13C-detected protonless NMR spectroscopy of proteins in solution

Progress in Nuclear Magnetic Resonance Spectroscopy 48 (2006) 25–45 www.elsevier.com/locate/pnmrs

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C-detected protonless NMR spectroscopy of proteins in solution

Wolfgang Bermel a, Ivano Bertini b,*, Isabella C. Felli b, Mario Piccioli b, Roberta Pierattelli b b

a Bruker BioSpin GmbH, Rheinstetten, Germany Magnetic Resonance Center and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy

Received 2 August 2005 Available online 20 December 2005

Keywords: 13C-detected NMR; Protonless NMR; Sequential assignment; Spin-state selection; Paramagnetic proteins

Contents 1. 2. 3. 4. 5. 6.

Why the need of hetero detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instrumental aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The problem of the 13C–13C coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A protocol for the assignment of backbone and side chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection of resonances in paramagnetic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Why the need of hetero detection The NMR determination of the structure of large biological macromolecules in solution is primarily limited by the fast transverse relaxation that broadens lines and reduces spectral resolution. Several steps ahead have been made recently to overcome this limitation. In particular, the constructive use of cross-correlated relaxation phenomena enables a reduction of the effective transverse relaxation rates of specific spins, such as backbone NH groups [1] and aromatic CH groups [2]. More recently, selected cross-correlation rates were exploited to obtain line narrowing for methyl [3] and methylene [4] groups. The other very efficient way to reduce transverse relaxation rates consists in 2H isotopic enrichment [5–8]. The lower gyromagnetic ratio of 2H compared to that of 1H contributes to a drastic reduction of dipole–dipole interactions thus providing less efficient relaxation mechanisms. Selective isotope labelling also simplifies crowded NMR spectra and allows the selection of specific resonances [9–11].

* Corresponding author. Fax: C39 055 4574271. E-mail address: [email protected] (I. Bertini).

0079-6565/$ - see front matter q 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.pnmrs.2005.09.002

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Direct-detection of heteronuclei, and of 13C in particular, offers a valuable alternative to 1H detection. The idea of 13C direct-detection has its roots in the early days of NMR and has been used for decades to study small molecules such as organic and inorganic compounds [12] or metabolites [13]. However, 13 C direct-detection has not been widely applied to study biological macromolecules. Thanks its large 1H gyromagnetic ratio, the 1H sensitivity is indeed intrinsically much higher than that of 13C, and proton-detected experiments are usually convenient. Moreover, most of the efforts to improve instrument technology have been devoted to increase the sensitivity of 1H detection rather than that of the heteronuclei. The availability of increasingly high magnetic fields and the advent of cryogenically cooled probeheads [14] has now moved the sensitivity of NMR spectroscopy into regions that were unforeseeable less than a decade ago. As a consequence, 13 C sensitivity has been dramatically increased up to a level suitable to turn 13C detected experiments on enriched samples into routine methods for biomolecular NMR applications. The features of 13C NMR spectra can be realized from Fig. 1, which shows a 13C–15N correlation experiment recorded on 13C,15N labeled reduced monomeric superoxide dismutase (SOD, 15 kDa) at 14.1 T. The map shows all expected cross peaks with excellent resolution, including those of Pro residues as well as those of Asn and Gln side-chains. The spectrum

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Fig. 1. The 2D CON-IPAP spectrum acquired on a 1.5 mM sample of C, N labeled reduced monomeric SOD with a 14.1 T Bruker Avance spectrometer equipped with a cryogenically cooled probehead optimized for 13C detection at 298 K. The pulse sequence used is reported in Appendix.

shows 161 cross peaks out of the expected 163 while 159 cross peaks out of the 169 expected are observed in the 1H–15N HSQC spectrum. The usual drawbacks of conventional 1H–15N HSQC experiments (e.g. reduced intensity for those NH in fast exchange, and solvent-suppression dependent artefacts) do not affect signal intensity in a C–N experiment. The interest in 13C NMR lies in the fact that the line narrowing due to the observation of a low-g nucleus allows systems with large molecular mass to be investigated. Other scenarios can be envisaged in which 1H may be difficult to detect and 13C detection can be helpful. This is the case for protein regions characterized by chemical/conformational exchange [15], regions that in many cases are important for the protein function, where the backbone NH protons could be difficult to detect, interrupting sequence specific assignment. Unfolded systems, where the chemical shift dispersion of 1H resonances may be unfavourable, can efficiently be characterized using 13C NMR. This is also true for paramagnetic systems, where the contributions to relaxation arising from the paramagnetic centre are automatically reduced by more than a factor of 10 by shifting from 1H to 13C thanks to the smaller gyromagnetic ratio. The transverse relaxation rates as well as coupling topologies constitute the main aspects to be taken into account in the design of 13C direct-detection experiments. Through 13C directdetection, 13C spins in 2H labeled proteins as well as quaternary carbons can be directly monitored without requiring several transfer steps as would be the case for NH detected experiments, opening more possibilities for designing experiments and also avoiding the need to suppress the solvent signal. The 13C nuclear spins with the most promising transverse relaxation properties at high fields and on systems of high molecular mass are aliphatic nuclei in deuterated molecules [16]. Indeed the dominant contribution to relaxation is given by dipole–dipole interactions, which are only marginally field dependent, and thus give the opportunity to best exploit the advantages in terms of sensitivity

Fig. 2. Transverse relaxation rates of C 0 , Ca and Ha nuclei calculated as a function of the overall rotational correlation time at two different magnetic fields (11.7 T, dashed lines, and 16.4 T, continuous lines). Ca relaxation rates were calculated both for a protonated (Ca(H)) or a deuterated (Ca(D)) protein. The relaxation contributions considered were: chemical shift anisotropy [98, 99], one-bond dipole–dipole interactions [23], and various dipole–dipole interactions with 1H (or 2H) to mimic the average proton density in a protein.

of high magnetic fields [17]. As an example of the line narrowing that can be obtained by shifting from 1H to 13C, transverse relaxation rates of Ha and of Ca are shown in Fig. 2 as a function of the rotational correlation time tr. Upon 2H labeling, Ca transverse relaxation rates become very small also for large values of tr. The disadvantage of aliphatic nuclei is that the carbon–carbon coupling topology yields complex multiplets in the direct acquisition dimension. The basic building blocks needed to fully rely on 13Ca detection, which is recommended for deuterated proteins and for large molecular weights, are presented. Carbonyl nuclei (designated C 0 ) are characterized by a large chemical shift anisotropy that constitutes the dominant effect on transverse relaxation, that significantly increases with increasing magnetic field (Fig. 2) [18–20]. However, they present a simpler coupling pattern, with just ONE strong carbon–carbon one-bond scalar coupling. A consistent set of experiments to perform complete resonance assignment can be done relying mainly on the detection of 13C 0 nuclei. These experiments can also be useful for assigning signals from nondeuterated proteins of medium size. The less efficient relaxation pathways for 13C relaxation compared to those of protons also cause a reduction of 13C longitudinal relaxation rates, especially in the case of 2H labeling. This effect, that on one-hand causes longer recovery times (i.e. an increase in the overall duration of experiments), on the other hand can also be used constructively in 13C–13C NOESY experiments. In the next sections we will briefly review the recent efforts to improve the technology of probes and the methods currently available for removing the signal splitting due to the presence of large 13C–13C couplings. Provided these two problems are tackled and dedicated pulse sequences are developed, high quality data can be obtained and a sequence-specific assignment strategy based only on heteronuclei can be proposed [21].

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This alternative strategy, that does not actively exploit protons and for this reason we called protonless NMR, offers an alternative approach to the standard triple resonance assignment strategy. Such an approach does not aim at replacing methods relying on NH-detected TROSY experiments, which combine favourable relaxation properties due to 2H labelling, constructive use of relaxation interferences and the sensitivity of 1H direct-detection [22]. However, the fact that protonless sequential assignment of proteins is nowadays feasible constitutes a breakthrough per se. 2. Instrumental aspects The sensitivity in NMR experiments is given by Eq. (1) 3=2 1=2 S=N zAgexc g3=2 obs B0 Nscan

(1)

were gexc and gobs are the gyromagnetic ratios of the excited and of the observed nuclei, respectively, A is the number of spins, Nscan is the number of scans and B0 is the strength of the applied magnetic field [23]. Therefore, neglecting relaxation, 1 H observed experiments have a sensitivity gain of a factor of 32 (i.e. (gH/gC)5/2) compared to the corresponding experiments starting and ending at 13C nuclei (i.e. HCCH COSY or HCACO [24] vs 13C–13C COSY or CACO experiments [25]). Recent developments in probe technology and the availability of NMR spectrometers operating at high magnetic fields hold promise for dramatic improvements in sensitivity, not only for 1H but for 13C as well. 13C sensitivity, at least in NMR spectrometers devoted to biomolecular NMR applications, has so far been sacrificed in favour of 1H sensitivity. Indeed, as far as probe design is concerned, it is generally necessary to achieve a compromise between the performance of different nuclei in terms of sensitivity and pulse lengths. Generally, optimal sensitivity for a specific nucleus is achieved for a 2-coil probe by using the inner coil for that nucleus, at the expense of the performance of the other nuclei being on the outer coil. Probeheads optimised for 13C are nowadays available, such as dual probeheads, with the inner coil optimised for 13C and with the possibility to pulse on 1H, and triple-resonance probeheads, where the inner coil is used to pulse on 13C and 15N. The evolution of 13C signal-to-noise (S/N) ratio for these probeheads over the years is shown in Fig. 3. Changing the focus from 1H to 13C has prompted manufacturers to concentrate their efforts in obtaining a new generation of probeheads in which the main goal is optimized sensitivity on 13C, coupled with the capability to excite and/or decouple 1H, 2H and 15N. Indeed, a prototype roomtemperature 13C-observe triple-resonance probehead has been built yielding a substantial increase in 13C sensitivity and has greatly contributed to the expansion of the set of experiments available [21,26,27]. A very efficient way to increase sensitivity consists in reducing the thermal noise that contributes to the observed signal, leading to the so-called cryogenically-cooled probeheads. This has recently been extensively exploited for 1H allowing an increase in proton sensitivity by a factor of about

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Fig. 3. The specified signal-to-noise ratio of 60% benzene-d6 in 40% p-dioxane (the so-called ASTM sample) for 13C-observe probes plotted as a function of the year when a probe was first introduced at a particular magnetic field (expressed as 1H operating frequency in MHz). Black dots denote the 13C sensitivity of conventional 13C-observe probes. For the cryogenically-cooled probes, the 13C sensitivity of 13C-detection probes are indicated with triangles, the 13C sensitivity of the triple-resonance probes with both cryogenically enhanced 1H and 13C capacities are denoted by diamonds and the four-nuclei heteronuclear-observe probe is indicated with square (adapted with permission from [14]).

3–4 [14]. The improvement in the sensitivity is mainly due to the reduced thermal noise achieved by decreasing the operating temperature of the coils and of the 1H preamplifier. Inclusion of a cryo-cooled preamplifier also for 13C without changing the probe design of a standard probe for triple-resonance experiments has given a significant increase in 13C sensitivity without needing to make any compromise on 1H. The behaviour of these probes in terms of S/N is shown in Fig. 3 (diamonds). Dual cryogenically-cooled probes optimised for 13 C sensitivity (Fig. 3, triangles) are also available but as yet they do not allow pulsing on 15N and, therefore, are mainly used to study small molecules. Obviously, a combination of the cryo-technology with the design of triple-resonance probes with improved 13C sensitivity is in principle possible and would contribute to further increase the application of 13C direct-detection experiments in biomolecular NMR. Furthermore, 13C detected experiments are less prone to sensitivity losses due to high salt concentrations, particularly relevant in cryogenically-cooled probeheads. It has been shown that 13C detection significantly extends the range of sample conditions under which 13C detection becomes competitive with proton detection [28]. In order to perform a triple-resonance experiment using 13C detection, we should also consider aspects other than sensitivity. The minimal hardware requirements in terms of transmitters and channels are the same as for standard tripleresonance experiments (1H, 13C, 15N and 2H in the case of deuterated proteins). In addition, filters may be required to prevent cross talk between the 13C and 15N channel. This may become an issue when 15N is decoupled during 13C detection. These filters could be part of a band-selective preamplifier or external devices. Additional hardware may also be needed to perform band-selective 13C homodecoupling. Band-selective homodecoupling (BH) of 13C 0 from 13Ca and vice versa may be desirable in some experiments, but requires some more consideration. During acquisition, data points are sampled at a speed given by the amount of oversampling.

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Fig. 4. Dwell cycle showing the acquisition status, the pulse and the dwell window on the time axis. The durations of the decoupling pulse tp, of the hdduty and of the dwell time are indicated. Time periods D are switching delays, which are predetermined fractions of the dwell time.

In any case the receiver is unblanked during the whole period. In order to achieve homodecoupling, radiofrequency has to be irradiated during a fraction of the nominal dwell time, which in turn is defined as the reciprocal of the spectral width of the spectrum. This fraction is called hdduty, the homodecoupling on/off ratio given as a percentage of the nominal dwell time. During hdduty as well as some short delays for switching from observe to transmit mode and vice versa, the receiver system is blanked (Fig. 4). This results in a reduction in S/N with respect to the theoretical factor of 2 according to [29]: rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi hdduty S=NðBHÞ Z 2S=N 1K (2) 100 A short value of hdduty would thus provide a higher S/N. However, a smaller hdduty value implies higher RF power, which may cause unwanted probe ringdown and sidebands. All recent reports have used a 20% hdduty, which corresponds to a maximum theoretical S/N of 1.79 [29–31]. Experimentally measured S/N values rarely achieve such an ideal value and show a significant frequency dependent variability. This prevents a quantitative comparison of peak intensities within the same spectrum, but does not prevent the use of homodecoupling for R1 or R2 experiments, or any other quantitative experiment. Following the convolution theorem in Fourier transformation, an irradiation period spaced by a time interval much longer than irradiation produces sidebands spaced by n/r, where n is an integer and r is the dwell time, (in the case where small hdduty values are used). Therefore, the sweep width and the value of hdduty should be chosen to avoid overlap of the signals of interest with sidebands arising from homodecoupling. To perform homodecoupling, adiabatic chirp pulses, with 10 ms pulse length, 25% smoothing and sweep from low to high field have been used [32]. 3. The problem of the

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C–13C coupling

In 13C direct-detection experiments we need to face the problem of the large homonuclear one-bond carbon–carbon couplings that evolve during the acquisition delay.

The presence of these couplings is, of course, beneficial for coherence transfer efficiency but is detrimental to resolution in the acquisition dimension. In addition to the large one-bond 13 C homonuclear scalar couplings (1JCCZ35–55 Hz), there are also a variety of smaller homonuclear scalar couplings, such as 2 JCC, 3JCC, and 4JCC, that are generally within the resolution of the experiments. For this reason, we will focus hereafter on the one-bond scalar coupling constants, i.e. the 1JC 0 Ca and the 1 JCaCb. In the case of experiments with detection of the 13C 0 nuclei, the splitting is quite uniform and leads to doublets whose lines are separated by about 55 Hz. The same coupling is responsible for the primary splitting of the Ca carbon signal, to which the 35 Hz splitting of the Cb-coupling is added. In the recent literature, several methods have been proposed to collapse 1JCC splittings from the spectra [21,29,33,34]. Band-selective homodecoupling is the oldest method of collapsing homonuclear couplings in the direct acquisition dimension. In principle, the removal of the splitting due to two coupled spins with BH allows an increase of the S/N of a factor of about 1.8 (see Section 2), although this is seldom achieved. Furthermore, even in the case of Ca carbon decoupling while acquiring C 0 , care should be taken in defining experimental parameters such as hdduty and spectral width in order to avoid irradiation of the region of interest by sidebands of the band selective homodecoupling. Besides causing a Bloch-Siegert shift [35] BH induces decoupling sidebands around the signals of interest, since it uses composite pulse decoupling with cyclic schemes [29,36]. The intensity of the induced sidebands increases with the strength of the decoupling RF field and, therefore, it is desirable to use the minimum necessary decoupling power. Changing the phase of the sidebands in a controlled manner by starting the decoupling sequence at different times prior to acquisition permits one to cancel out to a large extemt those that are due to the cyclic scheme [29,36]. An elegant solution for ‘virtual’ decoupling is the use of spin-state selective schemes, such as in-phase anti-phase (IPAP) [37–39] and spin-state selective excitation (S3E) [39,40], widely used for the measurement of heteronuclear J couplings [41,42], and recently applied for removal of 13C–13C J-coupling in solid-state samples and in liquids [21,43]. In the IPAP approach (Fig. 5A,B), the removal of the splitting is accomplished by recording two FIDs for each increment, one for the anti-phase and one for the in-phase components. The IPAP scheme relies on complete interconversion between in-phase and anti-phase coherences and thus has duration of 1/(2JCC), where JCC is the relevant coupling. For C 0 acquisition, the shortest possible duration of the IPAP block is thus 9 ms. The in-phase and anti-phase components are stored separately and are combined to yield the two multiplet components. These are then shifted to the centre of the original multiplet (by JC 0 Ca/2 Hz) and summed to obtain a singlet [21,44], as shown in Fig. 5C. With the S3E scheme (Fig. 5D) two different experiments are acquired and store separately in which one component is absortive while the other is dispersive and which differ in the sign of one of the two components [21,45]. The sum and the difference of the two FIDs stored separately gives the

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Fig. 5. The IPAP and S3E approaches for C 0 direct-detection to remove the large Ca–C 0 splitting in the direct acquisition dimension. They are illustrated for the simple case of 1D experiments, but can be implemented in any experiment based on C 0 direct-detection, as discussed in the text. Band-selective 13C pulses are denoted by shapes (narrow and wide ones represent 90 and 1808 pulses, respectively). Panels A and B report the two variants of the pulse scheme for the IPAP approach to acquire C 0 (A for the in-phase and B for the anti-phase components, respectively). The results of the two experiments reported in panels A and B on a setup sample of 13C, 15N labeled alanine in D2O are shown in panel C and indicated with IP and AP, respectively. For 13C 90 and 1808 pulses, Q5 (or time reversed Q5) and Q3 shapes were used [100]. Decoupling of 1H and 15N was applied with waltz-16 [101] and garp-4 [102], respectively. The 13C 0 , 13Ca, 1H and 15N pulses were applied at 175, 55, 4.7 and 118 ppm, respectively. Panel C also shows schematically the approach employed to treat the data. Panel D shows the pulse scheme for the S3E approach to acquire C 0 . The two FIDs necessary to separate the in-phase and anti-phase components can be obtained by changing the phases of the pulses as indicated below. The data are treated as described in the text. The delay D is 1/(2JCaC 0 ) (9 ms). The phases are: (A) fIPAPZx,Kx and frecZx,Kx; (B) fIPAPZKy, y and frecZx,Kx; D) fS3E(1)Z458, 458; f1Zx, y; f2(1)Zx, y; frecZx,Kx and fS3E(2)Z458, 2258; f1Zx, y; f2(2)ZKx,Ky; frecZx,Kx where (1) and (2) are the two experiments required to separate the in-phase and anti-phase components. It is worth noting that the S3E requires half the time with respect to the IPAP approach.

absortive and dispersive component, respectively. The dispersive line in then phase-shifted by 908 and both components are frequency shifted to the center of the original multiplet [21,44]. The important difference with the IPAP approach is that the overall duration of the building block amounts to 1/(4JCC), where JCC is the relevant coupling to suppress, and thus is half of that of the IPAP approach. For C 0 acquisition, the block is 4.5 ms long. These building blocks (IPAP and S3E) can be implemented in any experiment based on C 0 direct-detection. In many of them they can be embedded within the last coherence transfer element already present in the experiment at no cost in terms of extra relaxation (i.e. it is not necessary to extend the overall duration of the experiment). In cases where the coupling to a 15N nucleus needs to be refocussed (in order to decouple 15N during acquisition) both building blocks can in principle be used. But here the use of an S3E element would not shorten the refocussing delay. So the IPAP approach is the preferred choice, since it is more robust than S3E with respect to small variations in the values of 1JCC [46]. Both versions can be implemented in those experiments that end with the refocusing of the C 0 –Ca antiphase signals (CACO, CBCACO, CCCO), or those in which the blocks necessary for spin-state are added at the end of the experiment prior to acquisition (NOESY, TOCSY). The S3E approach, due to its shorter duration, may have some advantages in terms of minimizing relaxation losses at high magnetic fields and with high molecular mass systems [45]. Along the same lines, Pervushin and coworkers proposed the acquisition of only the C 0 anti-phase component [47]. The absolute value of the anti-phase component then looks like an in-phase component and can be treated in an analogous way to

that described for the IPAP approach (see Fig. 5). A possible drawback of this method is that the positive and negative components of different signals in overlap will cancel out causing loss of information for systems characterized by extensive resonance overlap. On the other hand, as pointed out by Do¨tsch et al., acquiring the anti-phase component allows the removal of the last delays necessary to refocus it to in-phase that inevitably causes some relaxation losses [28]. Along the same lines as TROSY, an additional approach to select one of the two multiplet components in the direct acquisition dimension of a C 0 –Ca correlation experiment (named COCAINE), has recently been proposed, and provides an alternative way for ‘virtual’ decoupling of C 0 from Ca [48]. Other methods rely on signal processing algorithms to deconvolute the C 0 –Ca splitting. As an example, deconvolution using maximum entropy reconstruction has been proposed for this purpose [33,49]. These processing algorithms can be applied to spectra where either the in-phase or the anti-phase C 0 signal component [28,33] is acquired. Direct-detection of Ca is more complicated due to coupling to 0 C and Cb with large one-bond scalar-coupling constants (1JC0 Ca, 1 JCaCb). Therefore, if compared to C 0 direct-detection, two large couplings should be quenched in order to simplify the Ca line and obtain high-resolution. This can be achieved in principle with the same approaches discussed for C 0 , i.e. band-selective homodecoupling, spin-state selection and deconvolution. Pervushin and coworkers proposed triple band-selective homodecoupling where the three regions irradiated during Ca acquisition are the C 0 and the two Cb regions [31]. The performance of this approach is satisfactory in terms of collapsing the multiplet to a singlet, but the gain in S/N

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achieved is much less than predicted. The loss of signal is attributed to heteronuclear interference coming from simultaneous 15N and 2H decoupling [31]. Spin-state selective methods for Ca, such as the described IPAP and S3E approaches, are sufficient only for those nuclei that are singly split (e.g. Ca of Gly), but for the removal of the additional large one bond coupling to Cb we must rely on a scheme where the spin-state selection is applied repeatedly [21]. Two IPAP blocks, one specific for the C 0 –Ca coupling and one for the Ca–Cb coupling can be combined, as suggested for solid state applications [39]. Actually, the two IPAP blocks can be concatenated into a shorter block in which the total duration is determined by the value of the smaller coupling to consider. Fig. 6 shows the double IPAP implementation (DIPAP hereafter) that has been proposed for Ca [21]. For each increment, four experiments are acquired that differ in the selectivity of the Ca pulse at the centre of the delay that determines the effective evolution of the Ca–Cb coupling during this block and the position of the C 0 pulses that determine the effective evolution of the C 0 –Ca coupling during this delay. These four experiments, which yield the four possible Ca multiplets, are stored separately, combined through linear combinations and then shifted to the centre of the original multiplet in order to remove the primary splittings. The total length of the block is determined by 1/ (2JCaCb) and, despite compacting the two IPAP blocks into one, the duration is rather long (14 ms). However, relaxation rates of Ca nuclei are drastically reduced upon 2H labelling so that this approach becomes convenient for large proteins at high magnetic fields. Implementation of a ‘double’ S3E approach to Ca directdetection is also feasible. The methods discussed for C 0 and Ca acquisition can in principle be applied also to other carbon nuclei. However, the diversity of the side-chains of aminoacids is responsible for the large chemical shift dispersion of Cb nuclei, the partial overlap

A 13

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The methods described above to detect selected 13C nuclear spins whilst collapsing the large one-bond homonuclear carbon–carbon scalar couplings, as well as the availability of probeheads with improved sensitivity for 13C [14,53], have opened the way for the use of 13C direct-detection experiments in biomolecular NMR. We summarize here a set of experiments based exclusively on heteronuclei that allows one to perform a complete sequence specific assignment of a 13 15 C, N labeled protein without using 1H excitation and detection. These experiments are based on the most efficient

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4. A protocol for the assignment of backbone and side chains

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with other carbon nuclei of the side-chain and the different coupling topologies. Therefore, a general method of detecting side-chain nuclei by removing large one-bond carbon–carbon couplings in a single spectrum is not feasible. Partial solutions can be employed for specific sets of spins. Multiple band-selective homodecoupling has been proposed [31]. Also the DIPAP approach has been implemented in the experiment to correlate Cb and Cg in aromatic systems [50]. Similar approaches can be designed for methyl groups, or adapted to specific aminoacid types, as for example was done with the MUSIC approach [51]. Spin-state selective methods retain the information on the effective C 0 –Ca splitting. The latter contains a contribution arising from partial orientation of the molecule that provides precious structural information. These experiments are, therefore, suitable to accurately measure 13C–13C residual dipolar couplings, also when protons are not detectable or absent due to perdeuteration. The possibility of determining 13 C–13C residual dipolar couplings from fully coupled 13 C–13C TOCSY spectra has recently been described by Vo¨geli et al. [52].

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Fig. 6. The DIPAP method for Ca direct-detection to remove the two large Ca–C 0 and Ca–Cb splittings in the direct acquisition dimension. It is illustrated for the simple case of 1D experiments and it can be implemented in any experiment based on Ca direct-detection. Band selective 13C pulses are denoted by shapes (narrow and wide ones represent 90 and 1808 pulses, respectively). The two lines labeled with Ca and Ca/b actually indicate band-selective pulses that only affect Ca spins, unselective with respect to Cb, or both Ca and Cb spins. For 13C 908 and 1808 pulses, Q5 and Q3 shapes were used, respectively [100]. Decoupling of 1H and 15N was applied with waltz-16 [101] and garp-4 [102], respectively. The 13C 0 , 13Ca, 13Ca/b, 1H and 15N pulses were applied at 175, 55, 39, 4.7 and 118 ppm, respectively. Panels A–D report the four variants of the pulse scheme to acquire and store separately the four components indicated with IP–IP, AP–IP, IP–AP and AP-AP, respectively. The results of the four experiments performed on the set-up sample of 13C, 15N labeled alanine in D2O are shown in panel E. The delay D is 1/(2JCaCb’) (14 ms) and z is 1/(2JCaC 0 ) (9 ms). The phases are: fDIPAP(B)Zx,Kx; fDIPAP(A)Zy,Ky; fDIPAP(C)ZKy, y; fDIPAP(D)Zx,Kx; frecZx,Kx.

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31

Table 1 Selection of the protonless NMR experiments available, with the correlations observed in each experiment and an estimate of the number of scans necessary to acquire them compared to a CACO acquired with N number of scans Experiment 2D CACO–IPAP/S3E CBCACO–IPAP/S3E CCCO–IPAP/S3E CON–IPAP CANCO–IPAP CBCANCO–IPAP 3D CBCACO–IPAP/S3E CCCO–IPAP/S3E CANCO–IPAP CBCACON–IPAP CCCON–IPAP CBCANCO–IPAP

Correlations observed

Scans

Reference

Cai KCi0 Cbi KCi0 ; Cai KCi0 KCi0 ; Cai KCi0 Cb;g;d;3 i 0 Ni KCiK 1 a 0 a 0 Ci KCiK 1 CiK1 KCiK1 b b a 0 a 0 0 0 Ci KCiK1 ; CiK1 KCiK 1 ; Ci KCiK1 ; CiK1 KCiK1

N N 2N 2N 16N 16N

[58,83] [21,58] [58] [58,80] [26,27] [58]

Cbi KCai KCi0 ; Cai KCai KCi0 KCai KCi0 ; Cai KCai KCi0 Cb;g;d;3 i a 0 a 0 Ci KNi0 KCiK 1 ; CiK1 KNi KCiK1 b 0 0 ; C KN KC CaiK1 KNi KCiK i 1 iK1 iK1 b;g;d;3 0 0 ; C KN KC CaiK1 KNi KCiK i 1 iK1 iK1 b 0 a 0 0 0 C KN KC ; Cbi KNi KCiK Cai KNi KCiK i 1 iK1 iK1 1 ; CiK1 KNi KCiK1

N 2N 16N 2N 4N 16N

[21] [58] [26,27] [58] [58] [58]

building blocks of the conventional triple-resonance experiments [54–57] and are described in detail in the Appendix. In Table 1 the list of the experiments, including a summary of the correlations observed and the relative sensitivity, is reported. This heteronuclear assignment strategy is then critically discussed in terms of advantages and drawbacks with respect to the conventional sequence specific assignment strategy and is compared to other proposed approaches that partly rely on heteronuclear direct-detection. The exclusively heteronuclear assignment strategy starts with acquisition and analysis of the most sensitive experiments based on C 0 acquisition and on the exploitation of the large one-bond coupling constants for coherence transfer (JC 0 Ca, JCaCb, JCC and JNC 0 , Fig. 7). The CACO experiment [29] yields the intraresidue Ci0 –Cai correlation (n correlations for a protein of n aminoacids) as well as the correlations of carbonyl/carboxylate spins and the attached carbon of Asp/Asn ðCbi –Cgi Þ, Glu/Gln ðCgi –Cdi Þ. The Ci0 –Cai correlation can be detected through a variety of different schemes, by acquiring the in-phase or the anti-phase C 0 components [28] and eventually using signal processing algorithms to deconvolute the splitting [33,47], by using one of the techniques described in Section 2 to eliminate the JC 0 Ca coupling in the direct acquisition dimension (BH [29], IPAP [21], S3E [21], COCAINE [48]), by evolving single-quantum or double-quantum coherences (or both). Some of the different experimental schemes used to detect the Ci0 –Cai correlation are outlined in Fig. 8. The next experiment to consider is the CBCACO sequence [21] (Appendix, panel 1 for the IPAP version and panel 2 for the S3E version). This exploits the Ca –Cb coupling (JCaCb) through in COSY-type approach and yields for each residue, in addition to the Ci0 –Cai , also the Ci0 –Cbi correlation. The Cb chemical shift dispersion, which is much larger than that of Ca, allows one to reduce the overlap and to identify the aminoacid on the basis of the Cb chemical shift. The remaining carbons of the side chains of non-aromatic residues can be identified through the CCCO experiment [58] (Appendix, panels 3 and 4 for the IPAP and S3E versions, respectively) where C 0 is correlated, through Ca and by means of an additional TOCSY

step, to the rest of the side chain. By changing the spin-lock time, the relative intensities of the correlations observed can be modulated to detect all expected correlations. It is worth noting that the analysis of these experiments also yields in a straightforward way the assignment of acidic and amidic side-chain resonances as each aliphatic carbon is correlated to the backbone carbonyl and to the side chain carbonyl/ carboxylate nuclei, making the assignment procedure very simple. When overlap is severe, the CBCACO and the CCCO experiments can be extended in a third dimension by evolving the chemical shift of Ca to resolve ambiguities due to C 0 resonance overlap [58]. For aromatic residues, the correlation with the aromatic ring carbons ðCbi –Cgi Þ can be detected

Fig. 7. A schematic representation of sequence specific assignment through protonless NMR spectroscopy. In each panel, the key correlations necessary to perform spin system identification and sequence specific assignment are shown. The inset reports a schematic representation of the backbone of a protein and the magnetization transfer pathways responsible for the observed correlations. The experiments based on C 0 acquisition are reported on the left side. The CBCACO and the CCCO can also be acquired in the 3D mode evolving Ca in the third dimension in order to remove ambiguities deriving from C 0 degeneracy. The CANCO has been designed as a 3D with N evolution. The CON experiment is reported in an oblique way to indicate that it can be combined with the other C 0 -based experiments by including N evolution and obtain the corresponding 3D experiment. The TOCSY experiment with Ca acquisition is reported on the right.

32

W. Bermel et al. / Progress in Nuclear Magnetic Resonance Spectroscopy 48 (2006) 25–45

A 13

φ2

∆

C´

B

C

α/β

BH .Dec.

φ1

13

φrec FID

φ -t 1 3 2

φ1 t 1 2

C α/β 1 H 15 N

13

∆

∆−t 1 2

φ2

∆−t 1 2

y

13

C´ H/15N .Ddecoupling

φ3

∆ 2

∆ 2

BH φrec FID

1

.decoupling

PFG C 13

C

α/β

φ2 φ1 y ∆+t 1 ∆−t1 2 2

IP

∆ 2

∆ 2

φIPAP

13

C´ 1 H/15N.Ddecoupling

φrec FID .decoupling

PFG AP

13

13

D 13

C

α/β

C

α/β

∆ ∆ ∆ ∆ 4 4 4 4 φIPAP

C´

φ2 φ1 ∆+t1 ∆− t1 y 2 2

13

C´ 15 H/ N .Ddecoupling

∆ ∆ 4 4 φ3 φ4 φS3E FID

1

φrec

.decoupling

PFG E 13

C

13 1

α/β

C´

φ1∆+t φ2 ∆−t y 1 1 2 2 φ3 φ rec FID

H/15N .Ddecoupling .Ddecoupling

PFG

Fig. 8. Some of the different experiments to detect the C 0 –Ca correlation: (A) with multiple quantum evolution during the indirect dimension and bandselective homodecoupling; (B–D) with single quantum evolution during the indirect dimension and various approaches to remove the Ca–C 0 splitting (BH, IPAP, and S3E, respectively); and (E) with detection of the anti-phase component. Band selective 13C pulses are denoted by shapes (narrow and wide ones represent 90 and 1808 pulses, respectively). Two different panels indicate the variants necessary to separate the in-phase and anti-phase components for the CACO–IPAP experiment. Experiments were tested on different spectrometers. Shaped pulses, 1H and 15N decouplings, position of carriers and data treatment for IPAP and S3E experiments have been described in the caption of Fig. 5. The delay D is 1/(2JC 0 Ca) (9 ms). In (B) and (E) the delay D can be shortened to reduce relaxation losses with paramagnetic or large systems at the expense of transfer efficiency. The phases are: (A) f1Zx,Kx; f2Zx; f3Z2(x), 2(Kx); frecZx, 2(Kx), x; (B) f1Zx,Kx; f2Z4(x), 4(y); f3Z2(x), 2(Kx); frecZx, 2(Kx), x,Kx, 2(i),Kx; (C) f1Zx,Kx; f2Z4(x), 4(y); fIPAP(IP)Z 2(x), 2(Kx); fIPAP(AP)Z2(Ky), 2(y); frecZx, 2(Kx), x,Kx, 2(x),Kx; (D) f1Zx,Kx; f2Z4(x), 4(y); fS3E (1)Z4(458); fS3E (2)Z2(458), 2(2258); f3Z 2(x), 2(y); f4Z2(x), 2(y); frecZx, 2(Kx), x,Kx, 2(x),Kx; (E) f1Zx,Kx; f2Z 4(x), 4(y); f3Z2(x), 2(Kx); frecZx, 2(Kx), x,Kx, 2(x),Kx.

through the CGCB–DIPAP experiment that employs the DIPAP approach for Cb spins to eliminate the splitting due to coupling with Cg and Ca spins [50] (Appendix, panel 5). The identification of 13C nuclei within each aminoacid is thus complete. Correlation with backbone nitrogen nuclei is performed using the CON-IPAP experiment [21,58] through the one bond C 0 –N coupling (Appendix, panel 6). The experiment yields the sequential Ci0 –NiC1 correlation (nK1 correlations for a protein of n aminoacids) and the correlations for side chains of Asn ðCbi –Ngi Þ and Gln ðCgi –Ndi Þ residues. This experiment is quite sensitive (the most sensitive one involving N nuclei) and characterized by a very good chemical shift dispersion for both N and C 0 (Fig. 1). Therefore, it can be combined with the other most sensitive blocks (CBCACO, CCCO) to yield 3D experiments (CBCACON-IPAP, CCCONIPAP) [58] with C 0 in the direct acquisition dimension, Ca (or Ca/b, or Ca/b/g.) in the second dimension and N in the third (Appendix, panels 7 and 8). With the above mentioned set of experiments, complete identification of spin-systems can be achieved and each residue can be correlated with the following backbone amide nitrogen. However, in order to perform complete sequence-specific assignment an additional correlation through the backbone is necessary. This correlation can be detected by using the CANCO-IPAP experiment [26,58]. In this experiment (Appendix, panel 9) magnetization is transferred from Cai and CaiC1 to NiC1 and then to Ci0 giving the two correlations (Cai NiC1 –Ci0 and CaiC1 –NiC1 –Ci0 ) necessary for sequence specific assignment (Fig. 7). Actually the experiment is optimised to detect the CaiC1 –NiC1 –Ci0 peak, which is the ‘sequential one’, with most sensitivity [26]. Two available variants of the experiment allow one to discriminate between the two correlations (Appendix, panel 10, for the most sensitive) [27]. A further variant of the CANCO-IPAP experiment includes the transfer to the Cb, allowing the exploitation of Cb chemical shift information as an aid for spin-system identification (Appendix, panel 11). Therefore, complete sequence specific assignment using protonless NMR can be achieved through C 0 direct-detection [58]. An example of the sequence-specific assignment strategy employing these experiments is shown in Fig. 9. The 3D experiments are necessary when dealing with large systems in order to increase the resolution. As mentioned in the previous paragraphs, methods used to collapse the large one-bond couplings in the direct acquisition dimension for Ca spins allow the design of experiments based on Ca direct-detection [21]. Indeed the Ci0 –Cai correlation can also be detected in the COCA experiment employing the DIPAP approach in the acquisition dimension (Appendix, panel 12). The Ca-TOCSY [21], a TOCSY experiment where DIPAP has been implemented prior to acquisition (Appendix, panel 13), yields the correlations of Cai with all the aliphatic 13 C nuclei in the side chain (Ca=b=g. ), providing an additional i way to assign all 13C spins in non-aromatic aminoacid sidechains. Sequential correlations can be detected through the CAN-DIPAP experiment in which each Cai is correlated to the one and two bonds distant backbone nitrogen nucleus (Cai –Ni , CaiC1 –Ni ) (Appendix, panel 14).

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33

Fig. 9. The assignment strategy based only on 13C direct-detection 3D experiments is shown for a fragment Gly 73-Pro 74-Lys 75-Asp 76 in 13C,15N labeled monomeric SOD. The portions of Ca/ali–C 0 planes of 3D spectra reported in the Fig. are taken from the following experiments recorded at 14.1 T: (A) 3D CANCO– IPAP, (B) 3D CBCACO–IPAP, (C) 3D CCCO–IPAP, (D) 3D CBCACON–IPAP, (E) 3D CCCON–IPAP. For each residue, the figure shows in panel A the region of the Ca–C 0 plane of 3D CANCO at the Ni chemical shift (129.2 ppm for Pro 74, 115.8 ppm for Lys 75), in panels B and D a portion of the Cali–C 0 plane of the CBCACO and CCCO at the Cia chemical shift (63.7 ppm for Pro 74, 54.9 ppm for Lys 75), and in panels C and E the portions of the Cali–C 0 plane of the CBCACON and CCCON at the NiC1 chemical shift (115.8 ppm for Lys 75, 121.5 ppm for Asp 76). All experiments were acquired using the IPAP approach that allows one to remove the effect of the large C 0 –Ca scalar coupling in the direct dimension.

The relative performance of experiments based on C 0 and Ca direct-detection, once the problem of the homonuclear coupling in the direct acquisition dimension is solved, strongly depends on the relaxation properties of the system, both in terms of longitudinal and transverse relaxation and on the chemical shift dispersion. Indeed, for medium sized proteins at intermediate fields and for non-2H labelled samples, the C 0 -based acquisition experiments are the preferred choice as transverse relaxation rates of C 0 are still tolerable. Actually these can be the preferred choice for unfolded systems to exploit the residual chemical shift dispersion of 13C 0 , especially in combination with that of 15N [59]. At high fields, with 2H labelled proteins, the relaxation properties of Ca and of aliphatic nuclei in general are favourable and thus experiments based on Ca detection offer valuable alternatives. Indeed, by focusing mainly on Ca or on other aliphatic 13C nuclei, experiments can be designed to minimize relaxation losses due to C 0 magnetization in the plane. A combination of the above-mentioned experiments, namely those based on C 0 and on Ca direct-detection, can also be used to provide redundant information useful in studying systems characterized by crowded spectra. A comment is due about alternative possibilities to the CANCO-IPAP [58] and CAN-DIPAP experiments used to obtain the sequential correlations in the protonless NMR sequence specific assignment strategy. Indeed in both cases described, the key correlations used to link aminoacid spin systems in a sequence specific manner are provided by the Ca–N scalar couplings. However, even if much smaller, there are also homonuclear C 0 –C 0 (about 3 Hz) and Ca–Ca (about 2 Hz) coupling constants that can be exploited for sequence specific

assignment by correlating a C 0 or a Ca of a specific residue with the previous and with the following one through a spin-lock on a very narrow bandwidth (C 0 or Ca) that thus requires a fairly low RF power [60]. The exploitation of 13C–13C cross-relaxation rates has been proposed as an alternative approach to detect correlations between directly bound 13C nuclei for large molecules using inverse-detected experiments [61]. With the increase in molecular mass and with the increase in percentage of 2H isotopic enrichment, it is actually more convenient to opt for experiments that start and end on 13C [62]. Indeed, the cross relaxation rate sCC increases, while the longitudinal relaxation rates decrease with increasing molecular mass. This makes dipolar-coupling based transfer competitive with scalarcoupling based transfer [61]. IPAP and DIPAP versions of the NOESY experiment have been implemented to increase resolution by removing complex multiplet structures in C 0 [43] and in Ca regions, respectively [63] (Appendix, panels 15 and 16). The onebond correlations are indeed observed with good sensitivity. With long NOESY mixing times, two and three bonds correlations have also been identified and explained by spindiffusion [43]. This effect can be exploited as an alternative approach used to extend the assignment to side-chains in large macromolecules. The identification of 13C–13C correlations in NOESY spectra between nuclei not directly bound and not mediated by spin diffusion would mean a breakthrough in solution structure determination of large macromolecules by providing distance constraints. An extra leap in sensitivity for 13C is required to be able to

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W. Bermel et al. / Progress in Nuclear Magnetic Resonance Spectroscopy 48 (2006) 25–45

detect truly long-range correlations to obtain 13C–13C distance constraints. Summarizing, the protonless NMR strategy in biomolecules, that was pioneered by Markley in 1988 [25,64,65], has been expanded to a large set of multidimentional experiments characterized by high sensitivity and simplified 13C patterns in the acquisition dimension thanks to spin-state selective methods and to homodecoupling. A hybrid approach, that combines 13C direct-detection with starting on 1H has been proposed by Do¨tsch and co-workers [53]. On the grounds that 13C has several advantages they proposed to ‘cut’ out-and-back versions of existing triple resonance experiments into ‘out-and-stay’ versions where the nuclei used to acquire are either C 0 (as in the example of HACACO [49]) or aliphatic nuclei (as in the case of HCC [49]). Improved versions of these experiments were later proposed by Pervushin and co-workers [66,67]. These additional experiments can also be added to the conventional triple-resonance assignment strategy. However, the drawback is the re-introduction of 1H transverse relaxation in one of the building blocks of the sequences. 5. Detection of resonances in paramagnetic proteins Paramagnetic systems are characterized by additional contributions to chemical shifts and nuclear relaxation arising from the so-called hyperfine interaction, i.e. the interaction between the nuclear spin I and the electron spin S [68]. This interaction occurs via two different mechanisms, a through-bond interaction, which depends on the amount of unpaired electron spin-density delocalized onto the investigated nuclear spin (contact interaction) [69,70] and a through-space interaction [71,72], which depends on the dipole–dipole interaction between nuclear and electron spins. Both effects contribute to chemical shifts and nuclear relaxation. The interplay between the two different contributions and their absolute and relative magnitudes depends

very much on the nature of the paramagnet and on its coordination sphere. The peculiarities of NMR spectroscopy of paramagnetic systems have been extensively discussed in the literature [73–76] and do not need to be re-introduced in this review. The essential feature that concerns us here is the signal broadening caused by paramagnetism-induced relaxation. Thus, the NMR signals of nuclei in the surrounding of the paramagnetic centre can be broadened beyond detectable limits. Independently of the mechanism that it originates from, paramagnetic relaxation depends on the square of the magnetogyric ratio. This is not the case for paramagnetisminduced chemical shifts. Therefore, 13C NMR studies of paramagnetic molecules provide in principle the same content of information as 1H studies, but the loss of information due to paramagnetism-induced line broadening will be much less effective. Actually, the applications on paramagnetic systems were the ones that promoted the revival of heteronuclear NMR in the recent years [75,77–81]. The keynote finding is that pseudocontact shifts and hyperfine relaxation provide metal-nucleus distance restraints [76]. These two observables, based on 13C nuclei, may actually provide structural information in regions of a protein where 1H resonances are broadened beyond detectable limits. Therefore, synergism between 1H and 13C NMR spectroscopy concerns not only the assignments, but also obtaining the structural constraints. Table 2 gives the contribution to relaxation rates due to the hyperfine interaction for 1H and 13C spins in the presence of different metal chromophores. It can be noted that the different metal ions present a variety of situations. While for some metal ions like Gd(III) or Dy(III), 13C signals may be still broad ˚ from the metal ion, beyond detection in a sphere as large as 8 A in other cases, like low spin Fe(III) or Ce(III) and Co(II), all 13 C signals can in principle be detected. In the latter case, nuclei that are 2–3 s bonds away from the metal centre can also be affected by the contact contribution to relaxation and thus

Table 2 Expected behaviour of different metal ions in terms of paramagnetism induced relaxation Metal ion

S, J

g, gJ

ts (10–12 s)

˚ ) R2 1H at d (A 1000 sK1

˚ ) R2 13C at d (A 1000 sK1

13

Fe(III) HS Fe(III) LS Fe(II) Co(II) HStetra Co(II) HSesa Cu(II) Gd(III) Ce(III) Dy(III) Tb(III) Yb(III) Tm(III)

5/2 1/2 2 3/2 3/2 1/2 7/2 5/2 15/2 6 7/2 6

2 2 2 2 2 2 2 6/7 4/3 3/2 8/7 7/6

100 1 1 10 1 3000 10000 0.1 0.5 0.3 0.3 0.5

7.5 3.0 6.0 5.2 5.1 7.0 13.3 3.8 9.9 9.3 5.6 7.9

4.8 1.9 3.7 3.3 3.2 4.5 8.4 2.4 6.3 5.9 3.6 5.0

430 130 30 190 60 170 45 29 15 15 25 20

C R1 sK1

Distances reported in columns 5 and 6 are the calculated distances corresponding to an R2 value of 1000 sK1 for 1H and 13C, respectively. Only dipolar and Curie contributions are taken into consideration, so the calculated values do not include the contributions provided by the contact interaction, i.e. the effects induced by through-bond effects. Column 7 reports the calculated R1 values corresponding to the distances of column 6. The values have been calculated considering trZ 10K8 s, TZ300 K and B0Z16.4 T (700 MHz for 1H, or 176 MHz for 13C). Electronic properties of the various metal ions are also reported.

W. Bermel et al. / Progress in Nuclear Magnetic Resonance Spectroscopy 48 (2006) 25–45

may become undetectable (this contribution is not included in the calculations as it depends on the molecular structure and on the electron spin delocalisation and thus is difficult to account for in a general way [68,75]). Direct-detection of 13C intrinsically offers a way to detect resonances close to the metal ion where 1H resonances are too broad to be detected. However, the 13C-based approach can be further optimised for paramagnetic systems by selecting the most efficient coherence transfer pathways and identifying those that are least affected by fast relaxation. In principle, the most sensitive experiments are those based on coherence transfer mechanisms mediated by large scalar couplings; however, when transverse relaxation is much faster than longitudinal relaxation, dipolar-based transfers can be usefully exploited, especially at higher fields. The 13C–13C COSY-based experiments were the first to be used for paramagnetic systems [77–79,81,82]. As an alternative, several other experiments based on coherence transfer via the C 0 –Ca scalar couplings, and also via the smaller N–C 0 scalar coupling, exploiting either the single quantum or multiple quantum coherence transfer can be used [29,80,81]. Signal losses due to fast relaxation can be reduced by shortening the coherence transfer delays [29] and even further by completely removing the building block in which the antiphase C 0 –Ca coherence is refocused and detecting directly the anti-phase component [28,83]. Several CACO schemes have been compared and the most suitable experiment for paramagnetic systems turned out to be the single quantum CACO experiment used without refocusing prior to C 0 detection (CACO–AP) [83], outlined in Fig. 8E. Fig. 10 shows the comparison of a standard CACO–IPAP experiment vs the single-quantum CACO–AP experiment for the Tm(III) substituted calcium binding protein Calbindin D9k [84]. Several additional peaks were observed in the experiment which was tailored for paramagnetic signals. This result is due both to the different pulse sequence used and to the choice of experimental parameters, such as optimized coherence-transfer and recycle delays [84]. As pointed out for large molecules, the 13C–13C NOESY experiment can be useful when the limiting factor consists in fast transverse relaxation [61]. Indeed in many paramagnetic systems the longitudinal relaxation rates are influenced to a smaller extent than the transverse relaxation rates. Table 2, column 6, reports the longitudinal relaxation rate for a given 13 C resonance with R2Z1000 sK1. The large variability of the values in column 6 provides an estimate of the potential interest in the use of homonuclear 13C–13C NOE-based transfer. For example, all lanthanides, including Gd(III) but especially the more far-shifting ones like Dy(III) or Tb(III), have a relatively small contribution to R1 arising from paramagnetic relaxation. For the above cases, the use of 13C–13C NOE can be a useful alternative to overcome the quench of scalar coupling based transfer. Of course, this will be particularly true on increasing the size of the molecule, as the magnitude of the NOE effect depends on tr. An interesting application of using 13C direct-detection to get closer to a paramagnetic centre is for those metalloproteins

35

Fig. 10. (A) CACO–IPAP and (B) CACO–AP spectra of 13C,15N labeled CaTmCb recorded at 16.4 T. The additional peaks observed in (B) are indicated by arrows. The CACO–IPAP experiment has been recorded using standard experimental conditions for the detection of diamagnetic resonances using the pulse sequence shown in Fig. 8C. The CACO–AP spectrum was acquired using the pulse sequence reported in Fig. 8E, with Ca–C 0 transfer delays shortened from 9.0 to 5.5 ms and a recycle period shortened from 1.0 to 0.3 s. Both spectra were acquired using a 13C-optimized triple-resonance probehead at 16.4 T and 300 K.

in which the metal ion is coordinated via carboxylate ligands. Indeed, for bound Asp or Glu residues, the carboxyl carbon atom is only two bonds away from the metal centre, while the closest 1H spin is four bonds away. Therefore, 13C–13C COSY has been successfully used in lanthanide substituted calcium binding proteins [78]. Furthermore, the intrinsic asymmetry of an homonuclear 13C–13C COSY experiment provides a clever way of identifying the coordinating residues of a paramagnetic metal ion and to discriminate between monodentate and bidentate carboxylate ligands [85]. It is the only NMR method available to distinguish between monodentate and bidentate coordinating side-chain CO groups. Finally, lanthanide substituted calcium binding proteins can also be used as examples to monitor the different performances of various type of experiments. This is graphically summarized in Fig. 11 for the Tm(III) substituted derivative of calbindin D9k [84]. The use of low g nuclei direct-detection provided the identification of all residues while only about 50% of aminoacids were

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W. Bermel et al. / Progress in Nuclear Magnetic Resonance Spectroscopy 48 (2006) 25–45

Fig. 11. Radii of the spheres within which signals cannot be observed in the case of CaTmCb. Different spheres are reported for each different set of NMR ˚ for standard 1H detected NMR experiments. Radii are as follows: 17.5 A 1 ˚ for ˚ experiments, 15 A for H experiments optimized to fast relaxing signals, 14 A ˚ for 13C detected experiments optimized to standard 13C detected experiments, 9 A ˚ from 13C 1D spectra, 4.2 A ˚ for 1D 15N spectra. fast relaxing signals, 5 A

assigned with standard 1H detected experiments. In this case, 15 N 1D spectra were also used to identify previously unobserved resonances. Indeed, the development of 15N optimized probes, characterized by a 15N selective inner coil, would certainly contribute to increase the S/N achievable for this nucleus, thus providing better quality 15N direct-detected experiments. 6. Conclusions We have shown that the combination of upgraded instrumental performances and the design of novel pulse sequences allows 13C direct-detected protonless NMR spectroscopy to be used to conveniently perform the complete 13 15 C, N assignment for proteins. Development of the exclusively heteronuclear NMR experiments summarized in this review has been stimulated by the need for a different approach to study paramagnetic systems, for which the main problems are fast transverse relaxation and difficulties with 1H detection. These two features, however, are common to large proteins, as well as to exchanging and partially unfolded systems. Heteronuclear protonless NMR can thus be useful for making assignments in a wide range of applications. When one aims at extending this strategy to protein structural determination, 13C-based restraints are needed.

The present state of the art allows one to fully exploit chemical shift values [86] as Ca, Cb, and CO chemical shifts are available from the present approach. Of course, the wealth of 13 C data available can be further exploited as a source of 13Cchemical shift structural restraints. 13 C–13C residual dipolar couplings can in principle be accurately determined through IPAP-type experiments and therefore residual dipolar coupling restraints may be available. Cross-correlation rates and J-values can also be determined using 13C direct-detection experiments, providing some dihedral angle restraints. Selected protonated amino acids can provide 1H–1H NOEs, which are most valuable within this strategy [11]. Of course, the measurement of long-range 13 C– 13C NOEs would be a significant breakthrough. In the case of paramagnetic systems, nuclear relaxation can be related to metal-nucleus distances [71]. This approach has already been exploited for 13C nuclei [81]. However, reports are available in the literature, which point to a break-down in the relaxation–distance relationship for heteronuclei far from the metal ion [87,88], presumably due to the occurrence of other phenomena not yet fully understood. Paramagnetic probes and spin-labels were recently used to exploit the paramagnetism-derived structural information in natively diamagnetic proteins [89–97]. The availability of further data via protonless NMR may provide additional information at shorter distances than those obtainable through 1H NMR. Pseudocontact shifts are also very valuable restraints [30]. Here a problem can be the determination of the diamagnetic reference to be subtracted from the paramagnetic chemical shift. One should check that the structural features of the two systems are absolutely the same, as the diamagnetic chemical shift of carbon nuclei is highly sensitive to dihedral angle variations. Another paramagnetic-based restraint is the cross-correlation between Curie-relaxation and 13C–13C dipolar relaxation. Further studies are needed to implement their use. The combined use of paramagnetic and diamagnetic-based restraints is expected to provide a good method for determination of the structure of proteins in solution. Technological advancements in 13C solid-state spectroscopy, where 13C is the nucleus of detection, have been beneficial to the development of direct-detection of heteronuclei in solution and it is likely that the present development will have a fall-out in solid-state NMR. Optimization of the hardware and the development of dedicated cryogenicallycooled probeheads may further establish heteronuclear protonless NMR as a routine spectroscopic approach for studying biomolecules in solution. Acknowledgements This work has been supported in part by the EC (Contract QLG2-CT-2002-00988) and by the Italian Ministero per la Universita` e la Ricerca (COFIN 2003).

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Appendix The pulse sequences used to acquire the experiments discussed in the review are described in detail here. In particular we report the experiments based on C 0 direct-detection with IPAP and/or S3E spin state selective methods to remove the effects of the C 0 –Ca coupling in the direct acquisition dimension (CBCACO–IPAP, CBCACO–S3E, CCCO–IPAP, CCCO–S3E, CBCACON–IPAP, CCCON–IPAP, CON–IPAP, CANCO–IPAP, CANCO–IPAP selective) and the experiments based on Ca direct-detection with the DIPAP approach to remove the C 0 –Ca and Ca–Cb couplings in the direct acquisition dimension (COCA–DIPAP, Ca–TOCSY–DIPAP, CAN–DIPAP). We also report the NOESY experiment with implementation of spinstate selective approaches to remove the one bond splittings in the direct acquisition dimension (CACO–NOESY–IPAP and Ca–NOESY–DIPAP) and the CGCB–DIPAP experiment for connecting the aromatic side-chains to the backbone. These experiments were tested using different instruments and on several proteins. We report here the parameters used with a 14.1 T Bruker Avance instrument, equipped with a cryogenically-cooled probehead optimized for 13C sensitivity, on 13C,15N labelled reduced monomeric superoxide dismutase. When acquiring experiments on a deuterated protein, 2H decoupling should be applied as indicated for 1H. In all the figures (unless otherwise specified), band selective 13C pulses are denoted by shapes. For 13C bandselective 908 and 1808 pulses Q5 (or time reversed Q5) and Q3 shapes [100] were used with durations of 320 and 256 ms, respectively except for the 1808 pulse indicated in grey (Q3, 1.0 ms) and for the 1808 pulse indicated in crossed stripes (adiabatic inversion pulse over the C 0 and Ca regions, smoothed chirp 500 ms, sweep width 60 KHz, 20% smoothing [103]). The rectangular wide and narrow pulses correspond to 180 and 908 hard pulses. Pulse field gradients (PFG line) are also indicated by shapes. All the gradients, used for purging and not for coherence selection, have a duration of 1.0 ms and a sine-shape. The 1H and 15N carriers were placed at 4.7 and 118 ppm, respectively. The change in the position of the 13C carrier (39 ppm for Cali, 55 ppm for Ca and 173 ppm for C 0 ) is indicated by vertical arrows. The RF power used for the 13C FLOPSY16 spin-lock was 10 kHz (applied for durations ranging from 10 to 22 ms in the 2D versions and 22 ms in the 3D version). Decoupling of 1H and 15N was applied with 2.9 kHz (waltz-16) [101] and 1.0 kHz (garp-4)[102] respectively. For experiments that employ the IPAP approach to suppress the C 0 –Ca coupling, the in-phase (IP) and anti-phase (AP) components are acquired and stored separately using the pulse schemes illustrated that differ only for the two panels indicated with IP and AP respectively. For experiments that employ the S3E approach to suppress the effect of the C 0 –Ca coupling, the two components that need to be acquired and stored separately differ by the phase fS3E and by a p increment of phase f4 in the CCCO-S3E experiment and f5 in the CBCACO-S3E experiment. For experiments that employ the DIPAP approach to suppress the C 0 –Ca and the Ca–Cb couplings, the four variants of the experiment that should be acquired and stored separately are shown in the four panels indicated with IP-IP, AP-IP, IP-AP, AP-AP respectively. The phase cycle, the method used for quadrature detection and the durations of the delays shown in the pulse sequences are reported case-by-case. Panel 1 CBCACO–IPAP: The delays are: DZ9 ms, D1Z8 ms. The phase cycle is: f1Zx,Kx; f2Z8x,8(Kx); f3Z2y,2(Ky); fIPAP(IP)Z4(x), 4(Kx); fIPAP(AP)Z4(Ky),4(y); frecZx,(Kx),(Kx),x,(Kx),x,x,(Kx). Quadrature detection in the F1 and F2 dimensions is obtained by incrementing f1 and f3 in a States-TPPI manner. Transfer pathway: F1(Ca/b, t1)/F1(Ca, t2)/F1(C 0 ,t3) Correlations observed : Cib –Cia –Ci0 ; Cia –Cia –Ci0

Panel 2 CBCACO–S3E; The delays are: DZ9 ms, D1Z8 ms and 3Z4 ms. The phase cycle is: f1Zx,Kx; f2Z8x, 8(Kx); f3Z2y, 2(K y); f4Z4x, 4(y); f5Z4x, 4(y); fS3E(1)Z4(458), 4(2258); fS3E(2)Z8(458); frecZx, (Kx), (Kx), x, (Kx), x, x, (Kx). Quadrature detection in the F1 and F2 dimensions is obtained by incrementing f1 and f3 in a States-TPPI manner.

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Transfer pathway: F1(Ca/b, t1)/F1(Ca, t2)/F1(C 0 ,t3) Correlations observed : Cib –Cia –Ci0 ; Cia –Cia –Ci0

Panel 3 CCCO–IPAP; The delays are: DZ9 ms, 3Zt1(0). The phase cycle is: f1Zx,Kx; f2Z2x, 2(Kx); fIPAP(IP)Z4x, 4(Kx); fIPAP(AP)Z4(Ky),4y; frecZx, (Kx), (Kx), x, (Kx), x, x, (Kx). Quadrature detection in the F1 and F2 dimensions is obtained by incrementing f1 and f2, respectively, in a States-TPPI manner. Transfer pathway: F1(Cali, t1)/F1(Ca, t2)/F1(C 0 ,t3) Correlations observed : Ciali –Cia –Ci0 ; Cia –Cia –Ci0

Panel 4 CCCO–S3E: The delays are: DZ9 ms, 31Zt1(0) and 3Z4 ms. The phase cycle is: f1Zx,Kx; f2Z4x, 4(Kx); f3Z2x, 2(y); f4Z 2x, 2(y); fS3E(1)Z4(458); fS3E(2)Z2(458), 2(2258); frecZx, (Kx), (Kx), x, (Kx), x, x, (Kx). Quadrature detection in the F1 and F2 dimensions is obtained by incrementing f1 and f2, respectively, in a States-TPPI manner. Transfer pathway: F1(Cali, t1)/F1(Ca, t2)/F1(C 0 , t3) Correlations observed : Ciali –Cia –Ci0 ; Cia –Cia –Ci0

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Panel 5 CGCB–DIPAP: The experiment to correlate the Cb of aromatic aminoacids with the quaternary Cg carbon that employs the DIPAP approach to obtain singlets in the direct acquisition dimension. Pulses on Cg and on Cb and Ca/b were centered at 130, and 33 ppm, respectively. The phase cycle is: f1Zx,Kx; f2Z8(x), 8(y), 8(Kx), 8(Ky); f3Z4(y), 4(Ky); frecZx, (Kx),(Kx), x,(Kx), x, x,(Kx), (Kx), x, x,(Kx), x,K(x),(Kx), x; fDIPAP(IP-IP)Z2(x), 2(Kx); fDIPAP(AP-IP, IP-AP)Z 2(Ky), 2(y); fDIPAP(AP-AP)Z2(Kx), 2(x). Quadrature detection is achieved by incrementing f1 in a States-TPPI manner. 1 H and 15N decoupling is applied during all the experiment, except during the relaxation delay at 7 ppm in the first part and at 3.5 ppm in the second part (1H) and at 197 ppm (15N). The duration of the delays is: DZ4 ms, D1Z14.4 ms, dZ 9 ms. Transfer pathway: F1(Cg, t1)/F1(Cb,t2) Correlations observed : Cig –Cib

Panel 6 CON–IPAP: The delays are: DZ9 ms, D1Z25 ms, 3Zt1(0). The phase cycle is: f1Zx,Kx; f2Z2x, 2(Kx); f3Z4x, 4(Kx); fIPAP(IP)Zx; fIPAP(AP)ZKy; frecZx, (Kx), x, (Kx), (Kx), x, (Kx), x. Quadrature detection in the F1 dimension is obtained by incrementing f1 in a States-TPPI manner. Transfer pathway: F1(C 0 )/F3(N, t1)/F1(C 0 , t2) 0 Correlations observed : Ni –CiK 1

Panel 7 CBCACON–IPAP: The delays are: DZ9 ms, D1Z25 ms, D2Z8 ms and 3Zt2(0). The phase cycle is: f1Zx,Kx; f2ZKy; f3Z2x, 2(Kx); f4Z4x, 4(Kx); f5Z8x, 8(Kx); fIPAP(IP)Zx; fIPAP(AP)ZKy; frecZ2(x, (Kx), (Kx), x), 2((Kx), x, x, (Kx)) . Quadrature detection in the F1 and F2 dimensions is obtained by incrementing f1 and f3, respectively, in a States-TPPI manner. Transfer pathway: F1(Ca/b, t1)/F1(Ca)/F1(C 0 )/F3(N, t2)/F1(C 0 , t3)

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Correlations observed : Cib –Ci0 –NiC1 ; Cia –Ci0 –NiC1

Panel 8 CCCON–IPAP: The delays are: DZ9 ms, D1Z25 ms, 31Zt1(0), 3Zt(0). The phase cycle is: f1Zx,Kx; f2Z2x, 2(Kx); f3Z 4x, 4(Kx); f4Z8x, 8(Kx); fIPAP(IP)Zx; fIPAP(AP)ZKy; frecZ2(x, (Kx), (Kx), x), 2((Kx), x, x, (Kx)). Quadrature detection in the F1 and F2 dimensions is obtained by incrementing f1 and f2, respectively, in a States-TPPI manner. Transfer pathway: F1(Cali, t1)/F1(Ca)/F1(C 0 )/F3(N, t2)/F1(C 0 , t3) Correlations observed : Ciali –Ci0 –NiC1 ; Cia –Ci0 –NiC1

Panel 9 CANCO–IPAP: The delays are: DZ9 ms, D1Z25 ms, D2Z32 ms. The phase cycle is: f1Zx,Kx; f2Z8x, 8(Kx); f3Z2x, 2(Kx); fIPAP(IP)Z4x, 4(Kx); fIPAP(AP)Z4(Ky), 4y; frecZx, (Kx), (Kx), x, (Kx), x, x, (Kx). Quadrature detection in the F1 and F2 dimensions is obtained by incrementing f1 and f3, respectively, in a States-TPPI manner. Transfer pathway: F1(Ca, t1)/F3(N, t2)/F1(C 0 , t3) 0 a a 0 Correlations observed : Cia –Ni –CiK 1 ; CiK1 –NiK1 –CiK1

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Panel 10 CANCO–IPAP ‘selective’: The delays are: DZ9 ms, D1Z25 ms, D2Z32 ms and 3Zt2(0). The phase cycle is: f1Zx,K x; f2Z8x, 8(Kx); f3Z2x, 2(Kx); fIPAP(IP)Z4x, 4(Kx); fIPAP(AP)Z4(y), 4(Ky); frecZx, (Kx), (Kx), x, (Kx), x, x, (K x). Quadrature detection in the F1 and F2 dimensions is obtained by incrementing f1 and f3, respectively, in a States-TPPI manner. Transfer pathway: F1(Ca, t1)/F3(N, t2)/F1(C 0 , t3) a 0 Correlations observed : CiK 1 –Ni –CiK1

Panel 11 CBCANCO–IPAP: The delays are: DZ9 ms, D0Z8 ms, D1Z25 ms, D2Z32 ms. The phase cycle is: f1Zx,Kx; f2Z8x, 8(Kx); f3Z2x,2(Kx); fIPAP(IP)Z4x,4(Kx); fIPAP(AP)Z4(Ky),4y; frecZx,(Kx),(Kx)x,(Kx),x,x,(Kx). Quadrature detection in the F1 and F2 dimensions is obtained by incrementing f1 and f3, respectively, in a States-TPPI manner. Transfer pathway: F1(Ca/b, t1)/F3(N, t2)/F1(C 0 , t3) b b 0 a 0 0 0 Correlations observed : Cia –Ni –CiK 1 ; CiK1 –Ni –CiK1 ; Ci –Ni –CiK1 ; CiK1 –Ni –CiK1

Panel 12 COCA-DIPAP: The delays are: DZ9 ms, D1Z14.4 ms. The phase cycle is: f1Zx,Kx; f2Z4x, 4y; fDIPAP(IP-IP)Z2x, 2(Kx); fDIPAP(AP-IP)ZfDIPAP(IP-AP)Z2(Ky), 2y; fDIPAP(AP-AP)Z2(Kx), 2x; frecZx, (Kx), (Kx), x, (Kx), x, x, (Kx). Quadrature detection in the F1 dimension is obtained by incrementing f1 in a States-TPPI manner. 0 Transfer pathway: F1(C , t1)/F1(Ca, t3) Correlations observed : Ci0 –Cia :

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Panel 13 Ca–TOCSY–DIPAP: The delays are: DZ9 ms, D1Z14.4 ms, 3Zt1(0). The phase cycle is: f1Zx,Kx; f2Z4x, 4y; f3Z8x, 8(Kx); fDIPAP(IP-IP)Z2x, 2(Kx); fDIPAP(IP-AP)Z2(Ky), 2y; fDIPAP(AP-IP)Z2y, 2(Ky); fDIPAP(AP-AP)Z2x, 2(Kx); frecZx, (Kx), (Kx), x, (Kx), x, x, (Kx), (Kx), x, x, (Kx), x, (Kx), (Kx), x. Quadrature detection in the F1 dimension is obtained by incrementing f1 in a States-TPPI manner. The experiment is generally performed with all the pulses before the spin-lock, as well as the spin-lock itself, to cover the aliphatic region. In this case, the experiment yields all the correlations of Ca with the remaining aliphatic carbon atoms within the side chain of the aminoacid. However, the experiment can also be performed with all the pulses before the spin-lock, as well as the spin-lock itself, that are selective over the Ca region and with a much longer duration of the spin-lock. In this case the experiment yields Ca–Ca sequential correlations. Transfer pathway: F1(Cali, t1)/F1(Ca, t3) Correlations observed : Ciali –Cia

Panel 14 CAN–DIPAP: The delays are: DZ13.6 ms, dZ3.6 ms, xZ2.25 ms, 3Zt1(0). The phase cycle is: f1Zx,Kx; f2Z2x, 2(Kx); fDIPAP(IP-IP)Z4x, 4(Kx); fDIPAP(AP-IP)Z4(Ky), 4y; fDIPAP(IP-AP)Z4y, 4(Ky); fDIPAP(AP-AP)Z4x, 4(Kx); frecZx, (Kx), x, (Kx), (Kx), x, (Kx), x. Quadrature detection in the F1 dimension is obtained by incrementing f1 in a States-TPPI manner.

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Transfer pathway: F1(Ca)/F3(N, t1)/F1(Ca, t2) a Correlations observed : Cia –Ni ; CiK 1 –Ni

Panel 15 CACO–NOESY–IPAP: The delays are: DZ9 ms, 3Zt1(0), tMZmixing time. The phase cycle is: f1Zx,Kx; f2Z4x, 4(Kx); fIPAP(IP)Z2x, 2(Kx); fIPAP(AP)Z2(Ky), 2y; frecZx, (Kx), (Kx), x, (Kx), x, x, (Kx). Quadrature detection in the F1 dimension is obtained by incrementing f1 in a States-TPPI manner. 0 Transfer pathway: F1(Ca, t1)/F1(C , t2) Correlations observed : Cia –Ci0 ; and other correlations arising from spin
diffusion

Panel 16 Ca–NOESY–DIPAP: The delays are: DZ9 ms, D1Z14.4 ms, 3Zt1(0), tMZmixing time. The phase cycle is: f1Zx,Kx; f2Z 4x, 4(Kx); fDIPAP(IP-IP)Z2x, 2(Kx); fDIPAP(IP-AP)Z2(Ky), 2y; fDIPAP(AP-IP)Z2y, 2(Ky); fDIPAP(AP-AP)Z2x, 2(Kx); frecZx, (Kx), (Kx), x, (Kx), x, x, (Kx). Quadrature detection in the F1 dimension is obtained by incrementing f1 in a States-TPPI manner. Transfer pathway: F1(Cb/ali, t1)/F1(Ca, t2). Correlations observed : Cib –Cia and other correlations arising from spin
diffusion

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