Accepted Manuscript Strategies for solid-state NMR investigations of supramolecular assemblies with large subunit sizes Pascal Fricke, Veniamin Chevelkov, Chaowei Shi, Adam Lange PII: DOI: Reference:

S1090-7807(14)00298-5 http://dx.doi.org/10.1016/j.jmr.2014.10.018 YJMRE 5545

To appear in:

Journal of Magnetic Resonance

Received Date: Revised Date:

3 September 2014 21 October 2014

Please cite this article as: P. Fricke, V. Chevelkov, C. Shi, A. Lange, Strategies for solid-state NMR investigations of supramolecular assemblies with large subunit sizes, Journal of Magnetic Resonance (2014), doi: http://dx.doi.org/ 10.1016/j.jmr.2014.10.018

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Strategies for solid-state NMR investigations of supramolecular assemblies with large subunit sizes Pascal Fricke, Veniamin Chevelkov, Chaowei Shi, and Adam Lange*

Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125 Berlin, Germany. *[email protected]

ABSTRACT Solid-state NMR is a versatile tool to study structure and dynamics of insoluble and noncrystalline biopolymers. Supramolecular protein assemblies are formed by self-association of multiple copies of single small-sized proteins. Because of their high degree of local order, solidstate NMR spectra of such systems exhibit an unusually high level of resolution, rendering them an ideal target for solid-state NMR investigations. Recently, our group has solved the structure of one particular supramolecular assembly, the type-iii-secretion-system needle. The needle subunit comprises around 80 residues. Many interesting supramolecular assemblies with unknown structure have subunits larger in size, which requires development of tailored solid-state NMR strategies to address their structures. In this “Perspective” article, we provide a view on different approaches to enhance sensitivity and resolution in biological solid-state NMR with a focus on the possible application to supramolecular assemblies with large subunit sizes.

KEYWORDS: dynamic

nuclear

polarization;

proton

supramolecular protein assemblies

1

detection;

homonuclear

recoupling;

Protein structures and functions are among the central subjects of biological research. Many mechanisms and structures within cells are not only made up of single proteins but are rather assemblies of several copies of the same protein or even assemblies of different proteins. Those structures often pose difficulties to researchers due to their inherent complexity. Many wellestablished methods such as X-ray crystallography and liquid-state NMR spectroscopy fail in the structure determination due to the insolubility of these supramolecular complexes, which makes them unsuitable for single crystal formation or dissolution. The variety of methods that currently allow for successful research on such structures in a close to in vivo state is very limited. One of them is solid-state NMR spectroscopy (ssNMR), as it enables to study the structure of solid samples that lack a long-distance order [1]. Often, the sample preparation can be carried out in such a way that the biological system under study resembles the in vivo state very closely, which makes this technique yield meaningful results.

Protein studies face the ssNMR spectroscopist with several difficulties: Depending on the size and dynamics of the system of interest, the spectra can lack resolution. Furthermore, proteins with many amino acid residues give rise to a multitude of peaks which possibly overlap severely. Additionally, NMR in general is known for its inherently low sensitivity, which makes the availability of a sufficiently large amount of isotopically labeled sample a necessity. All these factors make the acquisition of high quality spectral data and the subsequent signal assignment comparably difficult.

Recently, there have been several advances in the field of ssNMR spectroscopy that can help to facilitate this process and broaden the applicability to a wider range of supramolecular systems. Our group has been working on the structural determination and dynamics of the type-III-secretion system (T3SS) needle of the bacteria Salmonella typhimurium [2, 3] and Shigella flexneri [4, 5], which are supramolecular assemblies of the needle proteins PrgI and MxiH, respectively. For this,

2

we employed several different methods of ssNMR spectroscopy that helped us to gain further insight into the atomic structure of the underlying system [6].

The structure determination via ssNMR is based on the collection of long-distance restraints that prove the spatial connection between certain atoms within the protein or assembly structure. A sufficient amount of such restraints in conjunction with secondary chemical shifts then allow calculating the atomic structure of the underlying protein assembly. Revealing these connections is carried out by long-range magnetization transfer, which is often made difficult by limited resolution and dipolar truncation. These problems can be solved by employing CHHC/NHHC [7-9] or PAR/PAIN [10] experiments and the use of sparsely labeled samples [11-13].

In this “Perspective” article, we will discuss three comparably new experimental methods that are of potential interest in respect to their impact on studying supramolecular systems using ssNMR. We will present the possibilities arising from the use of these methods together with their limitations.

3

1 Dynamic Nuclear Polarization

NMR spectroscopy is inherently insensitive due to the unfavorable Boltzmann distribution at ambient temperature and at typical magnetic field strengths. A considerable research effort has been put into finding ways to overcome this limitation. In the hyperpolarization approach, the aim is to increase the population difference of the NMR spin levels to an off-equilibrium state which results in higher signal intensities. To date, dynamic nuclear polarization (DNP) is the only hyperpolarization method that was shown to have widespread applicability, high polarization factors and a robust hardware implementation at the same time. Its usefulness in a variety of fields has been shown in numerous publications (e.g. [14-17]).

DNP takes advantage of the much higher polarization of the electron spins. In ssNMR at high fields, this is usually done by adding a biradical to the sample. The electron spin frequency is then saturated by microwave irradiation resulting in a polarization transfer to the nuclei close in space and subsequent spin-diffusion. The hyperpolarization of the nuclei can then be directly exploited by a regular NMR measurement [18, 19].

At typical magnet field strengths for ssNMR, the needed uniform and stable microwave frequency with high output power is produced by a gyrotron oscillator (e.g. 395 GHz microwave frequency in an NMR spectrometer with 600 MHz 1H Larmor frequency). The microwaves are directed into the probe via a corrugated wave guide and remain activated throughout the whole NMR experiment. For DNP to be efficient, the sample must be cooled to cryogenic temperatures (typically ~ 100 K), which is achieved by a sophisticated system that individually controls the pressure and temperature of the three nitrogen gas flows (bearing, drive, cooling gas) towards the solid-state DNP probe [20].

The enhancement factor of DNP is calculated by comparing the signal intensities of the

4

resulting NMR spectra with the microwaves being switched on and off. There are several radicals published in the literature that have shown a considerable enhancement. Especially the use of the biradical TOTAPOL is reported in numerous DNP-related publications. Its enhancement factor at a magnetic field strength corresponding to 600 MHz 1H Larmor frequency is about 30 [21]. Nevertheless, TOTAPOL has recently been superseded by AMUPol [22], which has similar or even better chemical characteristics but exhibits an enhancement factor of around 120 under otherwise identical conditions (Fig. 1, data recorded at MPI Göttingen).

Fig. 1: Comparison of DNP enhancement factors ε using different radicals to polarize the reference substance [13C, 15N]labeled proline in a 600 MHz wide-bore NMR spectrometer equipped with a DNP setup (MPI Göttingen). Lower spectrum: proline sample with TOTAPOL and microwaves (MW) switched off. Center spectrum: proline sample with TOTAPOL and MW switched on. Upper spectrum: proline sample with AMUPol and MW switched on. The intensity of the upper spectrum was scaled in such a way that the amplitudes for both samples were equal when measured with MW off.

To ensure a uniform distribution of the radical and to cryoprotect the supramolecular assembly from possible damage by crystal formation at cryogenic temperature, the sample is usually diluted in a matrix that forms an amorphous glass after freezing. A matrix consisting of a mixture of approximately 60/35/5 (v/v/v) glycerol-d8/D2O/H2O has proven to meet these requirements. The sample and the biradical (in mM quantities) are then mixed with the matrix [23].

5

Using a 600 MHz wide-bore ssNMR-DNP setup, we studied in our lab uniformly [13C, 15N]labeled MxiH needles with TOTAPOL as the biradical, for which we observed an enhancement factor of 23. The system is known to show excellent resolution at room temperature. As expected, the resolution at cryogenic temperature was found to be considerably worse (approximately 1 ppm carbon line width at half maximum, Fig. 2). Nevertheless, the resolution is among the best ever published for ssNMR DNP spectra of biological samples. It was possible to assign 46 % of the backbone resonances and 32 % of the side-chain resonances [24].

Fig. 2: 2D 13C-13C PDSD NMR spectrum of uniformly 13C-15N-labeled MxiH T3SS needles with TOTAPOL as polarization agent. Some of the corresponding atom assignments are shown. The dotted line indicates the chemical shift position at which one 1D slice of the NMR spectrum was extracted, which is shown on top. The signal-to-noise ratio (S/N) and the full width at half height (FWHH) are given for the peak marked with an asterisk. Figure from [24].

The line-broadening is attributed to the fact that the mobility of the protein structure is very limited at cryogenic temperature, so that the different conformations “freeze out” causing overlapping (broad) signals [25]. The severity of the line broadening – or in the extreme case the absence of the resonance altogether – allows for conclusions with respect to the dynamics of this part of the protein structure. Parts that are mobile at room temperature are more prone to have broad

6

signals or to even vanish at low temperature, while the well-resolved peaks most likely belong to a more rigid region.

Simply evaluating the number of cross peaks for a given amino acid residue in a set of 2D and 3D NMR spectra giving short-range connections, we observed that this number is considerably higher for the residues which belong to the inner part of the needle structure. This finding allows for the deduction that the outer part of the needle has a higher mobility than the inner part, which is assumed to be due to the constraining effect of the tightly packed inner helices of the needle protein increasing the rigidity in this area.

In this study, we combined the increase in sensitivity gained by DNP with the evaluation of the effects of the cryogenic freezing. The line-broadening at low temperature is usually considered an unwanted side-effect of DNP in ssNMR, but could help in this case to draw conclusions with respect to protein dynamics. Diluting the sample in a matrix is counter-intuitive when high signal intensity is desired. Nevertheless, it is essential to maintain the integrity of the supramolecular structure of the sample during the freezing process. Without the signal enhancement by DNP, the needed NMR spectra could not have been acquired in a reasonable timeframe under otherwise identical conditions.

In ssNMR, the peak resolution of biomolecular samples even at ambient temperature and under optimal conditions is a critical factor. For many systems it is too poor to allow for an exhaustive peak assignment and subsequent analysis. Causing further line-broadening by cryogenic freezing and the addition of a radical can easily render the resulting NMR spectra useless. The DNP enhancement can only partly make up for the loss in resolution. Because of this, the simple enhancement factor can easily be misleading and conveys a too optimistic view on the expected practical enhancement. Better ways to quantify the actual enhancement have been published and

7

help to get a better insight into what can be realistically expected [26].

Only due to the outstanding spectral resolution of our system at ambient temperature a meaningful analysis was still possible under “DNP-conditions”. Nevertheless, the percentage of assigned resonances dropped significantly. That is why DNP is not yet a general solution to sensitivity problems concerning biological samples including supramolecular assemblies. Prior to subjecting the possibly limited amount of sample to studies using DNP (and making it difficult or even impossible to recover from the matrix), it should be carefully evaluated whether the foreseeable loss in spectral resolution will make this a worthwhile endeavor.

Recently, there has been considerable improvement in this field. The introduction of much more efficient radicals (AMUPol [22]) and new sample preparation methods[27] address the main problems of DNP in combination with ssNMR. Even better sensitivity will allow for the acquisition of NMR spectra of high dimensionality (4D) further reducing the demand for very high peak resolution, making the technique better suited for samples giving rise to highly crowded spectra. Although the current results do not yet promise a general applicability of this method for supramolecular assemblies, we are positive that this may change in the future.

8

2 Deuteration and proton detection

Currently MAS NMR is mainly based on correlating

13

C and

13

C detection to obtain multidimensional spectra

N resonances. On the other hand, 1H has a high gyromagnetic ratio γ, high

15

density and almost 100% natural abundance. These properties make protons highly suitable for long-range distance restraint determination. Also, proton detection significantly enhances spectral sensitivity compared to 13C detection. Unfortunately, very strong proton-proton dipolar interactions in the solid state significantly broaden proton lines making proton NMR ineffective. In MAS NMR of biomolecules the most efficient approach to attenuate proton-proton dipolar couplings is extensive deuteration and/or high frequency sample rotation. Proper proton dilution in proteins can be achieved by using perdeuteration with subsequent proton reintroduction on hydrogen exchangeable sites employing a buffer containing a H2O-D2O mixture. Reif and coworkers obtained proton-detected, ultra-high resolution spectra using a perdeuterated α-spectrin SH3 domain recrystallized from a 10% H2O buffer [28]. Even without high power decoupling proton and nitrogen line widths were on the order of 20 Hz and 12 Hz at MAS rates in the range of 12-24 kHz. Ultrafast MAS rates up to 60 kHz and high external magnetic fields can yield well resolved proton detected spectra of fully protonated samples [29] and samples at 100% proton back-exchange level [30]. Nevertheless, the achievable spectral quality also depends on the sample homogeneity.

These remarkable results stimulated the application of extensive deuteration and proton detection to many biological systems in different physical states, especially noncrystalline samples [31-33] and large molecular machines [34]. As an example, Fig. 3 shows the high resolution protondetected (H)NH 2D HETCOR spectrum of the insoluble, non-crystalline T3SS needle of Salmonella typhimurium. For this sample, the average proton and nitrogen linewidths are 47 Hz and 16 Hz, respectively, which are larger than the values observable in

9

-SH3.

Fig. 3: A) Proton detected 15N,1H correlation spectrum of highly deuterated PrgI needles. The spectrum was obtained on a 600 MHz spectrometer at an MAS rate of 28 kHz. Prior to Fourier transform the data in the proton dimension were apodized with a 36°-shifted squared sine bell window function. The protein was perdeuterated and back-protonated using 20% H2O buffer. The dashed line indicates the

15

N chemical shift of 120.4 ppm. Figure modified from [23]. B) 1D

1

row along the H dimension from 2D HN correlation spectrum presented in panel A. The projection was extracted at the 15

N chemical shift of 120.4 ppm. The proton line widths (FWHM) of selected resonances are indicated.

The first essential step in NMR studies is the assignment of resonances. In spite of the relatively short history of proton detected ssNMR, already a plethora of assignment protocols was created and many biomolecules were assigned. Advantages of these experiments are:

high

dimensionality of spectra, observation of three types of nuclei, efficient inter-nuclei polarization transfer, long magnetization lifetime and no need for high power decoupling. All assignment approaches analyze a set of proton detected 3D experiments correlating H,N,CA,CO and CB spins

10

from residues i and i-1. Below we would like to focus on methods providing highest performance. The most efficient approach for magnetization transfer between different types of nuclei is crosspolarization, while magnetization transfer between directly bound carbons can be achieved by recoupling of the homonuclear dipolar interaction optimized for one bond transfer [32, 33], or by pure through-bond INEPT transfer, utilizing relatively high CA-CO or CA-CB scalar couplings [29, 31]. In such experiments the choice between INEPT and homonuclear recoupling is determined by sample properties and experimental conditions. The INEPT element comprises a relatively long transfer period and a train of selective and/or hard pulses. Thus it is preferable at low dephasing rates of transversal magnetization and at a high performance of the applied RF pulses. For a number of proteins, these experimental protocols allowed to obtain different types of correlations within short experimental time: CAiNiHi, CAi-1NiHi, COiNiHi, COi-1NiHi, CAiCOiHi, COi-1CAi-1Hi, CBiNiHi, and CBi-1NiHi. These spectra were used for a sequential walk along the backbone by matching CA, CO and sometimes also CB frequencies. Due to the high resolution and sensitivity, reliable connectivities between subsequent residues can be readily established giving the final protein assignment [29, 31-33]. Most of the assignments were obtained using samples with 100% reprotonation on exchangeable sites, which required ultrahigh MAS rates (40-60 kHz) and high external magnetic fields (750 - 1000 MHz). On the other hand, employing the more widely available 2.5 mm and 3.2 mm probes with relatively slow MAS rates demands a low reprotonation degree to obtain high resolution proton-detected spectra. In such systems, establishing the correlation between CAi-1 and (HN)i (or between COi and (HN)i) is a challenging task because of the low proton density. Initially polarizing CAi-1 by direct magnetization transfer from Hi is inefficient due to the long distance and dipolar truncation, while the direct transfer from Hi-1 is reduced due to the low probability of proton occurrence in both positions (i and i-1) simultaneously. At the same time, correlations between CAi-1 and (HN)i as well as between COi and (HN)i are crucial for backbone assignment. Thus these experimental conditions require the development of new and the revision of existing experimental protocols. Recently, our group presented two efficient

11

experimental approaches for the backbone assignment of partially reprotonated PrgI needles at very fast MAS (25-28 kHz) and moderate external magnetic field (600 MHz 1H Larmor frequency), employing only dipolar-based magnetization transfers. The first method exploits an “out-and-back” 13

CA-13CO element. Initial polarization is propagated from Hi to CAi-1 via COi-1 and then back to

COi-1, then it is transferred via Ni to Hi for detection. The protocol is optimized specifically for magnetization transfer between directly bound nuclei. It yields high sensitivity and high resolution (H)COCA(CON)H, (HCO)CA(CO)NH and (H)CACO(CAN)H 3D spectra with unambiguous information about inter-spin connectivity. The second approach employs long range H-CO and HCA magnetization transfers to obtain high sensitivity (H)COCAH 3D spectra, containing both CAiCOiHi and COi-1CAi-1Hi correlations. Combined with trivial (H)CANH and (H)CONH 3D correlations those spectra readily allow to assign the protein backbone. Fig. 4 illustrates a sequential walk along the backbone between residues A73 and V65, based on the first method. Inter-residue connectivity is established by a set of five 3D correlations: CAiNiHi, CAi-1NiHi, COi-1NiHi, COi1CAi-1Hi,

and CAiCOiHi. Recently, we extended this scheme to four dimensions resulting in data

that are ideal input for (semi-)automatic assignment approaches and also paving the way to assign supramolecular assemblies with larger subunit sizes compared to the needle protein PrgI [35].

12

Fig. 4: A) Schematic representation of CAiNiHi, CAi-1NiHi, COi-1NiHi, COi-1CAi-1Hi and CAi-1COi-1Hi-1 inter-spin connectivities obtained by proton-detected 3D experiments. Arrows represent spin polarization transfer pathways. B) Strip plots for a sequential backbone walk between the residues A73 and V65 using the correlations presented in panel (A). Color coding of the peaks is given according to the experimental type, as presented in panel (A). Signals from CAiNiHi, CAi-1NiHi and COi-1NiHi experiments are shown in the upper row, whilst the signals from COi-1CAi-1Hi and CAi1COi-1Hi-1

experiments are in the lower row. Figure from [32].

Inter-nuclear proximities are estimated from dipole-dipole couplings, which make protons well suited to obtain distance restraints because of their high gyromagnetic ratio. The dilution of the proton network removes trivial short range contacts and gives better access to long-range distance restraints. 3D and 4D correlation experiments, incorporating a proton-proton dipolar recoupling element, were employed for high quality structural elucidation of several deuterated biomolecules [30, 36-38].

Solid state NMR allows to study internal protein dynamics in the absence of overall molecular tumbling, which is an advantage compared to solution state NMR. However, due to the strong

13

anisotropic interactions, the influence of local structural fluctuations on spin evolution might be difficult to quantify. Extensive deuteration and fast MAS rates (20-60 kHz) can almost completely average out anisotropic interactions. High sensitivity and resolution at such experimental conditions give a great possibility to study protein dynamics with single residue resolution. For small proteins such as α-spectrin SH3 and ubiquitin numerous relaxation parameters were obtained, including 15N T1 relaxation, 1H–15N dipole and

15

N CSA cross correlated relaxation rates, and 1H,15N dipolar

order parameters [39, 40]. Extensive data sets were analyzed within the framework of the extended model-free Lipari–Szabo theory to account for motions at two different time scales. The variety of results obtained by proton detected ssNMR demonstrates the power of this approach to elucidate structure and dynamics of complex biomolecules.

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3 Improvement in classical carbon-detected experiments by band-selective homonuclear CP transfer

The sequential resonance assignment is the basis of all further ssNMR spectroscopic studies [41-44]. An important step was the development of efficient 15N-13C SPECIFIC-CP transfer which is widely used today [45]. Another crucial part in many protein sequential assignment schemes is the magnetization transfer between CO and CA nuclei. Recently, robust and efficient band-selective homonuclear (BSH) cross-polarization between CO and CA spins was achieved in our group in both highly deuterated [46] and protonated protein samples [47]. This approach is specifically designed for high external magnetic fields and moderate magic-angle spinning rates. In turn, these conditions are favorable to study biomolecules with widely available 3.2 mm probes and high external magnetic fields.

Based on a detailed quantum-mechanical description of two dipolar-coupled homonuclear spins in a powdered solid under MAS and continuous RF irradiation, it can be seen that recoupling takes place at various conditions when the modulations of the spatial and spin parts of the spin Hamiltonian mutually cancel each other (Fig. 5a). At the given experimental conditions (e.g. 850 MHz of proton resonance frequency and 21 kHz MAS rate), recoupling of the flip-flop and flopflop terms that correspond to n=1 ZQ (zero-quantum) and n=2 DQ (double-quantum) transitions, respectively, occurs when the difference or sum of the effective fields acting on CO and CA is equal to one or two times the spinning rate [47]. The most efficient recoupling is achieved when the sum of effective radio-frequency fields on CO and CA resonances equals two times the spinning rate, yielding up to 50 % of magnetization transfer efficiency in highly deuterated proteins and 33% in protonated proteins.

Using PEG precipitated ubiquitin as an example, the performance of BSH-CP was compared to widely used proton-driven spin diffusion (PDSD [48]) and MIRROR [49] in NCOCX experiments

15

at MAS rates of 21 kHz on an 850 MHz wide-bore spectrometer (Fig. 5b). The contact time of the BSH-CP step in the NCOCA experiment was 4.4 ms with a ramp down to 80 % of the carbon RF field from an initial value of 15.1 kHz, the mixing time of PDSD was 100 ms, and the CO-CA transfer by MIRROR was achieved in 25 ms recoupling time with a 14.9 kHz proton CW recoupling RF field which yields maximal CA signal. The net CA amplitude in the BSH-CP NCOCA experiment is about 57 % higher than in the PDSD NCOCX experiment and 14 % higher than in the MIRROR NCOCA experiment.

Fig. 5: a) Magnetization vectors for CO-CA 2D correlation experiments based on BSH-CP transfer. b) Comparison of PDSD-based NCOCX, MIRROR and BSH-CP based NCOCA spectra of uniformly [13C, 15N]-labeled ubiquitin.

Additionally,

different

from

the

second-order

phenomena

of

the

often

applied

PDSD/DARR/MIRROR methods, BSH-CP employs a first-order recoupling effect which is desirable in assignment experiments because unwanted sequential or long-range magnetization

16

transfers are suppressed by dipolar truncation [47, 50]. Two-dimensional PDSD NCOCX and MIRROR NCOCA spectra were compared to the BSH-CP-based NCOCA spectrum. A considerable number of cross peaks which are not interresidual correlations between Ni and CAi-1 nuclei are present in the PDSD NCOCX spectrum (Fig. 6a). Some of these long range and relay correlation cross peaks also appear in the MIRROR-based NCOCA (Fig. 6b) spectrum. The intensity ratios of 58 unambiguously assigned NiCAi-1 cross peaks are presented in Fig. 6c, and the average value relative to BSH-CP is only ~62 % for MIRROR and ~50 % for PDSD.

Fig. 6: Comparison of BSH-CP NCOCA(red), PDSD NCOCX (blue) and MIRROR NCOCA (green) spectra of uniformly 13

[ C,

15

N]-labeled ubiquitin. 2D spectra were overlaid in a) and b), and the intensity (I) ratios compared to BSH-CP

NCOCA of 58 unambiguously assigned NiCAi-1 cross peaks are presented in c).

The DREAM scheme is also suitable for homonuclear recoupling, but it only works well when the spinning rate considerably exceeds the isotropic chemical shift difference of CO and CA spins [51, 52], and thus it is more suitable for small rotor sizes with small active sample volume, which

17

may not be favorable if enough sample is available to fill larger rotors (e.g. 3.2 mm outer diameter) [53].

In general, our BSH-CP approach is very well-suited for some experimental conditions which are favorable to study supramolecular assemblies, such as high external magnetic fields, e.g. 600, 700, 800, 850, 900, 1000 MHz and MAS rates of 15, 17, 20, 21, 22, 24 kHz, respectively. One should mention that BSH-CP is not very flexible and not applicable at very high external magnetic fields and at low spinning rates. It still works at lower spinning rates, e.g. 11 kHz on a 600 MHz NMR spectrometer equipped with a 4 mm probe, but the transfer efficiency is highly dependent on the frequency offset of each spin due to the significantly lower spinlock RF strength (data not shown).

We applied BSH-CP in a complete set of 3D protein sequential resonance assignment experiments, namely NCOCA, NCACO, and N(co)CACB in addition to standard 3D NCACB and CANCO experiments [54]. Due to the excellent transfer efficiency provided by BSH-CP, the complete set could be recorded in a short time frame (less than one week) with high sensitivity and resolution. Reliable and unambiguous protein backbone assignments can be obtained easily with this set of five 3D NMR spectra.

18

Conclusion Supramolecular assemblies with large subunit sizes are challenging to study using ssNMR spectroscopy. Due to their complex structure they give rise to a manifold of peaks and, at the same time, sensitivity is often an issue, especially if higher dimensional NMR spectra are to be acquired. Recent advances in the field of ssNMR spectroscopy have shown to considerably increase the number of methods that can help to overcome these problems and thus facilitate the study of biologically interesting systems.

The here presented approaches are, in principle, not only applicable to supramolecular assemblies but a broad range of sample types because many aspects of data acquisition and data analysis for biological samples do not differ very much from system to system. Nevertheless, the methods were chosen because they have proven to be worthwhile in our group and because they represent interesting new advances that should be taken into account when a supramolecular assembly is to be studied.

Hyperpolarization using DNP drastically enhances the sensitivity of an NMR measurement by transferring the high polarization of the electron spins to the nuclei. These experiments are typically carried out at cryogenic temperatures in a glass-forming matrix. This has the unwanted side-effect to considerably broaden the NMR lines, which can render the resulting spectra challenging to analyze, depending on the nature of the sample and the resolution at ambient temperature. Nevertheless, we found that in conjunction with the reduced dynamics at very low temperature, one can make deductions with respect to the dynamics at room temperature of certain parts of the assembly structure.

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Proton detection is another way to increase the sensitivity and resolution of an NMR measurement drastically. As a prerequisite, the strong dipolar couplings between the protons must be overcome by perdeuteration and either partial back-protonation, or by full back-protonation together with ultrafast MAS. The resulting resolution of the 2D, 3D and 4D NMR spectra was shown to be excellent. There are now established protocols and pulse sequences that take advantage of several magnetization transfer pathways along the peptide chain and allow for a complete backbone resonance assignment. This approach has proven to be extremely effective for the purpose of elucidating the structure and dynamics of large biological macromolecules.

Magnetization transfer is an essential part of every NMR pulse sequence to reveal the connectivity and structure of the sample. Transfer from CA to CO (and vice-versa) can be achieved using different approaches (PDSD, DARR, MIRROR, BSH-CP, DREAM), where the effectivity of each method depends on the spinning speed and strength of external magnetic field. For moderate spinning speeds (15-24 kHz) BSH-CP has proven to be the most effective way to carry out the transfer and therefore supersedes other methods. We therefore strongly recommend replacing the formerly most effective approach (MIRROR) by BSH-CP. The increase in sensitivity can help to reduce the measurement time considerably and consequently facilitates the study of large biomolecules.

The presented approaches represent new strategies to study supramolecular assemblies by ssNMR. They facilitate the data acquisition by increasing the sensitivity of the measurement, which reduces either the needed amount of sample or results in a better signal-to-noise ratio at a given measurement time. DNP and proton detection even allow for a view on the sample structure from another standpoint as they are carried out at different measurement conditions and focus on nuclei which were so far not regularly used for detection purposes, respectively.

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ACKNOWLEDGEMENTS This work was supported by the Max Planck Society, the Leibniz-Institut für Molekulare Pharmakologie (FMP), the Deutsche Forschungsgemeinschaft (Emmy Noether Fellowship to A. L.), and the Fonds der Chemischen Industrie (Kekulé Scholarship to P. F.).

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DNP 1

H Detection

Homonuclear CP

Highlights

• • • • •

Supramolecular assemblies ideal targets for solid-state NMR New approaches that increase sensitivity and resolution Dynamic nuclear polarization to enhance signal intensity drastically Proton detection to increase resolution and sensitivity Recoupling techniques for optimal homonuclear magnetization transfer

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