for sequence-specific assignment focus on C0 or on Ca direct detection [11–14] while the other side-chain nuclei are correlated to the backbone ones in the indirect dimension. This means, for example, that there is a variety of ways to detect the C0 –Ca correlations and that there are several options for designing experiments for backbone sequence-specific assignment since the starting and ending points may be either C0 or Ca. The relative performance of the experiments based on C0 or Ca direct detection therefore strongly depends, in addition to usual features such as the relaxation properties and the chemical shift dispersion of the system, on the performance of the approach in removing the large one-bond carbon–carbon couplings in the direct acquisition dimension [15]. Heteronuclear decoupling is in general easier to perform both in the direct and in the indirect dimensions, simply by applying one of the many decoupling sequences available, or by using 180° pulses in indirect dimensions. For the homonuclear case, when performing decoupling in indirect dimensions of NMR experiments, it is quite straightforward to decouple C0 from Ca and vice versa by using band-selective pulses [16,17]. Alternatively, if transverse relaxation permits, constant-time versions of the experiments can be used to refocus the evolution of homonuclear couplings in indirect dimensions of NMR experiments. The additional complications related to performing homonuclear decoupling in the direct acquisition dimension derive from the fact that we need to give radio-frequency pulses at a specific frequency which is very similar to the one that we would like to sample, while keeping the receiver open for acquisition. Therefore, it is not trivial to filter one from the other. One solution [18–20], initially proposed for selective homonuclear decoupling in small molecules [21], consists in sharing the acquisition time between ‘‘acquisition-mode’’ and ‘‘decoupling-mode’’. This alternating switching should of course be very quick, faster than the sampling speed that we want to achieve, in order to allow time for decoupling between acquired datapoints.

In the case of 13C detection, if the frequency ranges of different types of spins are well separated and far from each other, such as for C0 and Ca spins, it is feasible to perform band-selective irradiation of Ca while acquiring carbonyls (and vice versa), removing in this way the large 13C–13C coupling affecting carbonyls (for all carbonyls at once) [22,23]. However, one of the drawbacks of this approach consists in the appearance of decoupling side-bands, and the expected gain in intensity is not achieved for all signals. This approach works fairly well for decoupling C0 from Cas and vice versa, whereas it is more difficult to implement complete decoupling of Cas from Cbs, which also share a large one bond coupling, due to the very close frequency ranges of these two kinds of nuclear spins [23]. Moreover, from a more technical point of view, this set-up may require the use of special hardware. More recently it has been proposed to use short p pulses during acquisition of the FID to perform 13C homonuclear decoupling [24], inspired by approaches proposed for homonuclear decoupling in the direct 1H dimension [25–28]. Another approach that has been proposed consists in eliminating the coupling using post-acquisition methods such as deconvolution using maximum entropy reconstruction [29]. However, the approaches that have become most extensively used so far, thanks to their good performance and easy implementation, are those based on spins-state-selective methods and are referred to as virtual homonuclear decoupling approaches.

3. Spin-state-selective methods for homonuclear decoupling The general common feature of these approaches consists in preserving the coupling and letting it evolve differently in alternate experiments, in order to sample the in-phase and the antiphase components of the signal or their different linear combinations [30–34]. These approaches are very elegant and do not require an additional radio frequency channel, since the different 13C nuclear spins involved in the scalar coupling can be selectively manipulated by using band-selective pulses. The general idea, initially proposed to determine one-bond 1 H–15N couplings [35], is illustrated for the most simple case, that

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Fig. 3. 1D versions of the pulse schemes to achieve C0 homonuclear decoupling through the IPAP (A–C) and the S3E (D–F) approaches. Narrow and wide vertical symbols represent p/2 and p band-selective pulses on the nuclei indicated on the respective line, and other symbols have usual meaning. Traces illustrating the method are extracted from spectra acquired on the cyclic octapeptide 13C, 15N labeled hymenistatin [15]. IPAP method: The two independent experiments to obtain the in-phase (A) and antiphase (B) C0 components with respect to Ca are shown, together with the manipulations necessary to separate the two multiplet components, shift them to the center of the original multiplet, sum them, and achieve virtual decoupling (C). The delay D is set to 1/2JC0 Ca = 9 ms. The phase cycles are: (A) /1 = y, y; /rec = x, x (B) /1 = x, x; /rec = x, x; other pulses have phase x. S3E method: The two independent experiments (D and E) to separate the two C0 multiplet components (with respect to Ca) through the S3E approach are shown together with the manipulations necessary to separate the two multiplet components, apply a p/2 phase correction to one of the two, shift them to the center of the original multiplet, sum them and achieve virtual decoupling (F). The delay D is set to 1/2JC0 Ca = 9 ms. The phase cycles are: (D) /1 = x, x; /rec = x, x; (E) /1 = y, y; /rec = x, x. Other pulses have phase x.

of direct detection of carbonyl carbons in 1D mode (Fig. 3A and B) [30–32,34]. The spin system that we consider is constituted by two spins, C0 and Ca, with offsets XC0 and XCa, mutually coupled through the one-bond scalar coupling 1JC0 Ca. The two experiments needed to acquire the in-phase and antiphase components of C0 are illustrated schematically in Fig. 3(A and B). After creating C0 transverse coherence (C0 x) through the first band-selective p/2 pulse on C0 , it is possible to allow the evolution of the C0 –Ca coupling for a selected overall time (D = 1/(21JC0 Ca)) to acquire the anti-phase (AP) component of the signal (2C0 yCa z, Fig. 3B)



C0x ! C0x cos p1 J C0 C a D þ 2C0y Caz sin p1 J C0 Ca D

The in-phase component could of course be acquired just by switching on the receiver right after the first p/2 pulse. However, using the same number of pulses and delays to acquire the inphase component is preferred (Fig. 3A). In this way, possible effects due to pulse imperfections or relaxation losses are expected to influence both components similarly without introducing differential effects on the intensity of the antiphase component with respect to the in-phase one. Therefore the in-phase component is acquired through a similar experiment in which the p pulses on Ca spins are shifted by 1/(41JC0 Ca), to refocus the overall evolution of the coupling during this pulse sequence element (Fig. 3A). It is worth noting that the in-phase and antiphase signals are orthogonal with respect to each other and thus they would require a phase correction that differs by p/2. This of course does not constitute a problem. Alternatively, to simplify subsequent processing of the data and use the same phase correction parameters for both components, one can simply change the phase of the first p/2 C0 pulse by p/2. Once the in-phase and antiphase components have been acquired, the linear combinations that should be performed to separate the two multiplet components are shown schematically in Fig. 3C. Proper separation of the two components provides a first indication of the performance of the homonuclear decoupling method. The two sub-spectra containing the two multiplet components are shifted to the center of the original multiplet to remove the effect of the large one-bond coupling on the spectra. These operations can in principle be performed either on time domain data or in the frequency domain. However, to reduce possible limitations due to digital resolution in the frequency domain they are generally performed on time domain data. Even though the overall duration of this pulse sequence element, determined by the magnitude of the coupling to be suppressed, is quite short, in some cases it might be useful to try to reduce this time to minimize losses due to transverse relaxation, or more generally to transverse dephasing. Therefore, instead of waiting for complete interconversion between the in-phase and antiphase components that requires D = 1/(21JC0 Ca), a method can be designed that only requires half of the time and exploits different linear combinations of the in-phase and antiphase components (Fig. 3D and E) [33,36,37]. This approach, referred to as S3E [38–41] is based on the reduction of the overall time in which the coupling is allowed to evolve to D/2 = 1/(41JC0 Ca), so that both components of the signal (in-phase and antiphase) are simultaneously present at the time of acquisition [33,37]. In this case, separation of the two multiplet components is achieved by acquiring two different linear combinations of in-phase and antiphase components by changing the relative sign of the two. The S3E idea, initially proposed as a variant of 2D 1H–15N heteronuclear correlation spectroscopy for determining 1JHN couplings [38–40], exploits a pair of 1H p/2 pulses before acquisition that selectively change the sign of one of the two orthogonal components of the signal (either the in-phase or antiphase) [39]. In the original implementation, the two 1H pulses have a total effect of either zero or p rotation on the antiphase component while leaving the in-phase component unaffected, providing in this way the two independent spectra needed to achieve separation of the two multiplet components. This strategy has been adopted for 13C homonuclear decoupling for solid state applications [36]. While dealing with 13C detection in solution it was realized that the same result could be obtained by using p pulses instead of pairs of p/2 pulses, an attractive feature when focusing on the homonuclear case rather than on the heteronuclear one, as bandselective pulses covering specific frequency ranges (C0 and Ca) are needed. Therefore it was proposed to use a p band-selective 13 C pulse right before (Fig. 3E) or right after (Fig. 3D) the scalar coupling evolution period to selectively change the sign of one of the two components (IP or AP) in alternate experiments [37].

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While at the beginning of the block (time point a) only in-phase magnetization is present and so the sign change affects both components deriving from the initial in-phase signal, at the end of the block (time point b) the two components are orthogonal with respect to each other, and so the p pulse changes only the sign of one of them while preserving the other. Analogously, the two independent components are then summed and subtracted in order to separate the two multiplet components. After applying a p/2 phase correction to one of the two, they are again shifted to the center of the original multiplet and summed to obtain virtual decoupling (Fig. 3F). Slightly different variants of this approach, have also been proposed [42–44]. For both approaches described (IPAP and S3E), the linear combinations needed to achieve homonuclear decoupling also have an undesired effect that impacts on the sensitivity of the experiments. While the expected gain in signal of a factor of two is observed experimentally, we have to consider that also the noise increases when summing together two different spectra. Therefore the final gain in signal-to-noise ratio obtained through the in-phase/antiphase homonuclear decoupling method should also take into account this feature that reduces the maximum expected increase in signal-to-noise ratio (to about 21/2). Another aspect that should be considered is that these virtual decoupling methods will not manage to eliminate contributions resulting from two-bond and three-bond 13C homonuclear couplings. Part of these could actually be eliminated through bandselective irradiation of a specific region, for example irradiating all aliphatic 13C signals when acquiring carbonyls. However, these additional homonuclear couplings are expected to be quite small, smaller than their 1H analogues. So the additional 13C homonuclear couplings are expected to influence the spectrum much less than 1 H homonuclear couplings influence spectra based on 1H detection. Therefore this potential limitation can be accepted, and its effect can hardly be noted in the spectra. Concluding, both IPAP and S3E methods perform very well for C0 homonuclear decoupling due to the fact that the C0 –Ca couplings are very uniform throughout a protein sequence, so the block can be optimized at once for all the signals. Good separation between the two components can be achieved thanks to the good performance of the band selective pulses and to the moderate relaxation losses during this pulse sequence element, two aspects that contribute to the excellent performance of this homonuclear decoupling approach.

Fig. 4. A schematic picture of how spin-state-selective methods for homonuclear decoupling can be implemented in multidimensional NMR experiments (A and B refer to the two independent components of the signal needed for homonuclear decoupling described in detail in Fig. 3).

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4. Implementation in multidimensional NMR experiments For simplicity, the principles on which these approaches are based have been described referring to the simple case of acquisition of carbonyl coherence in the 1D mode. Therefore this block can be added at the end of any pulse sequence in which in-phase carbonyl coherence is acquired (Fig. 4, top panel). In principle the same results can also be obtained starting from antiphase coherence. For example, in case of the IPAP approach, it is sufficient to invert the two blocks: one refocuses the evolution of the coupling yielding the antiphase component (Fig. 3A), and the other the in-phase component (Fig. 3B). This property is particularly useful considering that many of the experiments in which scalar couplings are actively exploited for coherence transfer do end with a refocusing step of antiphase carbonyl coherence (either respect to Ca or N or to both). In these cases virtual decoupling methods can be implemented without adding additional pulse sequence elements (additional pulses and delays) by simply modulating the extent of the evolution of the C0 –Ca coupling in the final coherence transfer step in which carbonyl coherence is refocused (Fig. 4, bottom panels). This can be illustrated taking as an example the two most simple 2D C0 detected experiments, CACO and CON. The minimal CACO experiment that provides C0 i–Ca i correlation can actually be implemented directly acquiring antiphase C0 coherence [45,46]. Of course, if possible, inclusion of an additional block to refocus antiphase coherence [2,9], and implementation of homonuclear decoupling [22] can improve the quality of the spectra. The IPAP approach for homonuclear decoupling can easily be implemented just by changing the position of the final Ca p pulses to modulate the evolution of the coupling in the final block and acquire IP and AP components of the C0 signal (Fig. 5) [32,34]. It is interesting to note that in the case of the CACO experiment, substitution of the IPAP approach with the S3E scheme provides a reduction of the overall duration of the pulse sequence (Figs. 4, bottom panels and 5) [15,37]. This strategy is therefore particularly

Fig. 5. The examples of CACO-IPAP, CACO-S3E. The figure shows the pulse schemes of two variants of the 2D CACO experiment that differ in the type of spin-stateselective approach used to obtain C0 –Ca homonuclear decoupling. The IPAP version is reported in panels A (IP component) and B (AP component). The S3E version is reported in panels C (one of the two experiments), D (the other). Narrow and wide vertical symbols represent p/2 and p band-selective pulses on the nuclei indicated on the respective line, pulsed field gradients are indicated with ‘‘PFG’’, and other symbols have usual meaning. The delay D is set to 1/2JC0 Ca = 9 ms. The phase cycle is: (A) /1 = x,x; /2 = 4(x), 4(y); /IPAP(IP) = 2(x), 2(x); /rec = x, 2(x), x, x, 2(x), x; (B) /IPAP(AP) = 2(y), 2(y); (C) /1 = x, x; /2 = 4(x), 4(y); /S3E = 2(x), 2(x); /rec = x, 2(x), x, x, 2(x), x; (D) /S3E = y, y; other pulses have phase x. Quadrature detection in the F1 dimension is obtained by incrementing /1 in a States-TPPI manner [98]. Transfer pathway: F1 (Ca, t1) ? F1 (C0 , t2). Correlations observed: Ca i – C0 i. Adapted with permission from Ref. [15].

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Fig. 6. The examples of CON-IPAP, CON-SE-DIPAP. The pulse scheme to acquire the CON-IPAP or the CON-SE-DIPAP experiment is shown. The two variants differ in the approach used for 15N decoupling during acquisition, with the key pulse sequence elements indicated in green: (1) in the CON-IPAP version, 15N decoupling is applied during acquisition of the FID while the last 15N p/2 pulse highlighted in green is omitted; (2) in the CON-SE-DIPAP variant, 15N RF irradiation during acquisition is omitted, while two independent experiments are acquired by changing by p the phase of the last 15N p/2 pulse, to acquire the two independent components of the signal for C0 –N virtual decoupling. C0 –Ca virtual decoupling is implemented by shifting the positions of the Ca pulses in the final C0 –N coherence transfer step to acquire the two independent components of the signal needed to obtain the inphase (IP, panel A) and antiphase (AP, panel B) C0 components with respect to Ca. Narrow and wide vertical black symbols represent p/2 and p band-selective pulses on the nuclei indicated on the respective line, the pulse with phase /2 is an adiabatic inversion pulse on C0 and Ca, pulsed field gradients are indicated with ‘‘PFG’’ and other symbols have usual meaning. The delays are: D = 9 ms (1/2JC0 Ca), D1 = 32 ms, e = t1(0). The phase cycle is: /1 = x, x; /2 = 2x, 2(x); /3 = 4x, 4(x); /IPAP(IP) = x; /IPAP(AP) = y; /rec = x, (x), x, (x), (x), x, (x), x; other pulses have phase x. In the SE-DIPAP variant /4 = 4(y), 4(y); the two independent components, linear combinations of in-phase and anti-phase coherences of C0 respect to N, are acquired separately with two experiments that differ in the phase of the last 15N pulse (/4 is incremented by p in the second experiment). This allows the recovery of both orthogonal components of the 15N signal that has evolved during t1, avoiding loss of one of the two. The manipulation of the data needed is described in detail in the original publication [48]. Quadrature detection in the F1 dimension is obtained by incrementing /1 in a States-TPPI manner. Transfer pathway: F1(C0 ) ? F3(N, t1) ? F1(C0 , t2). Correlations observed: C0 i–Ni+1 The Figure also shows in panels C and D a portion of 2D CON spectra acquired on human securin, an intrinsically disordered protein of 202 amino acids, with the two pulse sequence variants that differ in the 15N decoupling method: (C) CON-IPAP, (D) CON-SE-DIPAP. A representative trace of the signals is also shown. Virtual 15N decoupling implemented in the CON-SE-DIPAP version performs well and enables us to acquire the FID as long as needed. This might be useful to improve the resolution in cases of long-lived C0 coherences. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

useful in implementing homonuclear decoupling when transverse relaxation or transverse dephasing is an issue [37,42,43]. The implementation of homonuclear decoupling methods in 13C direct detection has also stimulated the development of a clever method to recover the component of the signal that is usually lost when implementing quadrature detection in 2D experiments, in other words a different variant of the so-called ‘‘sensitivity

improvement’’ approach [47]. Indeed, simple modifications of the pulse sequence, essentially a p/2 pulse right before the acquisition of the FID on the ‘‘passive’’ nuclear spins involved in the final coherence transfer step, enables us to recover the DQ/ZQ term that is usually lost and convert it into observable antiphase coherence. This antiphase term can thus be used to achieve homonuclear decoupling, in combination with an in-phase term, as suggested in the version of CACO referred to as COCAINE [47]. This approach has also been adopted for the heteronuclear case, as discussed in more detail below. The other very useful 2D experiment based on C0 direct detection is the CON (Fig. 6) that provides the C0 i–Ni+1 correlation [8,22,33]. In this case, the implementation of homonuclear decoupling is even more straightforward since 1JC0 N coupling is smaller than 1JC0 Ca. Therefore the final coherence transfer step necessary to refocus the 1JC0 N coupling is well suited to implementing C0 –Ca homonuclear decoupling just by changing the position of the p Ca pulses, to modulate the evolution of the C0 –Ca coupling during the final step. In this case the final step is long enough to enable complete inter-conversion between the in-phase and antiphase terms and thus the IPAP variant is generally implemented. Finally, even if many more possibilities are available to achieve heteronuclear decoupling during acquisition, spin-state-selective approaches can be implemented and have interesting properties that may provide some advantages in specific cases. For example, in cases of pronounced overlap and long lived C0 coherences, such as are encountered for many unfolded and/or intrinsically disordered proteins, implementation of spin-state-selective C0 –N heteronuclear decoupling enables us to acquire the FID as long as needed without suffering possible limitations deriving from heating caused by 15N RF irradiation (Fig. 6). The CON-SE-DIPAP variant [48] of the CON experiment that incorporates C0 –Ca as well as C0 –N virtual decoupling, and recovers the orthogonal component deriving from 15N quadrature detection to increase the sensitivity [49], has been implemented [48]. 5. What if more than one homonuclear coupling is to be suppressed? The extension of these ideas to the situation of a spin sharing two scalar couplings is conceptually straightforward (Fig. 7). In principle two blocks, each one optimized for the specific coupling to be suppressed, can be designed exploiting either the IPAP or the S3E approach. They can be implemented one after the other, as initially proposed for solid state applications [30,31] in which two IPAP blocks were applied for Ca detection, one optimized for the C0 –Ca coupling and the other for the Ca–Cb coupling (Ca spins are indeed characterized by two coupling partners, C0 and Cb, except for glycines). Alternatively the two IPAP blocks can be

Fig. 7. A schematic picture illustrating how the application of spin-state-selective methods for homonuclear decoupling can be extended to the case of two homonuclear couplings.

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Fig. 8. 1D versions of the DIPAP and DS3E methods for Ca direct detection to remove the two large Ca–C0 and Ca–Cb splittings in the direct acquisition dimension. They are illustrated for the simple case of 1D experiments and can be implemented in any experiment based on Ca direct-detection. Narrow and wide vertical black symbols represent p/2 and p band-selective pulses (generally with Q5 or time reversed Q5 and Q3 shapes) [99] on the nuclei indicated on the respective line (C0 , Ca and Ca/b) and the other symbols have the usual meaning. The traces shown to illustrate the methods are extracted from spectra acquired on 13C, 15N labeled cyclic octapeptide hymenistatin [15]. DIPAP approach: Panels A–D report the four variants of the pulse scheme for acquiring and storing separately the four components indicated with IP–IP, AP–IP, IP–AP and AP– AP respectively. The results of the four experiments, are shown in panel E; the final results are shown. The latter is scaled relative to the other traces in order to have the same noise intensity. The delay D0 is set to 1/2JCaCb = 14 ms and D to 1/2JC0 Ca = 9 ms. The phases are: /DIPAP(A) = x, x; /DIPAP(B) = y, y; /DIPAP(C) = y, y; /DIPAP(D) = x, x; /rec = x, x; other pulses have phase x. DS3E approach: The four variants of the 1D experiment that are necessary for separating, through linear combinations, the four components of the Ca multiplet are reported in panels E–I. The delays a, b and c are set to allow for the evolution of the one bond Ca–C0 and Ca–Cb couplings for a total evolution time of 1/4J each (a + b + c = 1/4JCaCb = 7.1 ms and a  b + c = 1/4JC0 Ca = 4.5 ms, with a + b = c). All the pulses, unless explicitly indicated, have phase x. The phase cycles are: /DS3E(1) = x, x, /DS3E(2) = y, y, /DS3E(3) = y, y, /DS3E(4) = x, x, /rec = x, x. The four different 1D spectra obtained are reported panel J; the same spectra after application of a phase correction of p/2 are shown in panel K; the final result is shown in panel L. Adapted with permission from Ref. [15].

merged reducing the total duration of the pulse sequence element needed to achieve homonuclear decoupling [34,37]. The merged duration is thus determined by the longer one of the two blocks tailored to the smaller coupling to be suppressed (the Ca–Cb coupling in this case) [34,37]. This approach is referred to as DIPAP [34] where ‘‘D’’ stands for ‘‘double’’ to indicate that two couplings are involved. In an analogous way, two S3E blocks can be combined; in this case the approach is referred to as DS3E [37]. The implementation for the case of Ca direct detection in 1D mode is illustrated in Fig. 8. The spin system that we consider is constituted by three spins, C0 , Ca and Cb, with offsets XC0 , XCa and XCb, with Ca coupled to C0 and Cb through the one bond scalar couplings 1JC0 Ca and 1JCaCb respectively.



Cax ! Cax cos p1 J C0 Ca D cos p1 J CaCb D0

þ 2C0y C0z sin p1 J C0 Ca D cos p1 J CaCb D0

þ 2Cay Cbz cos p1 J C0 Ca D sin p1 J CaCb D0

 4Cax C0z Cbz sin p1 J C0 Ca D sin p1 JCaCb D0

The picture seems more complicated because four different variants of the basic experiment are necessary to separate the four different components of the multiplet and achieve virtual decoupling. However, the underlying ideas are the same. A technical complication derives from the fact that, due to the proximity of Ca and Cb chemical shifts, it is very difficult to selectively invert Cbs while leaving Cas unperturbed. A possible solution to this problem consists in the employment of 13C p band-selective pulses only on Cas or, alternatively, on both Cas and Cbs, providing a tool to achieve the discrimination needed. In the case of the DIPAP approach [34] the length of the block is determined by the Ca–Cb coupling (Fig. 8A–D). The central pulse that is either selective on Ca (and thus prevents evolution of the Ca–Cb coupling, Fig. 8A and B) or that also inverts Cb (and thus allows for evolution of the Ca–Cb coupling, Fig. 8C and D) is used to detect the IP and AP components of the Ca–Cb coupling. Changing the positions of the p pulses on C0 enables us to acquire the IP (Fig. 8A and C) and AP (Fig. 8B and D) components respect to the C0 –Ca coupling. The four independent components acquired are shown in Fig. 8E. Their linear combinations enable us to separate

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the four multiplet components. They can then be shifted to the center of the original multiplet to eliminate the large one-bond homonuclear 13C couplings involving Ca. Different linear combinations of the data also yield properly decoupled Ca signals also for glycines, which lack the Cb, without the need to acquire a second set of data. Separation of the four different lines in the multiplet is very efficient and leads to the simplification of the original multiplet following removal of the two large couplings. The DS3E approach [37], illustrated in the bottom of Fig. 8(F–L), is based on simultaneous acquisition of the four independent components of the signal, changing their relative signs, to enable subsequent separation through the appropriate linear combinations. The approach exploits the use of band-selective pulses either before or after the block for the appropriate spins. A change in phase of p/2 for two out of the four components is necessary before the final shift of the different components to the center of the multiplet to achieve homonuclear decoupling, as illustrated in panel K. The overall length of this pulse sequence element is ½ of that of the DIPAP approach and for this reason it may provide a valuable alternative when fast transverse relaxation or dephasing constitutes a limiting factor. Summarizing, what are the major differences and potential complications of Ca direct detection compared to C0 direct detection? As mentioned above, it is technically more difficult to selectively invert Ca with respect to Cb, a key requirement for spin-state selective methods. Although pulses with improved performance are in continuous development, the Ca and Cb chemical shift ranges are quite close to one another. Therefore some compromises in the choice of RF pulses are necessary primarily affecting residues whose Ca and Cb chemical shifts are close. Ca–Cb couplings are quite uniform, but less than Ca–C0 couplings, another aspect that may influence the performance of the method. The many linear combinations needed to achieve homonuclear decoupling also introduce additional noise, reducing the final gain in sensitivity. Despite these potentially adverse features, the two approaches (DIPAP and DS3E) can easily be implemented in experiments ending with in-phase Ca magnetization (for example in 13C–13C NOESY or 13C–13C TOCSY experiments), or in experiments ending with a C0 –Ca or N–Ca coherence transfer block [15]. The methods were initially tested on ubiquitin and then used to study different kinds of proteins (paramagnetic, very large multimeric assemblies, intrinsically disordered proteins) [34,37,50]. The approaches described above in detail for C0 and Ca direct detection can obviously also be implemented for other kinds of spins (methyl groups, aromatic Cbs) [37,51]. Finally, dedicated isotopic labeling strategies have also been developed to chemically eliminate the need for homonuclear decoupling, simply removing the directly-bound 13C spins. This definitely simplifies the problem of homonuclear decoupling [52,53]. However, it also means that the one-bond carbon–carbon couplings are not available for coherence transfer, and so a limited set of experiments can be acquired on a specific sample and such samples need to be prepared specially. Of course, in case of complex systems the cost of an extra sample can be worth it!

6. Spin-state-selective methods to measure couplings Before a glimpse of the wide array of experiments based on 13C direct detection that have become generally used thanks to the good performance of spin-state-selective methods for homonuclear decoupling in the direct acquisition dimension, a few comments are due on the fact that these experiments can also be used to determine the values of coupling constants. The various tools described above (IPAP, S3E) are widely used nowadays for

Fig. 9. Selected examples of the spectra acquired on a standard 13C, 15N labeled ubiquitin sample at 700 MHz using variants of the (HACA)CON experiment for the determination of the one-bond couplings [57]. (A) Portion of the (HACA)CON spectrum recorded with the experiment modified to determine the C0 –N coupling; the two components on the left (top: IP, bottom: AP) are summed and subtracted in order to obtain the two components of the doublet in two different spectra, on the right (top: up component, bottom: down component). (B) Portion of the (HACA)CON spectrum obtained with the experiment modified to determine the Ca–C0 coupling through the ‘‘accordion’’ principle [59,99]. For clarity only the IP component is shown from spectra in which the evolution of the coupling was scaled by 0.5, 1 and 2 times with respect to chemical shifts (from left to right). Adapted with permission from Ref. [57].

the determination of one-bond couplings [35,40,54] and thus can also be used to determine C0 –Ca and Ca–Cb couplings. As an example, the experiment by Clore [55] has actually been proposed for this purpose. The digital resolution that can be achieved in the direct dimension is definitely an appealing aspect, and can be exploited for the determination of the C0 –Ca coupling [55,56]. The great interest in the determination of the large one-bond couplings, with the aim of extracting residual dipolar couplings to achieve structural and dynamic information, has also stimulated the implementation of spin-state selective approaches in a more general way in simple 2D 13C detected experiments to be used in all cases in which 13C detection provides advantages [57]. As mentioned above the C0 –Ca coupling can be determined by simple inspection of the two sub-spectra containing the two multiplet components necessary for C0 virtual decoupling. However, the implementation of the IPAP or S3E methods in indirect dimensions of NMR experiments actually offers even more possibilities. Several different couplings can be determined; the evolution of the coupling with respect to that of chemical shifts in indirect dimensions can be modulated [58,59] as schematically indicated in Fig. 9, and different contributions to the signals can be more easily suppressed/refocused than in the direct acquisition dimension. Minor modifications of simple 2D 13C detected experiments such as CACO and CON, can thus be used to determine essentially all the desired one-bond couplings involving backbone nuclear spins. For example the (HCA)CON version of

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the CON experiment was modified in various ways exploiting the IPAP approach in the indirect dimension to provide the values of the C0 –Ca, Ca–Ha, C0 –N, HN–N couplings [57]. As an example, Fig. 9 shows how the apparent value of the splitting can be modulated by appropriate selection of the incremented delay in which the desired scalar coupling evolves.

7. Implementation in solution and solid-state multidimensional NMR experiments The tools described above for homonuclear decoupling can in principle be implemented in any multidimensional experiment based on 13C direct detection of 13C labeled proteins. As discussed above, the building blocks described can be added at the end of experiments based on either C0 or on Ca direct detection. Alternatively, if the final coherence transfer step is a refocusing block, small variants enable homonuclear decoupling to be achieved without introducing additional pulses and delays into the pulse sequences. Finally, when the last coherence transfer step consists of refocusing of the coupling to be suppressed, implementation of the homonuclear decoupling approach (S3E) may even contribute to reducing the overall duration of the pulse sequence, as schematically indicated in Fig. 4. The implementation of homonuclear decoupling methods in 13C detection for biomolecular NMR applications has contributed a wide array of experiments to study proteins, as briefly summarized here, grouping the different experiments into several broad classes (coherence transfer type, C0 or Ca detection, starting polarization source, implementation of longitudinal relaxation enhancement techniques, solution/solid-state). The possibility to acquire narrow C0 lines enabled the development of a complete set of triple resonance experiments in which carbonyls are detected and a variety of different correlations are acquired and used to perform complete sequence specific assignment [33,48,60–64]. The set of experiments exploits scalar couplings for coherence transfer (3D CBCACON, 3D CBCANCO, 3D (HN)CANCO, 3D COCON, 4D and 5D experiments). When only one-bond couplings are exploited, one obtains the set of experiments (3D CBCACON, 4D HCBCACON) to identify the correlations for each amino acid. This enables us to count the spin systems, but not to achieve sequence specific assignment. To achieve this, additional experiments exploiting either two- or three-bond scalar couplings are necessary: these include the 2JNCa, 3JC0 C0 couplings that enable the complementary correlations to be detected for sequence-specific assignment. The set of 3D experiments based on C0 detection is now routinely used for several applications [65–71]. More recently, thanks to the development of new hardware (more sensitive instruments optimized for 13C detection) and software (non-uniform sampling and corresponding processing tools), 4D and 5D experiments based on C0 detection have also been proposed and contribute to expanding the suite of experiments available to study systems characterized by extensive overlap and relatively long coherence lifetimes, as in the case of unfolded proteins and intrinsically disordered protein (IDPs) [72– 76]. Analogous experiments based on Ca direct detection have been developed, including the COCA, CAN, CACA, CACAN, and CAN-inter [52,53]. These are more limited in number because of the more demanding requirements for homonuclear decoupling respect to the one for carbonyls. Therefore, as a general rule for solution applications, when C0 detection works, it is definitely the first choice. Ca detection becomes important for those cases in which C0 detection is not applicable or when complementary information is needed [34,37]. For example, with high molecular mass at high fields, Ca detection in deuterated proteins may

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become the only possible solution, stimulating the design and application of Ca detected experiments, as indeed has already happened [37,52,53]. Alternatively, direct detection of Ca may also be appealing for the large Ca chemical shift dispersion, as recently proposed for IDPs [50]. Depending on the kind of system to which they should be applied, various different flavors of these experiments have been proposed that differ mainly in the starting polarization source (13C or 1H) [2,9,48,63,64,77] and in whether the nuclear spins are manipulated in a non-selective manner or in a selective way, to exploit where possible longitudinal relaxation enhancement techniques [61,72,78,79]. The final class of experiments, in general acquired through 13 C-start, 13C detection methods, consists of 13C–13C NOESY experiments that can provide an alternative in cases where coherence transfer mediated by scalar couplings fails [22,32,80,81]. These are quite extreme situations, in which only a limited amount of information is available through solution NMR experiments. Therefore it is worth noting the contribution of 13C detected experiments to accessing information. Homonuclear decoupling can also provide efficient line narrowing in these cases to improve spectral quality, provided that care is taken to minimize relaxation losses. The implementation of DS3E for Ca detection and of S3E for detection of C0 and of methyl groups has been demonstrated for the case of ferritin [37]. With improvements in instrument technology and sample preparation, the linewidths of the 13C signals observed in uniformly labeled biosolids have also narrowed down to numbers that are comparable to the scalar 13C–13C couplings. Therefore it makes sense to try to implement homonuclear decoupling in the solid state too [30,31,36,42–44,82,83]. It is worth noting that the approaches to virtual homonuclear 13 C decoupling described above, were initially proposed for solid state NMR spectroscopy of biological systems [30,31,36], where 13 C has been, at least until recently, the nucleus of choice for direct detection. They have been extended and further developed for solution NMR applications [15,34], and subsequently reintroduced into solid state NMR [42–44,82,83] providing an interesting example of how effective cross fertilization between these two fields can stimulate fast progress. Indeed the methods were first implemented for 13C–13C 2D solid-state MAS correlation experiments using the IPAP approach [30,31]. A further step forward consisted in implementing the S3E method for acquisition of doublecross-polarization CON and CAN 2D experiments [36,43]. Further improvements in line narrowing, due for example to the higher

Fig. 10. A schematic illustration of an interesting property of virtual decoupling approaches. The top panel reports the multiplet structure expected for 13C signals of C0 and (non-GLY) Ca in a uniformly 13C labeled protein. Decoupling can be performed by either applying radio frequency irradiation decoupling schemes to one of the two spins involved in the coupling (middle panels) or by exploiting spinstate-selective approaches (bottom panel). In the last case, mutual decoupling of the two signals can be achieved.

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spinning frequencies available, have also contributed to the implementation of scalar-type experiments for solid state biomolecular NMR applications, as shown by several experiments that are very similar to those applied in solution [42,44,83,84]. These include the CON, CAN, and CANCO as well as INADEQUATE and various COSY variants. In all these cases the implementation of homonuclear decoupling in the acquisition dimension brings a significant improvement in resolution, and in most cases also a parallel increase in sensitivity. One interesting property of the virtual decoupling methods that emerged while implementing these techniques in solid state NMR experiments, where many of the commonly used experiments are based on the acquisition of both C0 and Ca, is that the coupling interaction is preserved, thus allowing decoupling of the two spins involved, without losing any of the information on the two (Fig. 10). This is a subtle but interesting difference from the methods in which radio frequency irradiation decoupling schemes are applied to one of the two spins involved in the coupling, where information on one of the two spins is lost. This property has been exploited in the solid state [43,83] and may also result useful for applications in solution.

8. Selected examples The principles exploited for virtual homonuclear decoupling in C direct detection have been described in detail and demonstrated on standard samples (13C uniformly labeled alanine and ubiquitin). Now we would like to show some examples of applications to real cases of general interest. The field in which 13C detected experiments have become most widely used is that of intrinsically disordered proteins (IDPs), which have recently attracted the attention of the scientific community thanks to their properties of high disorder and flexibility that provide functional advantages complementary to those of structured proteins, a property unexpected at least until a few years ago [85]. The lack of a stable 3D structure and the solventexposed protein backbones have, as major consequences, a dramatic reduction in chemical shift dispersion and pronounced

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amide proton exchange broadening, both factors influencing protons to a larger extent than 13C spins [14,86]. On the other hand, the high flexibility is also responsible, in the cases of IDPs amenable to NMR, for long coherence lifetimes enabling us to plan quite complex experiments. Needless to say, the advantage of performing heteronuclear direct detection depends critically on the performance of homonuclear decoupling, otherwise doubling (or even quadrupling) the number of signals would definitely cancel the advantages provided by the favorable 13C chemical shift dispersion (Fig. 1). The good resolution provided by 13C detection is readily evident (Fig. 11). This is maintained even when approaching physiological conditions (temperature and pH), as shown in Fig. 11, where often amide proton signals become too broad to be detected because of exchange broadening (and detection of Ha protons in the 2D mode is affected by quite significant overlap) [68,69,87]. The combined application of homonuclear and heteronuclear spin-state-selective decoupling, described in the previous section (Fig. 6), can provide further improvements in resolution, making C0 detected NMR experiments a very useful tool for the study of IDPs [86]. 13 C detected experiments also find application in studying systems where very efficient relaxation processes do constitute the limiting factors, either because of paramagnetic contributions or from slow tumbling (e.g. in paramagnetic proteins or very large multimeric protein assemblies) [22,37,81,88–92]. In both cases, protons are not detectable either because they are not there (2H isotopic labeling is necessary for the study of very large multimeric proteins assemblies) or because they relax too fast (due to the presence of a paramagnetic center or to very slow tumbling). Therefore 13C detection offers a way to recover information that would otherwise be lost. Also in this case, implementation of homonuclear decoupling methods enables better resolved spectra to be acquired provided that relaxation losses are tolerable. The S3E/DS3E homonuclear decoupling approaches were indeed developed for this purpose to be able to use them for this kind of rapidly relaxing systems [37]. Paramagnetic tags are becoming widely used to access longrange information through the determination of a variety of paramagnetic effects, in particular paramagnetic relaxation enhance-

Fig. 11. 2D spectra correlating the backbone amide nitrogen either with the directly bound amide proton (1H–15N HSQC) or with the directly bound carbonyl (13C–15N CON). (Left panels) 1H–15N HSQC and (right panels) 13C–15N CON acquired on 13C, 15N labeled a-synuclein (1.0 mM in 20 mM phosphate and 200 mM NaCl at pH 7.4 with 5% D2O for the lock signal), a 140 residue human intrinsically disordered protein implicated in neurodegenerative diseases, are shown as a function of increasing temperature (from left to right, for each type of spectrum): 285.7 K; 295.5 K; 304.8 K. Each spectrum was acquired with one scan per increment, and the same spectral resolution (in Hz) was chosen for the two experiments. Adapted with permission from Ref. [87].

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Fig. 12. (H)NCO-S3E (A) and (H) NCA-S3E (B) and J-(H)N(CA)CO-S3E (C) and J-(H)N(CO)CA-S3E (D) spectra of diamagnetic microcrystalline [U–2H, 13C, 15N]-(E, ZnII)-SOD 100% back exchanged at the amide sites. All peaks (intra- and inter-residue) are labeled with respect to their nitrogen shift. (E) Overlay of regions from the four spectra, illustrating a backward walk along the backbone of the protein providing sequential assignment (color coding of the cross peaks is the same as in panels A–D). The experiments were run on a 1 GHz spectrometer at 60 kHz MAS. Adapted with permission from Ref. [83]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

ments (PREs). 13C detected protonless experiments were initially proposed to improve the methods for studying paramagnetic systems, and thus also find application in this field, providing additional information [68,90,92]. Immobilization of proteins in biosolids eliminates the problem of slow tumbling for large proteins, offering the possibility to acquire complementary information through solid state NMR [93]. As mentioned above, fast spinning, high magnetic fields and sample preparation strategies have dramatically reduced the intrinsic line width in 13C detected spectra of biosolids stimulating the routine use of spin-state-selective homonuclear decoupling approaches. As an example of the quality of the spectra that can be obtained, Fig. 12 shows the case of dimeric SOD [83].

Finally, 13C detected methods have also been tested for in-cell studies of proteins [95–96]. While for folded proteins 1H detection is in general preferable because 2D HN correlation spectra are quite well resolved and provide a satisfactory picture of the status of a protein in the cell, the situation is more complicated in case of IDPs, where the chemical shift overlap and increased signal linewidth deriving from the in-cell environment significantly reduce the number of signals for which atomic resolution information can be obtained from simple 2D HN correlation spectra. Therefore it was nice to see that 2D CON maps can be acquired in a short time, comparable with the lifetime of in-cell samples [94–96], and that 13C signals are less affected by inhomogeneous broadening than 1H ones (Fig. 13) [97]. Needless to say, this application

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Fig. 13. Selected regions of 2D 1H–15N (2D HN SOFAST) and 2D 13C–15N (2D Ha-flipCON) correlation spectra acquired on a-synuclein overexpressed in Escherichia coli cells (magenta contours) and on cell lysates (blue contours). The regions selected include those containing correlations involving GLY nitrogen nuclei (all spectra) as well as the one containing correlations involving PRO nitrogen nuclei (2D Ha-flipCON only). The traces of representative cross peaks extracted from the spectra are also shown: (A) HN GLY 73, (B) HN GLY 67 (left peak), (C) HN GLY 86 (left peak), (D) C0 ALA 107, (E) C0 VAL 66 (right peak), (F) C0 ALA 85 (13C traces are extracted from 2D Ha-flipCON spectra). Their comparison shows that the broadening effect produced by the in-cell environment is larger for 1H than for 13C. Adapted with permission from Ref. [97]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

is only possible because of the good performance of homonuclear decoupling.

partially supported it. Tomáš Hošek is acknowledged for comments on the manuscript.

9. Conclusions

References

Spin-state-selective methods for homonuclear decoupling of C0 and Ca have significantly contributed to converting 13C detected NMR experiments into a useful tool for biomolecular applications. Initially proposed for solid state NMR applications, they were implemented for a wide array of solution NMR experiments and then further improved for solid state applications, providing an example of how common and complementary aspects of solution and solid state NMR can stimulate faster progress. Their good performance also enables their application to the study of IDPs in cells. Acknowledgements The EC Projects BioNMR (Contract # 261863) and IDPbyNMR (Contract # 264257) contributed to stimulating this work and

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