Journal of Magnetic Resonance 253 (2015) 36–49

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High-resolution proton-detected NMR of proteins at very fast MAS Loren B. Andreas a, Tanguy Le Marchand a, Kristaps Jaudzems b, Guido Pintacuda a,⇑ a b

Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques, UMR 5280/CNRS, ENS Lyon, UCB Lyon 1, Université de Lyon, Villeurbanne, France Latvian Institute of Organic Synthesis, Riga, Latvia

a r t i c l e

i n f o

Article history: Received 29 October 2014 Revised 23 December 2014

Keywords: 1 H detection Fast magic-angle spinning Resonance assignment Protein structure Protein dynamics Paramagnetism

a b s t r a c t When combined with high-frequency (currently 60 kHz) magic-angle spinning (MAS), proton detection 1 boosts sensitivity and increases coherence lifetimes, resulting in narrow H lines. Herein, we review methods for efficient proton detected techniques and applications in highly deuterated proteins, with 1 an emphasis on 100% selected H site concentration for the purpose of sensitivity. We discuss the factors affecting resolution and sensitivity that have resulted in higher and higher frequency MAS. Next we describe the various methods that have been used for backbone and side-chain assignment with proton 13 13 detection, highlighting the efficient use of scalar-based C– C transfers. Additionally, we show new 13 1 spectra making use of these schemes for side-chain assignment of methyl C– H resonances. The rapid acquisition of resolved 2D spectra with proton detection allows efficient measurement of relaxation parameters used as a measure of dynamic processes. Under rapid MAS, relaxation times can be measured in a site-specific manner in medium-sized proteins, enabling the investigation of molecular motions at high resolution. Additionally, we discuss methods for measurement of structural parameters, including 1 1 measurement of internuclear H– H contacts and the use of paramagnetic effects in the determination of global structure. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Due to impressive progress in instrumentation and methodology, atomic resolution structural studies of biomolecules by solid-state NMR have advanced from single amino acids and small peptides to functional proteins of several hundred residues [1]. Successful application of solid-state NMR depends critically on the resolution, which determines the size of the protein target through the number of resolvable peaks in an n-dimensional spectrum, and on the sensitivity, which determines the required measurement time. In powdered solids, resolution is hampered by large anisotropic interactions between nuclei. For many years, biomolecular solid-state NMR has developed through the detection of nuclei 13 15 with lower gyromagnetic ration (c), such as C and N, for which 1 magic-angle spinning (MAS) in combination with strong H dipolar decoupling is sufficient to recover narrow lines. However, the detection of lower-gamma nuclei sacrifices sensitivity. The direct detection of proton coherences is the most straightforward avenue to sensitivity in magnetic resonance due to the high gyromagnetic ratio, the high natural abundance and

⇑ Corresponding author. E-mail address: [email protected] (G. Pintacuda). http://dx.doi.org/10.1016/j.jmr.2015.01.003 1090-7807/Ó 2015 Elsevier Inc. All rights reserved.

1

the ubiquitous presence of Hs in biomolecules. Over the last decade, methods for resolved and sensitive proton spectra in the solid-state have been developed, through the combination of suitable labeling strategies and the advent of probes capable of very fast (60 kHz and above) MAS. As illustrated in the present review, proton detected NMR of deuterated samples at very fast MAS has now matured into a technique that can reliably be applied to proteins of several 10s of kD in size, for the assignment of resonances, and for the determination of structure and dynamics.

2. Sensitivity and resolution The advantage in sensitivity of proton detection was recognized early by the solution NMR community. Linewidths and other factors being equal, the sensitivity of detecting a polarization improves with c3/2 [2]. Therefore proton detection is approximately 8 or 31 13 15 fold more sensitive compared with C or N, assuming that initial 1 polarization was on H in either case [3]. In solid proteins, however, a network of dipole-coupled protons results in severe homogeneous broadening that is difficult to overcome using MAS alone, due in part to the narrow chemical shift 1 range in the H dimension, which is not well separated even at the highest magnetic fields available [4]. Nevertheless, the last two decades have witnessed an increase in the MAS frequency

L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49

from 10 to 100 kHz and an increase in the magnetic field to up to 1 GHz, both of which have driven a significant improvement in spectral resolution required for investigation of global protein structure. The effect of MAS and magnetic field are illustrated in 15 1 Fig. 1 on a N– H dipolar-based heteronuclear single-quantum correlation (CP-HSQC) for the Single-Stranded-DNA Binding (SSB) protein from Escherichia Coli [5]. An increase in MAS rates causes 1 a linear improvement in H coherence lifetimes, resulting in narrower lines (Fig. 1a–c and h), while by increasing the magnetic field, nearby protons are separated in frequency, which results in less efficient spin flip-flops, and further narrowing the homogeneous line width (Fig. 1d–f and h). While resolved spectra can be acquired for fully-protonated microcrystals [5,6], for highest resolution at currently available fields and spinning frequencies, MAS needs to be combined with dilution of the proton content in the sample [7,8]. In proteins, proton dilution is conveniently achieved by expression in perdeuterated media and then by exchanging the amides using buffers with appropriate H2O/D2O ratios to achieve protonation at a controlled level. Resolution enhancement following protein deuteration was first demonstrated at an MAS rate of 10–20 kHz on samples with full protonation of the amide sites [9,10], and Reif’s group showed that a further improvement in resolution was achieved by lowering the levels of amide protonation (typically 10–40%, see Table 1) [11,12]. With 1 faster MAS however, the network of H dipolar interactions is more 1 efficiently averaged and less H dilution is necessary to achieve the same narrow line widths (Table 1) [13,14]. In fully back-exchanged 1 2 13 15 (100% H at exchangeable sites) and otherwise [ H, C, N]-labeled 1 microcrystalline proteins, high-quality CP-HSQCs (with H linewidths of typically 50 Hz) can be obtained in a very short time (typically few minutes) on very small amounts of samples, typically 2–3 mg at 60 kHz [14], or even less than 1 mg at 100 kHz [15]. These high-quality correlation spectra can be acquired on samples of a different nature and aggregation state [16], with resolution similar to that recorded at slower MAS with much higher proton dilution, and requiring larger amounts of sample [17]. Fig. 2 shows examples on two small microcrystalline domains, a sedimented nucleocapsid, and two a-helical and b-barrel membrane proteins. The high resolution of these correlations requires a high degree of sample homogeneity, which is often achieved by preparing crystals (or 2D crystals in the case of membrane proteins), although these preparations need not possess macroscopic order. For example, non-crystalline, sedimented proteins and fibrils can exhibit high-resolution spectra due to a high degree of local order. Such samples have little inhomogeneous broadening, making them ideal for biological investigation, as well as for systematic measurement of factors affecting homogeneous resolution. The resolution achievable with deuteration (Fig. 1g) is significantly higher than that achievable on a fully protonated sample, even at the highest fields and MAS rates available today, indicating that further increase in the MAS frequency will lead to further line narrowing for fully-protonated proteins. The inhomogeneous linewidth is approximately 100 Hz for the samples in Fig. 1, suggesting that a spinning frequency of about 120 kHz would be needed for the inhomogeneous and homogeneous components of the line to be equal, following the trend in Fig. 1h. In order to achieve faster MAS rates, the rotor size and, correspondently, the sample volume, needs to be reduced. At first glance, this may appear as a limitation, given the loss in sensitivity that occurs with a reduction in the amount of sample. However, although the sample volume decreases by a factor of about 12 at 60 kHz MAS using a 1.3 mm rotor compared with 20 kHz MAS in a 3.2 mm rotor, the resulting loss in sensitivity is not as large as might be expected due to more effective inductive coupling of the sample to the smaller coil (see Table 2). Without considering

37

changes in the number of turns in the coil, or the spacing, the sensitivity of a typical-sized NMR coil scales as the inverse of the diameter (d) [18]. Thus the 12-fold reduction in sample is compensated by a 2.5-fold improvement in sensitivity per mg of sample, or only about a 5-fold reduction in sensitivity overall. Without considering changes in line width, the sensitivity of proton detection in a 1.3 mm coil is still nearly twice as sensitive than carbon detection in a 3.2 mm coil. This benefit is realized for microcrystalline samples at 60 kHz MAS, as indicated in Table 2. Of great importance for biological samples, this ten-fold reduction in sample volume allows the investigation of only 1–2 mg of sample, extending MAS NMR to the study of proteins that are difficult to produce in large amounts without compromising sensitivity. As shown in Table 2, at full proton back-exchange in deuterated samples, maximal sensitivity is found near 40 kHz MAS (without a drastic reduction in resolution). This implies that 1.9 mm rotors are ideally suited for low dimensionality spectra involving few magnetization transfers. When additional coherence transfers are considered, higher sensitivity is observed with faster rotation in 1.3 mm rotors [19]. Additionally, above 40 kHz MAS, both CP and heteronuclear decoupling are efficiently performed with low-power irradiation [20,21]. This produces a further increase in sensitivity by enabling a reduction in the interscan delay, in order to obtain highly resolved spectra without unwanted heating and subsequent sample deterioration. Also, an increase in coherence lifetimes at faster MAS allows coherences between neighboring spins to be easily refocused, and thus opens the way to the efficient exploitation of J-transfer based experiments. For example, limiting decoupling to 100 kHz, an increase of the MAS 13 13 rate from 10 to 60 kHz improves the efficiency of a C– C through-bond transfer by a factor of 3 [22], which means that this scheme becomes a competitive alternative to dipolar-based methods. Once again, overall these gains in sensitivity offset the loss in sample volume when using small diameter rotors for high frequency MAS. 3. Resolution in protein side-chains In the case of fully-protonated samples, aliphatic protons exist in an even denser network of homonuclear couplings. Nevertheless, when recorded at 1 GHz and spinning at 60 kHz, linewidths as narrow as 160 Hz have been observed for a microcrystalline domain of E. coli DNA polymerase [5] (Fig. 3a), demonstrating the potential for a constructive use of side-chain resonances. To increase the resolution further for general application to large targets, however, dilution of the strongly coupled proton network is needed, similar to the case of the amide protons. While amide protons can be exchanged with deuterium to control the proton concentration, introduction of protons (and deuterons) at aliphatic sites takes place during protein expression. This has the principal advantage of eliminating the need of unfolding and refolding, which is a necessary step to introduce amide proteins in proteins that do not exchange easily such as large globular domains or some integral membrane proteins. Reif and coworkers realized that residual protonation in highlydeuterated peptides and proteins is non-negligible at aliphatic sites [8], and recorded the first high-resolution spectra of side-chains in solid proteins [23]. This was further refined into an approach coined Reduced Adjoining Protonation (RAP) [24,25], in which the protein is expressed in a medium containing deuterated glucose and a low level of H2O in D2O. Although this method produces a mixture of isotopomers, at low levels of about 5% protonation the primary species of methyl and methylene groups have only a single proton and are populated at 14% and 10%, respectively. Under these conditions, and moderate MAS of 20 kHz Asami et al. showed that a proton resolution of 25–60 Hz could be recorded for microcrystalline

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L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49

1

Fig. 1. CP-HSQC spectra as a function of spinning frequency (a–c) at a H frequency of 800 MHz (18.8 T), and as a function of magnetic field (d–f) at 60 kHz MAS. In (a–f), the sample is a fully protonated sample of SSB protein. In g, the protein is perdeuterated with amide protons exchanged to 100% protons, and the spectrum recorded at 60 kHz 1 MAS and 1 GHz. In h are plotted the MAS-rate dependence at 800 MHz (red, dashed lines) and the field strength dependence at 60 kHz MAS (blue, dotted lines) of H NMR 13 15 1 1 linewidths for two signals (Ala64 and Thr108) of [ C, N]-SSB. In the same plot, bulk H T2’s are depicted (, red, dashed lines) as a function of MAS-rate at a H frequency of 800 MHz. Readapted with permission from Ref. [5]. Copyright Ó 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

39

L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49 Table 1 Linewidths and experimental conditions found for various proteins under conditions used for proton detected NMR spectroscopy. Sample

Labeling

B0 (MHz)

10% H 10% HN 100% HN 100% HN 100% HN 100% 10% HN 60% HN 100% HN 10% HN 5% RAP

60 24 60 40 24 60 24 24 24 8–24 20

1000 1000 1000 1000 1000 1000 400 400 400 600 600

100% VLa 100% HN

55 100

850 850

N

SH3

Ubiq.

a

MAS (kHz)

1

1

H T02 (ms)

95 ± 3.1 62.6 ± 9.5 19 ± 0.7 12.7 ± 0.3 3.4 ± 0.1 1.5 ± 0.1 60.6 29.8 11.4

15

13

15

N ( C) T02 (ms)

H LW (Hz)

39 ± 10

146.9 ± 6.7 93.0 ± 7.5 31.1 ± 1.9 15.8 ± 0.4 5.7 ± 0.4 5.6 ± 0.3 75.8 23.6 9.6

49 ± 17 61 ± 23 117 ± 55 253 ± 123 19.0 ± 3.3 32.5 ± 10.3 58.3 ± 20.5 17–35 25–60 (aliphatic)

Source Lewandowsky et al. [13]

11.3 ± 5.4 25.1 ± 13.5 41.2 ± 14.4

40 (methyl) 19–73

13.5

13

N ( C) LW (Hz)

Akbey et al. [12]

(110)

Chevelkov et al. [11] Asami et al. [24]

(30) 30–68

Huber et al. [29] Agarwal et al. [15]

SOD

100% HN

60

1000

100

Knight et al. [14]

e186 (A83)

100%

60

800 1000

220 180

Marchetti et al. [5]

SSB

100% HN 100%

60 40 60

1000 800 800

M2

100% HN

60

1000

100

bR

10% HN

20

700

>30

OmpG

30% HN 100% HN

20 60

850 1000

9.2

101 ± 31 130–180

Ab

25% HN

20

600

8.2

170 ± 47

10 0.85 1.5

100 350 ± 75 225 ± 75

(12.5–40)

Marchetti et al.[5] Barbet-Massin et al. [16] >9

Linser et al. [17]

11.1

53 ± 23

Linser et al. [17] Barbet-Massin et al. [16]

13.5

38 ± 10

Linser et al. [17]

Methyl labeling of V and L produced methyl groups labeled as CHD2.

(a)

(b)

(c) N46 K101

(d)

E96 S49

F28

100

E43

N103

A109

A87

G43

A42

L44

V104

Q77

T86

G51

T22

T26

V17

105

(e)

V74 E92 A39 T154 S206 H205 R45 N38 T89 F129 Y243 Y241 A40 L48 T156 V170

L32

R169

A123

A30 L33 W97 Q50

T92

V53

110

δ (15N) / ppm

V58 R21 T37 A56 D40

115

S19

sc

V9

G29

V23 sc sc Y57

F56

N35

S33

G28

Y63

Q50 D14

K18 F52 Y15 T32 S11

I30 L8 E7 M25 K39 D29 E17 V44 K27 E22 K43 W41 W42 L10 K26 V46 L61 L34

S36

125

N38 K60 A11 sc

D62

L12

N42

A55

L31

H13 S88 L39 S52 R97 E74 R12 E77 L40 N24 E44 C25 K41 D34 A79 Y67 I35 S55 F22

Q16

sc

130

sc

L33

F70 Q8

R81

L54

Q2 D38

8

6

7

10

9

8

R23

N46

L26 R45 I42 H37 L38 F48 L26’ H37’ A30’ A30,L36 L43’ A29 I39’ V28’ V27’ L43 L40’ I32’ K49 I42’ V27 V28 L36’ L38’ A29 I33’ W41 D44 I33 I35 I35’ W41’

I51

D149 T144 L88 M14 Y97 A108

S252 T208

Y147 W44

E73 N171

R93

H75

Y76

Y173 A167

L200

L40

A47

I124 L93

9

W132

I46

D20

E153 L202

K122 Y51

Q77

165T 13A L142

Y160

A68

10

G12 G203

G90 G157 E164 N143 L168 Y197 S134

D118 Y210 Y50 F37

A66

6

N163

G242

I71

W95 Y10 I7 I1 R45 L87

7

sc

N31’

V47

R34 L24

N83 V9 N21 K6 V85 F56 V37

D96

T106

R102 A113

E47

V93

I39

N89 E98 K94 F117 D67 M6 N76 E88 V125 C65 E75 L90 V70 S84 L119 A95

E69 L65 K94

Y26 V82

F47 sc

T99

A122

A15

V27

9

N45

N31

L115

S31

E36

K91 I92 I46

T27

F109

D76

H84

Y66

10

Q7

S20

Y78 T4 R3

T145 T124

S50

G40 N74

S79

G131 G146

N109 S34 V35

I32

G34

S28

S19 N112

S28

L64

C89

sc

Q89

H31

C80

R49

K59 E45

120

F30

T71

S57

T24

G95

K75

A116

G91

S110 sc

T73

G34’

T121

T68

sc

R151 N204 G274

S25 Y13

A196

R195

G114

8

7

6

10

9

8

7

6

10

9

T150 L158

8

7

6

δ (1H) / ppm 15

1

1

1

Fig. 2. N– H CP-HSQC measured at a H frequency of 1 GHz and MAS rate of 60 kHz on five perdeuterated [100%- HN]-labeled protein samples. (a) Microcrystalline a-spectrin SH3 domain, (b) microcrystalline b2-microglobulin, the light chain of the Class I major histocompatibility complex (b2m), (c) sedimented viral nucleocapsid of Acinetobacter phage 205 (AP205), (d) conductance domain from influenza A M2, (e) outer membrane protein G (OmpG) from Gram-negative bacteria. Reprinted with permission from Ref. [16]. Copyright Ó 2014 American Chemical Society.

40

L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49

Table 2 1 Characteristics of Bruker NMR rotors, and their calculated relative sensitivity for 1D H spectra (scaled to the 3.2 mm rotor at full protonation). The inhomogeneous linewidth Dmin of SH3 (39 Hz) at 1 GHz was used along with reported T2 values from Table 1. Where a measured T2 was not available, the value was linearly extrapolated. Stated Full-Width-at! rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  2ffi . The sensitivity of SH3 samples was Half-Maximum (FWHM) were calculated as the geometric sum of inhomogeneous and homogeneous contributions ðDmin Þ2 þ p1T 2 calculated as proportional to the inverse square root of the linewidth [Ref 3]. Rotor outer diameter (mm) Operating frequency (kHz) Sample volume (lL) Relative detection sensitivity per unit sample (au) Relative detection sensitivity (overall) lL ⁄ sens. per. lL 10% HN 100% H

FWHM (Hz) Rel. sensitivity FWHM (Hz) Rel. sensitivity FWHM (Hz) Rel. sensitivity

N

Fully protonated a

4 18 70 0.8

3.2 24 30 1

2.5 35 12 1.3

1.9 40 10 1.7

1.3 60 2.5 2.5

0.8a 95 0.7 4

1.9

1

0.51

0.56

0.20

0.09

39 0.69 130 3.8 708 1.6

39 0.37 101 2.3 532 1

39 0.19 48 1.7 365 0.62

39 0.21 46 1.9 320 0.72

39 0.08 42 0.73 220 0.32

39 0.03 40 0.34 140 0.18

This rotor system was built by Ago Samoson and recently reported [15,64].

0

Δ = 160 Hz (0.16 ppm)

(a)

Δ = 213 Hz (0.27 ppm)

(b)

not resolved

20 10

Δ = 147 Hz (0.18 ppm)

30

Δ = 388 Hz (0.48 ppm) Δ = 164 Hz (0.16 ppm)

30

40

δ (13C) / ppm

δ (13C) / ppm

20

40

50

Δ = 147 Hz (0.15 ppm)

50

60

60

70

Δ = 95 Hz (0.12 ppm) Δ = 287 Hz (0.36 ppm)

6

5

4

3

2

1

0

6

4

3

2

1

0

δ ( H) / ppm

δ ( H) / ppm 1

5

1

1

13

1

Fig. 3. (a) Proton-detected H– C correlation spectrum of fully protonated e186 from E. coli DNA polymerase III at a H frequency of 1 GHz (23.5 T) and 60 kHz MAS. (b) 1 13 1 1 Superimposition of H– C correlation spectra of perdeuterated V, L H cloud ubiquitin (in red) and fully protonated ubiquitin (in blue) at 800 MHz H frequency (18.8 T) and 60 kHz MAS. Adapted with permission from Refs. [5] and [32]. Copyright Ó 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim and 2014 American Chemical Society. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 2

chicken alpha spectrin SH3 [24]. Together with H decoupling, this 1 13 allows the acquisition of 2D H, C correlations of exceptional resolution, which serve as the base for the identification of aliphatic-aliphatic contacts that define a protein fold. While RAP labeling results in highly resolved aliphatic proton spectra, the site concentration is significantly diluted. Using E. Coli protein expression with the addition of metabolic precursors, methyl groups of I, L, and V can be labeled as isolated CH3 or CD2H [26,27], and also with stereo and regio-specificity [28]. With 60 kHz MAS, selective CH3 labeling in a deuterated background improves the resolution, while labeling with CD2H results in spectra with similar resolution as the RAP samples, but with near 100% site protonation [29]. This is illustrated in Fig. 4 in

spectra of the sedimented nucleocapsid AP205 labeled on methyl groups with CH3 and CD2H using a-ketoacid precursors [27]. While methyl groups can provide tertiary contacts defining a protein fold, these methyl-labeling approaches do not allow detection of other aliphatic side chain positions, such as CA. In this context, Stereo-Array Isotope Labeling (SAIL) offers a tailored combination of proton dilution with 100% site incorporation, which has been applied by cell-free expression methods to the labeling of several proteins for solution NMR [30]. Under MAS, this approach was shown to reduce the proton line widths of the amino acid valine by a factor of 2–7 spinning at 27 kHz, but has not so far been generally implemented due to cost [31].

41

L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49

(a)

(b)

H

CA

δ (13C) / ppm

10

C′

(a)

15

N H

N

CA 20

(H)CB(CACO)NH x yx y

1

H

35 Hz

60 Hz

25

t2

15

N

1

0

-1

δ (1H) / ppm 1

1

CB

0

t1/2

t1/2

13

t2,max-t2

τsat

t1,max-t1

-1

δ (1H) / ppm

13

Fig. 4. H, C CP-HSQC (a) and J-HSQC (b) of perdeuterated AP205 nucleocapsids, 13 13 where methyl groups of I, L and V residues are labeled as CH3 (a) and CHD2 (b). 1 Spectra were acquired at a magnetic field corresponding to a H frequency of 800 MHz, under 60 kHz MAS.

At 60 kHz MAS, it was recently shown that incorporation of fully protonated amino acids in an otherwise deuterated protein provides significant improvement in linewidths, an approach termed proton-cloud labeling [32]. For protein microcrystals of ubiquitin at 60 kHz MAS, cloud labeling of Valine and Leucine reduces the line width of aliphatic sites by a factor of 2–3. These results are reproduced in Fig. 3b, highlighting several narrow line widths as sharp as 95 Hz for Ca. The approach was also demonstrated for a large membrane protein (BamA, 52 kDa), resulting in linewidths of about 300 Hz.

13

CA

13

CO

H

CA

C′

(b) N

H

N

CA

(H)(CA)CB(CACO)NH x yx y

1

H t2

15

N

13

CB

3.1. Backbone resonance assignment

13

Assignment of resonances is an essential prerequisite for the measurement of structural and dynamical parameters at atomic resolution. This step is done in NMR by designing magnetization transfer schemes that are well defined in relation to the primary sequence. In solids, these procedures depend on the availability of large sample quantities (several mg), are extremely timeconsuming (weeks to months of acquisition times), and yield spectra that require laborious expert analysis. The availability of 1 resolved H resonances offers the possibility to acquire multidimen1 13 15 sional correlations involving H, C and N spins in the protein chain, shortening the spectral acquisition times, and accelerating the assignment process similar to the strategies used in solution NMR. Correlation experiments for assignment in solution rely on coherence transfers via the J couplings, which result in predictable magnetization pathways along the primary sequence through chemical bonds [33]. This is also the case of solid samples with highly diluted pro1 tons, where long coherence lifetimes were demonstrated for H, 15 13 N and C, allowing the direct application of pulse sequences developed for solution NMR [34]. These early reports were 1 extremely important to motivate further development of H detection in MAS NMR. For fully amide protonated samples, however, up to spinning frequencies of about 40 kHz, J transfers are typically inefficient due to short T2’s relative to the strength of the decoupling, and instead (short) dipolar transfers are the method of choice such that efficient transfer takes place between nearby (bonded) neighbors [35,36]. For example, the combination of the CP-HSQC sequence with H/C and N/C cross-polarization (CP) steps results in 3D spec1 15 tra such as (H)CONH and (H)CANH which correlate H, N and the 13 13 neighboring CO/ CA [37]. More complex experiments (such as shown in Fig. 5a) can be designed by the addition of homonuclear

13

CA

CO

δ3

δ3

t1

δ3

τsat

δ3

δ2

δ2 δ1

δ1

Fig. 5. Pulse sequences for two versions of a 3D HiNiCBi1 correlation experiment, together with the representation of the corresponding coherence transfer path13 13 13 13 ways. Transfers between CA and CB, and CA and CO are highlighted by green 13 and red boxes, respectively. In (a), coherence are transferred between C spins by two consecutive DREAM recoupling periods [36] and in (b) by evolution of the scalar couplings [16]. Narrow and broad black rectangles indicate hard p/2 and p pulses, while selective inversion pulses are indicated by half ellipses.

carbon–carbon dipolar recoupling periods, resulting in correlations 1 15 13 between a given H, N pair with the resonances of C spins from adjacent residues [36,38], or with DQ coherences involving CO–CA 1 and CA–CB pairs [39]. At 40 kHz MAS, low power H decoupling can already be used during indirect chemical shift evolutions, but high 13 13 15 power decoupling was used during C selective pulses, C to N CP 13 and C homonuclear recoupling [36]. At 60 kHz and above, the requirements for homonuclear dipolar recoupling become more severe for many sequences due to matching of the RF to a multiple of the rotor frequency. While a range of dipolar correlation experiments have been successfully developed for fast MAS [29,40–44], a simpler approach is to take advantage 13 of the long C coherence lifetimes and adapt J-based transfer schemes from solution NMR in a straightforward manner. In this spinning regime, resonance assignment can be performed by 1 13 13 15 means of experiments combining H to C and C to N CP with 13 13 1 C– C scalar evolutions under low-power H decoupling (an exam13 13 ple is reported in Fig. 5b). These C– C schemes involve either ‘‘full-transfer’’ blocks, where single-quantum coherence is fully transferred between two coupled spins [14,45,46], or ‘‘out-andback’’ blocks, where the chemical shift of a second spin is recorded via an antiphase coherence in order to minimize losses due to T2 decoherence and passive couplings [46].

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L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49

In the case of a microcrystalline sample at 60 kHz MAS, trans13 verse coherence lifetimes are extremely long for both CA and 13 CO spins (20 and 45 ms, respectively). High sensitivity is therefore observed even after the addition of one, two or three carbon– carbon transfers, with efficiencies that range from 0.5 to 0.1 with 1 respect to the basic H-detected CP-HSQC module (Fig. 6a). In these conditions, a full set of six experiments (i.e. three pairs of 3D interand intra-residue HNC correlations involving each CO, CA and CB spin) can be recorded in 1.5 (SH3) to 2.5 (b2m) days on less than 2 mg of sample [16]. Sequence-specific resonance assignment follows combined ana1 15 lysis of pairs of 3D spectra, where each H, N pair is correlated 13 with a C spin of the same or of the previous residue (Fig. 6b). 13 Notably, the simultaneous availability of three independent C chemical shifts enables to establish sequential connectivity with a high level of redundancy, and the complete data analysis can be performed rapidly by the application of unsupervised computational backbone assignment algorithms such as UNIO-MATCH [47], essential to accelerate the assignment of resonances and to render the procedure impartial. 13 C coherence lifetimes are shorter in the case of membrane pro13 teins, resulting in less efficient experiments with multiple C homonuclear coherence transfers. Nevertheless, the whole set of six spectra was acquired in 14 days for two proteins (M2 and

OmpG), where the approach delivered the complete assignment of the transmembrane and amphipathic helices of the former, and of considerable portions of the beta-barrel of the latter [16]. 4. Assignment of side-chain protons Assignment strategies for side chain resonances typically involve matching the side chain frequencies to the assigned back13 bone resonances. For example, in C-detected NMR, the carbon frequencies of side chain resonances (Cx) are often recorded in the direct dimension, with the residue identified in two indirect dimensions such as N and Ca. With this 3D N–Ca–Cx spectrum, it is possible to assign many side chain resonances in 10–30 kD sized proteins [48]. Methods for side chain proton assignment under MAS are currently under development. In an example of ILV methyl protona13 1 tion, assignment was carried out based on the C– H correlation 1 13 spectrum in the solid, the solution H shifts and the C shifts determined in the solid [29]. Additionally, long-range transfer from amide protons was used to identify methyl protons in ubiquitin. However, this strategy is likely to result in incorrect assignments since a given methyl may be farther from its own amide proton than to other amides. While the above side chain assignment methods were sufficient for microcrystalline model proteins, this

(a)

(b)

D76 E77 Y78 A79 C80 δ(15N) δ(15N) 118.4 ppm 120.9 ppm

δ(15N) δ(15N) 124.3 ppm 121.0 ppm

δ(15N) 119.4 ppm

20

25

30

10

8

ppm

10

(H)CONH H

H

H

N

10

ppm

H

CA

N

C

C

H

N

CA

8

(H)CO(CA)NH

CA

N

C CA

ppm

(H)CANH

CA

N

8

H

N

CA

δ (15N) / ppm

35

40

45 (HCA)CB(CA)NH (HCA)CB(CACO)NH

50

(H)CANH (HCO)CA(CO)NH

55

60

10

8

10

ppm

8

ppm

10

8

ppm

10

8

ppm 170

(H)NH

(H)CONH (H)CO(CA)NH

(H)(CO)CA(CO)NH (H)(CA)CB(CACO)NH (H)(CA)CB(CA)NH H N

CA

H N

C N

H

H

H

9.1

N

8.7

CA

9.4

N

8.4

CA

175

CA

N

7.1

CA

H

CA

C

δ (1H) / ppm Fig. 6. (a) Transfer pathway diagrams of a suite of 3D experiments for resonance assignment, as in Ref. [18]. The efficiency of the experiments is illustrated by proton 1D 1 spectra acquired with no indirect evolutions on a microcrystalline sample of perdeuterated [100%- HN] SH3, relative to the first FID of a 2D CP-HSQC (‘‘(H)NH’’). (b) Strips from the above experiments, acquired on microcrystalline b2m. The dashed lines indicate sequential connectivities for residues D76-C80. Readapted with permission from Ref. [16]. Copyright Ó 2014 American Chemical Society.

43

L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49

issue is particularly important for methyl dense proteins, such as membrane proteins. Extension of side chain assignment to larger and less ideal protein samples requires further development. 13 1 Ideally, a resolved C– H correlation must be matched to a resolved and assigned backbone resonance. In the case of RAP labeled samples, this was implemented using a 13 13 1 13 13 3D C– C– H spectrum with C– C RFDR, and matching the CH 13 planes to known C shifts [49]. This approach can be extended to ILV-methyl labeled samples, as shown in Fig. 7 for AP205 capsids. Here, a new (HXCX)CYCXHX experiment was designed for fully 13 C-labeled LV-methyl-protonated sidechains, incorporating an 13 13 1 13 out-and-back CX– CY scalar transfer into a H– C correlation (Fig. 7a–c). The resulting (HXCX)CYCXHX spectrum correlates a 13 1 13 13 CX HX methyl pair with the neighboring CY resonance. If the C spin system is identified in a different experiment (e.g. a 2D 1 DARR, Fig. 7d), sequence-specific methyl H assignment follows straightforwardly. 13 1 Ideally, a resolved C– H correlation could be directly matched 15 1 to the amide N– H correlation by an efficient through-bond or relayed-dipolar transfer. However, the experiment involves a

minimum of 4 transfers for alanine, and 6 for leucine. Additionally, this experiment requires deuterium decoupling for 1 13 2 15 optimal resolution, implying use of a 4-channel H– C– H– N probe [50], which is not yet available in many laboratories. Without such a probe, approaches are either limited to doubleresonance correlations such as those illustrated in Fig. 7, or must be applied without deuterium decoupling.

5. Site-specific relaxation Nuclear spin relaxation parameters are powerful reporters of the dynamical processes experienced by every atom in a protein, and their determination is a key step for the understanding of the link between structural flexibility and function in biological systems. The measurement of relaxation processes is of particular interest in solid samples such as microcrystals, hydrated sediments, and membrane-embedded systems, where functionally relevant internal motions are preserved, while the overall rotational tumbling, which dominates relaxation in solution, is abrogated.

(a) δHC

1

WALTZ-16

H δHC

13

C

2

x y x y

δHC

δHC δCC

δCC

t1

δCC

δHC

CW

δCC

t2

τw

δHC

δHC

δHC

t3

WALTZ-16

H

WALTZ-16

(c)

(b) 25

δ (13C) / ppm

δ (13C) / ppm

10

30

15

20

35 25

1

0

-1

35

δ (1H) / ppm

30

25

20

δ (13C) / ppm

δ (13C) / ppm

(d)

20

40 60

40

20

δ (13C) / ppm Fig. 7. Dipolar- and scalar-based carbon homonuclear transfers can be used for assignment of methyl protons, as demonstrated for the sedimented nucleocapsids of AP205. 13 1 13 13 13 (a) Pulse timing diagram for an ‘‘out-and-back’’ 3D (HXCX)CYCXHX correlation between methyl protons and its two C neighbors. (b) H– C and (c) C– C projections from the 13 13 13 above (HXCX)CYCXHX experiment. (d) C-detected C– C DARR correlation. The blue square corresponds to the region displayed in (c). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

44

L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49

However, contrary to solution NMR, where very sophisticated experiments enable the rapid determination of several dynamical restraints for each residue of a protein, traditional detection methods in ssNMR have difficulty to provide sufficient sensitivity for measuring widespread multiple site-specific relaxation parameters. As a result, most site-specific relaxation studies in the solid state have been limited to small model proteins (GB1, 1 SH3, ubiquitin) [51]. H–detected multidimensional experiments at ultra-fast MAS combined with perdeuteration represent a way to overcome this problem, providing simultaneously the resolution and sensitivity required for the efficient determination of sitespecific relaxation decays in larger proteins. This point was recently illustrated by the study of microcrystalline Cu+, Zn2+-superoxide dismutase (SOD), a homodimeric 15 1 enzyme of 153 residues. Here, a N– H CP-HSQC correlation acquired in about 15 min (on 3.5 mg of sample) on the GHz spectrometer was able to identify 136 (of which 103 baseline-resolved) resonances, and the combination of this dipolar correlation module 15 15 with a N inversion-recovery block allowed sampling of the N R1 decay (8 time points) of 126 amide groups in 2.5 days [52]. As a comparison, more than 2 weeks on a 700 MHz spectrometer were 13 15 necessary by C-detected methods to sample N R1 decays (4 time points) in microcrystalline Crh, a dimeric protein of 85 residues [53]. In that study, despite the smaller protein size and the larger 15 amount of sample necessary, only 30 N rates were determined with sufficient accuracy. In addition, the approaches in the very fast MAS regimes have the advantage of alleviating potential interference of coherent contributions to the relaxation parameters. This opens the way to the 15 13 site-specific measurement of parameters such as N R1q or CO R1, which are susceptible to residual coherent effects in fullyprotonated samples [54,55]. For example, MAS frequencies >40 kHz and a spin-lock nutation frequency >15 kHz are required 15 to accurately measure N R1q in solids [55]. On deuterated samples, the requirement on the spin-lock nutation frequency is further weakened [52], with the possibility of observing R1q relaxation dispersion in the window below 5–10 kHz spin-lock field [56], where this parameter is particularly sensitive to isotropic chemical shift fluctuations associated to microsecond to millisecond motions.

β1

15

N R1 (s-1)

0.8

β2

β3 β4

α1

β5

β6

β7

In the case of microcrystalline SOD, the incorporation of a vari15 able N spin-lock period into the CP-HSQC pulse sequence allowed 15 the measurement of N R1q decays (9 time points) for 125 residues in a total of 12 h of measurement time [52]. Fig. 8a shows the set of 15 15 N relaxation rates obtained. The combined analysis of these N R1 and R1q data with a Gaussian axial fluctuation (GAF) model lead to the complete description of the site-specific motions of the enzyme in the ns-us scale. Site-specific order parameters and timescales determined in this way are depicted on the dimeric structure of SOD in Fig. 8b. Notably, this analysis revealed motions with effective correlation times of tens of ns, a timescale that is generally inaccessible by solution NMR due to overall rotational diffusion, which occurs on a timescale of 25 ns for dimeric SOD. 5.1. Structure Once amide and methyl resonances are assigned, high-quality HN and HC spectra can conveniently be extended to include long-range mixing periods in order to determine distance restraints. At very fast MAS rates, efficient polarization transfers are obtained by applying first-order recoupling such as Radio Frequency-Driven Recoupling (RFDR) [57] and Dipolar Recoupling Enhanced by Amplitude Modulation (DREAM) [58], or alternatively by spin diffusion and spin diffusion in the rotating frame [15]. 1 1 Contrary to traditional solid-state methods, where H– H prox15 13 imities are recorded indirectly as proton-mediated N– C or 13 13 C– C correlations, these contacts can be measured directly in deuterated proteins, where amide and/or methyl sites are either sparsely or completely protonated. The first report by Zilm and coworkers introduced a 3D (H)NHH 1 1 experiment, comprising a H– H dipolar mixing period to correlate 15 nearby proton spins, and N editing to provide good resolution, as demonstrated on microcrystalline ubiquitin at 25 kHz MAS [10]. 1 1 Rienstra and coworkers employed H– H mixing in combination 13 15 with two high-resolution C and N dimensions in a 3D CONHH sequence [37]. The constraints recorded on microcrystalline GB1 at 40 kHz MAS were sufficient to refine the structure of the protein. This approach was then pushed to investigate significantly larger targets by exploiting the resolution enhancement offered by high

α2 β8

(c)

(a)

1

0.6 0.8 0.4 0.2 0.6 0

15

N R1ρ (s-1)

(b) 15

(d)

0 ns

10 5

500 ns

0 0

20

40

60

80

100

Residue number 15

120

140 153 1000 ns

Fig. 8. Site-specific N longitudinal relaxation rates (R1, a) and longitudinal relaxation rates in the rotating frame (R1q, b) for microcrystalline Cu+, Zn-SOD. The secondary structure elements are depicted in top of the plot. Dynamical parameters obtained from 1D-GAF model analysis; in (c) timescales of internal motion and in (d) order parameters are depicted on the dimeric structure of SOD (PDB code: 1SOS). Readapted with permission from Ref. [52]. Copyright Ó 2012 National Academy of Sciences of the USA.

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L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49

(a)

(c)

(b)

G114

R115

6

T116

L117 V5 γ2

20

D52

V26 γ1

7

V17 γ1 S111 I113

8

I149

I149

9

G147 I149

I149

F50/L117

δ (13C) / ppm

δ (1H) / ppm

R115

120

22 130 L43 δ2 L15 δ2

24

10

9

8

7

δ (1H) / ppm

L67 δ1

G114

(e) L43 δ1

H48/G51

10

δ (15N) / ppm

110

20 22

0.6

0.8

0.4

0.6

0.8

129

130

112

113

118 119

112

113 114

δ (15N) / ppm

L15 δ1

0.4

26

δ (1H) / ppm

24

7 HN

δ (1H) / ppm

HN

2.7Å

A64 Hβ

A64

V66 Hβ

2.3Å

A67

8

A67 Hα

A67 V66 9

A67 Hβ

V66 Hβ

Q82 N31

Q82 Hβ N31 Hβ

8

7

1

28 30 32

36 4

3

δ (1H) / ppm 1

26

34

δ1 (15N) 120.3 ppm 9

δ (13C) / ppm

(d)

1

2

1

2.8 2.3

2.8 2.3

0

-0.5

δ (1H) / ppm 1

Fig. 9. H-detected measurement of H– H contacts for structure determination. (a) 2D strips from a 3D (H)NHRFDRH spectrum of perdeuterated [100%- HN] ZnII SOD (3.3 ms 13 1 mixing time). Non trivial contacts (|i  j| > 1) are shown on the structure of CuII, ZnII-SOD [14]. (b) 2D C– H HSQC (left) and the V26 Cc1 2D plane from a 4D Hmet–Hmet 1 1 1 DREAM experiment (right) of perdeuterated V, L H cloud-labeled ubiquitin. Red contours represent positive diagonal peaks and blue contours negative cross-peaks. H– H structural restraints involving V26 are depicted on the structure of ubiquitin [29]. (c) A 2D slice from a 4D HN(HRFDRH)NH spectrum of hydrophobin rodlets with diagonal 1 15 compensation (black contours). The non-compensated spectrum is shown in grey, and the H– N CP-HSQC correlation in blue. The hypothetical structure for the hydrophobin rodlets is depicted above the spectra [60]. (d) 2D plane from a 3D (H)NHRFDRH spectrum of fully protonated SSB (533 ls mixing). Assigned amide-aliphatic proton correlations 1 are indicated by dashed lines. The arrows in the spectrum correspond to the contacts depicted in the molecular model [5]. (e) 2D plane from (H)CHPSDH spectra of V, L Hcloud-labeled ubiquitin at 55–60 kHz MAS (25–75 ms mixing time). Dashed lines indicate long-range contacts involving V17b and V26b and L50d2, as depicted on the crystal structure (PDB: 1UBQ) [32]. Adapted with permission from Refs. [5,14,29,32,60]. Copyright Ó 2011 and 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim; 2014 American Chemical Society.

magnetic fields and fast MAS. At 1 GHz magnetic field, the resolution and sensitivity of a 3D (H)NHH enabled identification of 297 1 1 H– H constraints and structure calculation of human SOD (153 residues) by using automatic tools implemented in UNIO [14]. Example strips from this experiment are shown in Fig. 9a. Within beta-sheet structures such as those found in beta-barrel proteins, 1 1 short H– H distances across beta-strands can be used to determine

long-range structural constraints. Within the b-strands of SOD, for 1 example, although only 20 contacts correspond to H pairs with a separation of more than 6 Å, a large number of long-range constraints were identified and were used to define the 3D fold for the molecule. In combination with dihedral-angle restraints 13 derived from C secondary chemical shifts, this resulted in a bundle with backbone root-mean-square deviation (RMSD) of 1.64 Å.

L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49

These experiments can be easily extended to higher dimension1 ality. Exploiting the long H coherence lifetimes stemming from 1 high H dilution, Reif et al. presented a set of 3D and 4D experiments that allow an unambiguous assignment of cross-peaks and an accurate quantification of peak volumes [59]. An increased number of distance restraints for solid-state NMR structural studies were determined by 4D correlations in which both the starting 1 and ending H spins can be uniquely identified. In addition, using low percentages of back-protonation of the amide sites (25%), spatial correlations could be measured for distances up to 13 Å. In this study, correlations to residual aliphatic protons were also accessed 15 13 by synchronous evolution of the N and C chemical shifts to encode valuable amide-methyl and methyl-methyl distance restraints. The method was demonstrated using microcrystalline SH3, and on average, six restraints per residue were obtained, half of which corresponded to long-range distances. More recently, these sequences were employed to monitor proximities between additional aliphatic sites in RAP-labeled samples [24]. While the high dilution levels used by Reif and coworkers allowed the measurement of long-range distances, the sensitivity of the 3D and 4D experiments is scaled quadratically with the site concentration 1 of Hs, resulting in extremely long acquisition times. 1 An approach to increasing the H concentration at selected sites, such as the ILV-methyl labeling developed for solution NMR, appears very pertinent in this context. Meier and coworkers used L67 R115

118

Cu+/2+,Zn2+-SOD δ (15N) / ppm

(a)

120 122

K128 R79

I17 V94 V14 D76 V119 A123 I113

D83

Cu

Q15

118 120 122

V94

R115

I17 V119 A123

(b)

E,Co2+/Zn2+-SOD

+

R79

V14 D76

I113

Cu

Q15

8.0

A60 L84

3D and 4D correlations to acquire 49 methyl-methyl distance restraints in ILV-labeled, deuterated ubiquitin (Fig. 9b) [29]. The measurement of the 4D experiment was performed with application of non-uniform sampling (NUS) taking only 14% of the data points to extend the sampling in all the indirect time domains while limiting the experimental time to about 3 days. In a-helices, 1 1 amide-amide contacts are primarily observed between a H and H of its neighboring residues up to 3 residues away, while long-range contacts are shielded by side-chains. Methyl–methyl contacts are therefore particularly strong restraints for structure calculations. This is also true for globular proteins, since these contacts can be found among side chains that are buried in the hydrophobic core. Fourier-transformed NUS data contain spectral artifacts, which can be minimized with non-linear processing methods. Artifacts can also be alleviated by the use of mixing schemes with lowintensity diagonal peaks (such as DREAM) [29], or by the design of pulse schemes that directly incorporate diagonal suppression. A recent report incorporated these ideas to record 4D time-shared experiments where it was shown that diagonal suppression allowed identification of unambiguous cross-peaks that are otherwise obscured by strong autocorrelations [60]. For example Fig. 9c shows that the removal of overlap is particularly relevant for hydrophobin functional amyloids, a system displaying large inhomogeneous broadening. Additionally, for deuterated SH3, diagonal suppression was shown to dovetail with non-uniform sampling

7.5

112

δ (15N) / ppm

46

2+

116

Zn

2+

H71

114

A145 H63

Co

T116 A140 D52 N131

112

116

T116 A140

2+ D52

7.0 6.5 6.0

7.0

δ (1H) / ppm

δ (1H) / ppm

(c)

(d) G51 15N R1

1 0.8 0.6 0.4 0.2 0 0

intensity

intensity

114

H71 N131 G72 A145 H63

10 20 30 40 50

0

time (s)

(e)

K9 15N R1

1 0.8 0.6 0.4 0.2 0

10 20 30 40 50

time (s)

(f)

(g)

(h) 15

Fig. 10. (a) CP-HSQCs from Cu2+, Zn-SOD and Cu+, Zn-SOD; (b) CP-HSQCs from Co2+-SOD and Zn-SOD; (c) spatial dependence of the PREs, and examples of N relaxation decays for Cu2+, Zn (blue points) and Cu+, Zn-SOD (red points); (d) spatial dependence of the PCS; (e–h) solid-state NMR structure bundles for SOD, calculated with diamagnetic restraints only (e), with Cu2+ PREs (f), with Co2+ PCS (g), and with both PCS and PREs (h). Adapted with permission from Refs. [52,61,62]. Copyright Ó 2012 National Academy of Sciences of the USA and 2013 American Chemical Society. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

L.B. Andreas et al. / Journal of Magnetic Resonance 253 (2015) 36–49

due to a lower dynamic range, allowing artifact-free ultra-sparse sampling down to only 2%. As discussed above, while for the currently available MAS and magnetic fields optimal resolution is obtained with high levels of 1 1 deuteration, it is already possible to record and identify H– H contacts in proteins labeled with higher proton concentrations. For example, amide to side chain cross-peaks were measured for microcrystalline E. coli’s DNA polymerase e (Fig. 9d) [5]. Additionally, selective labeling of proteins with protonated amino acids embedded in a perdeuterated matrix (‘proton clouds’) provides general access to long-range contacts between nonexchangeable side chain protons at 100% site-labeling. For example, cross-peaks were identified between Leu and Val proton-cloud labeled ubiquitin defining the hydrophobic core of the protein (Fig. 9e) [32]. 5.2. Paramagnetic effects The sensitivity provided by direct acquisition of proton spectra represents an asset for the quantitative measurement of paramagnetic relaxation enhancements (PRE) and pseudo-contact shifts (PCS) induced on the surrounding nuclei by a paramagnetic ion [61]. These effects represent powerful long-range restraints for the study of structure and dynamics of metalloproteins, but the insufficient sensitivity without proton detection methods made them difficult to measure quantitatively. As for the dynamics studies, the measurement of site-specific PREs requires monitoring of relaxation decays by the acquisition of long series of 2D correlation spectra, and measurement of PCS requires high-resolution 3D maps for both a diamagnetic and a paramagnetic form of a protein. Direct acquisition of proton spectra is a way to accelerate acquisition, as demonstrated for the PREs and PCS produced by Cu2+ and Co2+ respectively in deuterated human SOD. In the case of the Cu-loaded enzyme, the availability of sensitive 15 1 and resolved N– H CP-HSQC ‘‘fingerprint’’ spectra (Fig. 10a) 15 13 enabled the site-specific measurement of hundreds of N and C longitudinal relaxation rates in a few days [52]. From the difference between relaxation rate constants for SOD in its Cu2+ state and Cu+ state (Fig. 10c), the paramagnetic relaxation enhancements were readily evaluated, providing more than one hundred distance measurements between 10 and 24 Å of the Cu ion. When the protein is loaded with Co2+, rapid acquisition of 3D maps such as (H)CONH and (H)CANH correlations enabled site1 specific PCS to be measured. Several hundred PCS, including H PCS, were easily obtained from direct comparison of these 3D spectra relative to an isostructural diamagnetic analog (here Zn-SOD), and their assignment was straightforward, given the systematic change in chemical shifts along parallel lines in the spectra characteristic of this effect (Fig. 10b and d) [62]. Notably, due to the absence of Curie relaxation [63], metal ions with fast electronic 1 relaxation times such as Co2+ do not shorten significantly H coher1 ence lifetimes, and contrary to a common expectation H-detected spectra were obtained for spins as close as 5 Å from the metal ion. As illustrated in Fig. 10e–h, the addition of these paramagnetic restraints produces a significant impact on the backbone RMSD of the calculated structure ensembles, which drops from 3.1 Å (diamagnetic distance and chemical shift restraints only) down to 1.6 Å (PREs), 1.7 Å (PCS) or 1.4 Å (PREs and PCS simultaneously). In particular, the backbone geometry determined using all constraints (the bundle shown in Fig. 10h) is extremely well defined in the proximity of the Cu2+- and Co2+-binding sites. 6. Conclusions and future perspectives 1

In summary, the concerted use of very fast MAS and H dilution by perdeuteration has enabled the development of efficient

47

methods for rapid resonance assignment and reliable measurement of structural and dynamical parameters in crystalline and non-crystalline proteins. The boost in sensitivity and resolution offered by these methods represents a major step forward for the routine investigation of proteins of biological and medical interest. There exists a multitude of proteins in the size range of about 10– 100 kD, such as ion channels, GPCRs, protein fibrils, and enzymes, which have been functionally characterized but for which detailed structures have not been determined. These proteins are just within reach of the current solid-state NMR technology for global structure investigation at an atomic level. A further improvement in sensitivity and linewidth is expected with the arrival of commercially available magnetic fields of 1.2 GHz and MAS frequencies above 100 kHz, which are currently under production. Additionally, there is much to develop such as tailored labeling schemes or MAS cryoprobes. These advancements will make it possible to target proteins of higher molecular weight, with the eventual goal of investigating large, non-crystalline, fullyprotonated samples. Acknowledgments 1

Work on H-detected NMR on biomolecular solids in our laboratory has been developed over the years with the help of Michael J. Knight, Andrew J. Pell, Amy L. Webber, Stefan Jehle, Emeline Barbet-Massin, Alessandro Marchetti, Michele Felletti, Andrea Bertarello, Torsten Herrmann, Anne Lesage and Lyndon Emsley, and has relied on several international collaborations involving Roberta Pierattelli, Isabella C. Felli, Claudio Luchinat, Ivano Bertini, Gottfried Otting, Nicholas E. Dixon, Stefano Ricagno, Martino Bolognesi, Vittorio Bellotti, Kaspars Tars, James J. Chou, Robert G. Griffin and Hartmut Oschkinat. We acknowledge support from the Agence Nationale de la Recherche (ANR 08-BLAN-0035-01 and 10-BLAN-713-01), from the CNRS (TGIR-RMN-THC FR3050), from a Marie-Curie ITN (FP7-PEOPLE-2012-ITN contract no. 317127 ‘‘pNMR’’) and a Marie-Curie incoming fellowship (FP7PEOPLE-2013-IIF contract no. 624918 ‘‘MEM-MAS’’) and from Joint Research Activity and Access to Research Infrastructures in the 7th Framework program of the EC (EAST-NMR n. 228461, BioNMR no. 261863). References [1] L. Emsley, I. Bertini, Frontiers in solid-state NMR technology, Acc. Chem. Res. 46 (2012) 1912–1913. [2] G. Bodenhausen, D.J. Ruben, Natural abundance N-15 NMR by enhanced heteronuclear spectroscopy, Chem. Phys. Lett. 69 (1980) 185–189. 15 [3] Y. Ishii, R. Tycko, Sensitivity enhancement in solid state N NMR by indirect detection with high-speed magic angle spinning, J. Magn. Reson. 142 (2000) 199–204. [4] M.M. Maricq, J.S. Waugh, NMR in rotating solids, J. Chem. Phys. 70 (1979) 3300–3316. [5] A. Marchetti, S. Jehle, M. Felletti, M.J. Knight, Y. Wang, Z.Q. Xu, A.Y. Park, G. Otting, A. Lesage, L. Emsley, N.E. Dixon, G. Pintacuda, Backbone assignment of 1 fully protonated solid proteins by H detection and ultrafast magic-anglespinning NMR spectroscopy, Angew. Chem. Int. Ed. Engl. 51 (2012) 10756– 10759. [6] D.H. Zhou, G. Shah, M. Cormos, C. Mullen, D. Sandoz, C.M. Rienstra, Protondetected solid-state NMR Spectroscopy of fully protonated proteins at 40 kHz magic-angle spinning, J. Am. Chem. Soc. 129 (2007) 11791–11801. 1 [7] L. Zheng, K.W. Fishbein, R.G. Griffin, J. Herzfeld, 2-Dimensional solid-state H NMR and proton-exchange, J. Am. Chem. Soc. 115 (1993) 6254–6261. 1 1 [8] B. Reif, C.P. Jaroniec, C.M. Rienstra, M. Hohwy, R.G. Griffin, H– H MAS correlation spectroscopy and distance measurements in a deuterated peptide, J. Magn. Reson. 151 (2001) 320–327. [9] V. Chevelkov, B.J. van Rossum, F. Castellani, K. Rehbein, A. Diehl, M. Hohwy, S. 1 Steuernagel, F. Engelke, H. Oschkinat, B. Reif, H detection in MAS solid-state NMR spectroscopy of biomacromolecules employing pulsed field gradients for residual solvent suppression, J. Am. Chem. Soc. 125 (2003) 7788–7789. [10] E.K. Paulson, C.R. Morcombe, V. Gaponenko, B. Dancheck, R.A. Byrd, K.W. Zilm, Sensitive high resolution inverse detection NMR spectroscopy of proteins in the solid state, J. Am. Chem. Soc. 125 (2003) 15831–15836.

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