Nuclear overhauser spectroscopy of chiral CHD methylene groups

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Journal of Biomolecular NMR ISSN 0925-2738Volume 64Number 1 J Biomol NMR (2016) 64:27-37DOI 10.1007/s10858-015-0002-0

Nuclear overhauser spectroscopy of chiralCHD methylene groups

Rafal Augustyniak, Jan Stanek, HenriColaux, Geoffrey Bodenhausen, WiktorKoźmiński, Torsten Herrmann & FabienFerrage

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ARTICLE

Nuclear overhauser spectroscopy of chiral CHD methylene groups

Rafal Augustyniak1,2,3 • Jan Stanek4 • Henri Colaux1,2,3 • Geoffrey Bodenhausen1,2,3,5 •

Wiktor Kozminski4 • Torsten Herrmann6 • Fabien Ferrage1,2,3

Received: 23 June 2015 / Accepted: 12 November 2015 / Published online: 27 November 2015! Springer Science+Business Media Dordrecht 2015

Abstract Nuclear magnetic resonance spectroscopy(NMR) can provide a great deal of information about

structure and dynamics of biomolecules. The quality of an

NMR structure strongly depends on the number of exper-imental observables and on their accurate conversion into

geometric restraints. When distance restraints are derived

from nuclear Overhauser effect spectroscopy (NOESY),stereo-specific assignments of prochiral atoms can con-

tribute significantly to the accuracy of NMR structures of

proteins and nucleic acids. Here we introduce a series ofNOESY-based pulse sequences that can assist in the

assignment of chiral CHD methylene protons in random

fractionally deuterated proteins. Partial deuteration sup-presses spin-diffusion between the two protons of CH2

groups that normally impedes the distinction of cross-re-laxation networks for these two protons in NOESY spectra.

Three and four-dimensional spectra allow one to distin-

guish cross-relaxation pathways involving either of the twomethylene protons so that one can obtain stereospecific

assignments. In addition, the analysis provides a large

number of stereospecific distance restraints. Non-uniformsampling was used to ensure optimal signal resolution in

4D spectra and reduce ambiguities of the assignments.

Automatic assignment procedures were modified for effi-cient and accurate stereospecific assignments during auto-

mated structure calculations based on 3D spectra. The

protocol was applied to calcium-loaded calbindin D9k. Alarge number of stereospecific assignments lead to a sig-

nificant improvement of the accuracy of the structure.

Keywords NMR spectroscopy ! Protein structures !Nuclear Overhauser spectroscopy ! Automatic structure

calculation

Introduction

Understanding protein function requires precise and accu-

rate information about structure at the atomic level. Alongwith X-ray crystallography, NMR spectroscopy has

become a tool of choice to obtain high-resolution structures

of proteins. The assignment of resonances is required toanalyze NMR data at an atomic level. A suite of NMR

techniques is nowadays routinely used for backbone and

side-chain resonance assignments. However, stereospecificassignments of substituents of prochiral centers are a

demanding task. This problem arises for the two diaster-

eotopic protons of methylene groups in backbones andside-chains, and for the two diastereotopic methyl groups

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10858-015-0002-0) contains supplementarymaterial, which is available to authorized users.

& Fabien [email protected]

1 Departement de chimie, Ecole Normale Superieure – PSLResearch University, 24 rue Lhomond, 75005 Paris, France

2 Sorbonne Universites, UPMC Universite Paris 6, 4 PlaceJussieu, 75005 Paris, France

3 UMR 7203 LBM, CNRS, 75005 Paris, France

4 Faculty of Chemistry, University of Warsaw, Pasteura 1,02-093 Warsaw, Poland

5 Ecole Polytechnique Federale de Lausanne, Institut desSciences et Ingenierie Chimiques, BCH, 1015 Lausanne,Switzerland

6 Institut des Sciences Analytiques, Centre de RMN a TresHauts Champs, Universite de Lyon/UMR 5280 CNRS/ENSLyon/UCB Lyon 1, 5 rue de la Doua, 69100 Villeurbanne,France

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J Biomol NMR (2016) 64:27–37

DOI 10.1007/s10858-015-0002-0

Author's personal copy

of isopropyl residues in valines and leucines. The advan-

tages of stereospecific assignments have been widelydocumented (Clore et al. 1990; Clore et al. 1991b; Driscoll

et al. 1989; Guntert et al. 1989; Kainosho et al. 2006). The

restraints that are most commonly used for structure cal-culations are derived from NOESY experiments (Kumar

et al. 1980; Neuhaus and Williamson 2000). In methylene

groups CHpro-RHpro-S rapid spin-diffusion between the twoprochiral protons reduces their utility in the interpretation

of NOE effects. Random partial deuteration leads to mix-tures of CHpro-RDpro-S and CDpro-RHpro-S groups where

spin-diffusion is suppressed (stereo-specific deuteration of

either Hpro-R or Hpro-S is also possible (Kainosho et al.2006; Takeda et al. 2012) but will not be considered here).

Such CHD groups may be called chiral methylenes, in

analogy to chiral methyl groups (Luthy et al. 1969). Evenwhen deuteration is random, it turns out that stereospecific

assignments are possible. When stereospecific assignments

are not available, distance restraints involving diaster-eotopic substituents can use so-called pseudo-atoms, which

are fictitious atoms that represent all constraints for both

diastereotopic substituents. Despite pseudo-atom correc-tion protocols, this leads to uncertainties in distance

restraints and to structures that lack precision (Guntert

et al. 1989). On the other hand, the use of restraints basedon proper stereospecific assignments can lead to a signifi-

cant improvement in the accuracy of the structures of both

backbones and side-chains (Driscoll et al. 1989). Theadvantages are especially important for Asp, Asn, Glu and

Gln side-chains, since their carboxyl and carbonyl groups

are frequently involved in hydrogen bonding and otherinteractions with ligands or metal ions, but lack NOE

restraints.

Although in silico tools can be employed either to cir-cumvent the lack of stereospecific assignments or to ret-

rospectively extract assignments based on preliminary 3D

structures, (Folmer et al. 1997; Guntert et al. 1998; Ortset al. 2013; Pristovsek and Franzoni 2006) it is obviously

preferable to use experimental assignments of diaster-

eotopic protons or methyl groups. In general, protons canbe assigned stereospecifically if one can determine vicinal

scalar coupling constants and/or intra-residual nuclear

Overhauser effects (NOEs). For CbH2 groups, 3J(Ha,Hb, pro-R) and 3J(Ha, Hb, pro-S) constants (Clore et al.

1991a; Emerson and Montelione 1992; Lohr et al. 1999;

Mueller 1987) can be combined with NOEs between eitherHN or Ha on the one hand and the two b-protons on the

other (Wagner et al. 1987). Knowledge of heteronuclear3J(13C0, Hb) and 3J(15N, Hb) can also contribute to stere-ospecific assignments (Grzesiek et al. 1992). The combi-

nation of 3J(Ha, Hb), 3J(15N, 13Cc) and 3J(13C0, 13Cc)

coupling constants is especially useful for non-native statesand for intrinsically disordered proteins (Hahnke et al.

2010). The protons of CcH2 groups can also be stere-

ospecifically assigned if the assignment of CbH2 protons isknown and at least some of the four 3J(Hb,Hc) couplings

can be measured. Note that 3J(Hb, Hc) couplings are dif-

ficult to measure in large proteins (Cai et al. 1995) whensignals overlap, although this problem may be overcome in

part by non-uniform sampling (NUS) (Kazimierczuk et al.

2008).Nuclear Overhauser effects alone can also be used to

obtain satisfactory stereospecific assignments. In particular,it is possible to apply rotating-frame Overhauser spec-

troscopy (ROESY) to partially deuterated samples, bearing

in mind that signals arising from spin-diffusion and directcross-relaxation have opposite signs (Clore et al. 1990).

However, rapid T1q(1H) relaxation makes it impossible to

use long mixing times that would help to obtain informa-tive long-range restraints. Finally, it appears that ‘exact’

nuclear Overhauser effects (eNOE’s) (Vogeli et al. 2009)

with short mixing times can yield reliable stereospecificassignments (Orts et al. 2013).

The incorporation of stereoselectively deuterated amino

acids into proteins (Gardner and Kay 1998; Lemaster 1990)(possibly via suitable precursors) allows selective substi-

tution of either Hpro-R or Hpro-S protons by a deuteron.

Stereospecific assignment can be achieved by comparingspectra of two samples that are stereoselectively deuterated

in a complementary manner. Such sophisticated biosyn-

thetic methods can provide reliable results, as shown forCbH2 protons in aspartic acid and asparagine (Lemaster

1987) or CaH2 protons in glycines.(Curley et al. 1994)

Prochiral methyl groups in leucine and valine can beassigned by stereoselective 2H and 13C labeling (Atreya

and Chary 2001; Kainosho et al. 2006; Neri et al. 1989;

Ostler et al. 1993; Plevin et al. 2011).Obviously, random fractional deuteration is less chal-

lenging than stereospecific labeling (Nietlispach et al.

1996). Partial deuteration is sufficient to improve NOESYspectra thanks to the reduction of spin diffusion pathways

(Gardner and Kay 1998; Lemaster 1990). This improves

the interpretation of NOEs (Lemaster 1987). Generally, ithas been shown that partial deuteration levels between 50

and 75 % increase the sensitivity of many NMR experi-

ments because of line-narrowing, despite the reduction ofthe concentration of protons (Gardner and Kay 1998). A

deuteration level of 50 % leads to mixtures of CH2,

CHpro-RDpro-S, CDpro-RHpro-S and CD2 each with overallprobabilities of *25 % (site-to-site variations may be

significant, as discussed below). Random partial deutera-

tion also makes it possible to implement isotopic filters tofocus on selected isotopomers (Gardner and Kay 1998;

Kushlan and Lemaster 1993; Muhandiram et al. 1995). The

use of such filters can improve spectral resolution (Vallu-rupalli et al. 2009).

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Here, we introduce two new pulse sequences for nuclear

Overhauser effect spectroscopy, CHD–NOESY–H(C)COand CHD–NOESY–HSQC. Each pulse sequence can be

run either in 3D or 4D fashion, depending on the extent of

overlap. These methods allow one to isolate the signals ofchiral CHD methylene groups in randomly deuterated

proteins. Both sequences rely on the use of ‘CHD filters’.

The CHD–NOESY–H(C)CO experiment allows one toobserve NOEs from neighboring ‘source’ protons to ‘tar-

get’ protons in CHD groups that are adjacent to carboxyl orcarbonyl groups in the side-chains of aspartic acid, aspar-

agine, glutamic acid and glutamine residues (Asp, Asn, Glu

and Gln) as well as in some backbone CaHaCO groups.The CHD–NOESY–CT-HSQC experiment (or its non-

constant-time variant) allows the detection of cross-relax-

ation towards all CHD methylene protons. If high-resolu-tion NMR or X-ray structures are available, our new NOE

methods allow one to obtain unambiguous stereospecific

assignments.For the sake of illustration, the stereospecific assignment

of a set of methylene protons in calbindin D9k has been

performed ‘manually’, based on known NMR (Kordel et al.

1997) and X-ray (Svensson et al. 1992) structures. The

stereospecific assignment and structure determination wereachieved simultaneously using a modified version of the

ATNOS/CANDID algorithms implemented in the UNIO

software package (Herrmann et al. 2002a, b). This auto-mated procedure, which can be applied directly to raw

NMR spectra, can provide reliable stereospecific assign-

ments and enhances the accuracy of the NMR structure.This is illustrated for calbindin by comparing with a stan-

dard UNIO–ATNOS/CANDID procedure that copes withthe lack of stereospecific assignments by ‘atom swapping’

during NOE assignment and simulated annealing (Folmer

et al. 1997).

Results and discussion

NMR experiments

The pulse sequence for the 3D version of CHD–NOESY–

H(C)CO is shown in Fig. 1. The initial 1H frequency labeling

and NOESY mixing time sm are followed by an H(C)CO

Fig. 1 Pulse sequence for the 3D CHD–NOESY–H(C)CO experi-ment. Narrow black and wide empty rectangles correspond to 90" and180" pulses, respectively. Unless otherwise mentioned, all pulses areapplied along the x-axis of the rotating frame. The 1H carrier wasplaced on resonance with the water signal (4.7 ppm), the 15N carrierwas chosen at 120 ppm and the 13C carrier frequency was switchedbetween 40 (13Cb/c) and 180 ppm (13Cc/d) as marked by arrows.Black bell-shaped pulses on the proton channel represent 90" waterflip-back sinc-shaped pulses with duration of 2 (first two pulses) and1.48 ms (last two pulses) for WATERGATE (Piotto et al. 1992).Wide rectangles on the 13C channel represent frequency-swept chirppulses (Bohlen and Bodenhausen 1993) with durations of 500 ls (asingle pulse is used for an inversion across the entire 13C spectrum).Narrow black and wide gray bell-shaped pulses on the 13C channelrepresent Q5 and Q3 pulses (Emsley and Bodenhausen 1990a) withdurations of 480 and 340 ls respectively. Highly selective inversionswere performed with 1.5 ms REBURP pulses (open bell-shapedpulses on the 13C channel) (Geen and Freeman 1991). ‘BS’ indicatespulses for compensation of Bloch–Siegert effects (Emsley and

Bodenhausen 1990b). 1H, 2H and 13C decoupling was performedwith DIPSI-2 (Shaka et al. 1985) (x1

H/2p = 3.1 kHz), WALTZ-16(Shaka et al. 1985) (x1

2H/2p = 1 kHz) and GARP (Shaka et al. 1988)(x1

C = 2.7 kHz) respectively. The lengths and peak amplitudes of thegradients in the x, y and z directions were respectively: g0 = 0.5 ms,(38, 38, 38) G/cm; g1 = 0.5 ms, (0, 0, 8) G/cm; g2 = 2.4 ms, (21,21, 35) G/cm; g3 = 1 ms, (28, 0, 28) G/cm; g4 = 0.5 ms, -49, -49,-49) G/cm. The phase cycling employed was: u1 = 4{x}, 4{- x};u2 = 8{- x}, 8{x}; u3 = 2{x}, 2{- x}; u4 = y, -y; uacq = x, -x,-x, x, -x, x, x, -x, x, -x, -x, x, -x, x, x, -x. Frequency signdiscrimination in the indirect dimension was achieved using Statesmethod (States et al. 1982). The delays were: NOESY mixing timesm = 200 ms, sa = 1.85 ms & |4JCH|

-1 (JCH & 135 Hz), sb =2.03 ms[ |4JCH|

-1, sc = 3 ms\ |4JCC’|-1, d’ = 500 ls. Note that

the adiabatic pulse with inverted sweep on the carbon-13 channel andthe inversion pulse on the nitrogen-15 channel during the delay d’were omitted in this study. They should be used to refocus theevolution under scalar couplings

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sequence (Yamazaki et al. 1993) adapted from our earlier

work (Paquin et al. 2008). Here, CHD groups are selected by

two 90" pulses applied to the 1H channel after a refocused1H–13C INEPT sequence. Phase cycling of the second proton

pulse /2 = ±x results in the alternation of the sign of 13C

coherences that are in anti-phase with respect to the proton innon-deuterated CH2 groups, while the in-phase 13C coher-

ences originating from CHD groups remain unaffected.

Adding the signals of CH2 groups thus leads to their sup-pression, while signals from CHD groups remain. The sen-

sitivity can be enhanced by deuterium decoupling in all

intervals where the coherences are associated with aliphatic13C nuclei (Grzesiek et al. 1993; Kushlan and Lemaster

1993). The H(C)CO sequence offers the advantage that only

a small subset of spin systems is selected, namely –CHDCO–groups in the side-chains of Asp, Asn, Glu and Gln as well as

some backbone CaHaCOgroups, so that resulting 3D spectra

do not suffer from signal overlap.

The information content of the 3D CHD–NOESY–

H(C)CO spectra and its 4D counterpart (see Fig. S2) is thus

limited to a subset of methylene groups. By contrast, the 3Dand 4D CHD–NOESY–CT-HSQC shown on Figs. S1 and 2,

respectively, can yield stereospecific distance constraints for

allmethylene groups in a protein. Frequency labeling by the1H chemical shifts is followed by a NOESYmixing time and

a constant-time HSQC experiment (Vuister and Bax 1992)

which is again modified by inserting a pair of 90" pulses onthe 1H channel to retain the signals of all CHD groups and

eliminate the responses of non-deuterated CH2 groups. The

selection of CHD groups is compatible with the sensitivity-enhanced scheme (Palmer et al. 1991). Composite-pulse

deuterium decoupling is applied in intervals where 13C

coherences evolve, except during pulsed field gradients.Four-dimensional (4D) versions of both H(C)CO- and

HSQC-based experiments (Figs. S2 and 2) allow one to

exploit the chemical shifts of 13C nuclei that have a scalar

Fig. 2 Pulse sequence for the 4D C,C-edited CHD–HMQC–NOESY–HSQC. Solid and open bars represent non-selective 90"and 180" pulses, respectively. All pulses are applied along the x-axisof the rotating frame unless indicated otherwise. 1H, 2H and 13Cdecoupling was performed with WALTZ-16 (Shaka et al. 1983),GARP (Shaka et al. 1983) and WURST-40 (Kupce and Freeman1995) respectively. Selective sinc-shaped 180" pulses, with cB1

adjusted for proper inversion of all C’ spins without affecting theCaliph spins, are represented by open sinc-shaped pulses. ‘BS’ denotesBloch–Siegert compensation pulses (Emsley and Bodenhausen1990b). Suppression of CH2 resonances (including CH2D, and inpart also CH3 signals) is accomplished by insertion of a pair of sadelays during 13C evolution (t2) in the HMQC block as discussed inthe text. The presence of these delays allows for the insertion of anadditional pair of gradients g1 without causing any losses. The delayss1, s2 and s3 are set as for 4D HMQC–NOESY–H(C)CO experiments.The delay e is set to minimize the time required for gradient labellingby g6. Provided that the duration of the gradient g6 satisfiestg6 B 0.5t3,max it can be set as follows: e = tG6 (1 - t3/t3,max). Thedelays are sb = 2sa = 3.57 ms & 0.5/JCH (JCH & 140 Hz),e = 0.65 ms, and the NOESY mixing time sm = 0.2 s. The delayssa and sa0 = 0.63 sa are used for the best compromise in transferefficiency for CH (CHD), CH2 (CH2D) and CH3 groups in the

sensitivity-enhancement sequence (Palmer et al. 1991). Echo andanti-echo signals in the t3 dimension were recorded in interleavedfashion by inverting the amplitude of gradient g9 and incrementing u4

by p accordingly. Quadrature detection in t1 and t2 is accomplished byaltering u1 and u2, respectively, according to the States-TPPIprocedure. The phase u3 and the receiver phase are inverted foreven-numbered points in t3 to achieve axial peak displacement in x3.The phase cycle is: u1 = x, -x; u2 = 2{x}, 2{- x}; u3 = x;u4 = -x; urec = x, -x, -x, x. The 1H, 13C and 15N carrierfrequencies are set to 4.77, 42.8 and 117.8 ppm, respectively. Thedurations and amplitudes of the gradients, which are all applied alongthe z axis, are: g1 = 1 ms, 8.9 G/cm, g2 = 2 ms, 17.7 G/cm,g3 = 2 ms, 14.2 G/cm, g4 = 0.5 ms, 1.8 G/cm, g5 = 0.5 ms,23.2 G/cm, g6 = 2 ms, -31.9 G/cm, g7 = 0.5 ms, 3.6 G/cm,g8 = 1 ms, 5.3 G/cm, g9 = 0.5 ms, ± 32.1 G/cm. A recovery delayof 1.2 s was used between scans. 11,000 sampling points (t1, t2, t3)were randomly chosen from a 84 9 66 9 110 grid according to aGaussian probability distribution p(t) = exp[- (t/tmax)

2/2r2] withr = 0.5. The maximum evolution times in the indirectly detecteddimensions were 12 (t1), 6 (t2) and 10 ms (t3). The spectral widthswere 7.0 (x1), 11 x2), 11 (x3) and 12 kHz (x4). The total duration ofthe 4D experiment was 149 h

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coupling 1J(CH) to the ‘source’ protons to minimize signal

overlap. Two 4D experiments were recorded using non-uniform sampling in all three indirect dimensions (Kaz-

imierczuk et al. 2010) and were modified in the manner of

HMQC (Muller 1979) to minimize signal losses byexploiting favorable relaxation properties of heteronuclear

proton-carbon multiple-quantum (MQ) coherences (Carlo-

magno et al. 2000; Kumar et al. 2000; Marino et al. 1997;Miclet et al. 2004). The additional delays of 4sa & JCH

-1 for

coherence transfer between 1H and 13C are combined withsemi-constant time evolution of 1H chemical shifts (Stanek

et al. 2012). In both 4D experiments, the phase cycle was

reduced to four steps in order to record as many samplingpoints as possible and thus minimize artifacts arising from

non-uniform sampling (NUS). This was achieved in part

through the use of different CHD filters where antiphase13C coherences in CH2 moieties are suppressed by pulse

field gradients, twice in 4D CHD–HMQC–NOESY-

H(C)CO (gradients G5 and G7 Fig S2) and once in 4DCHD–HMQC–NOESY–HSQC (gradient G2 Fig. 2). In the

latter case, the selection is achieved in the HMQC part, by

adding a delay 2sa during which MQ coherences in CH2

groups are allowed to evolve under 1JCH. Thus the 1H

magnetization of the CHD groups, selected before the

mixing time, acts as ‘source’ of cross-relaxation, whereasin all other experiments, the selection is performed on the

CH2 ‘target’ groups.

Applications to calbindin D9k

All pulse sequences were applied to a calcium-loadedsample of the P43G mutant of calbindin D9k with uniform15N and 13C labeling and 33 % random average deuteration(evaluated by mass spectrometry), which is expected for a

sample obtained from expression in Escherichia. coli in a

minimal medium with protonated glucose and 50 % D2O(Leiting et al. 1998). The assignment of the calcium-loaded

form of calbindin D9k P43G is complete (Oktaviani et al.

2011) albeit without systematic stereospecific assignments.The stereospecific assignments of the Hpro-R and Hpro-S

protons in 37 out of 56 CbH2 groups had been determined

from scalar couplings and 1H–1H cross-relaxation networks(Kordel et al. 1993). We have evaluated the distribution of

isotopomers in our sample by comparing signal intensities

of CH2, CHpro-RDpro-S and CDpro-RHpro-S groups in CH2-

and CHD-filtered constant-time HSQC spectra. Results are

Fig. 3 Strip-plots extracted from a 3D CHD-filtered NOESY–H(C)CO spectrum of the P43G mutant of calbindin D9k recordedwith the pulse sequence of Fig. 1 showing signals of the CcHD groupsof (a) Glu35 and (b) Glu65. The chemical shifts in the x2(

13C)dimension of the 3D spectra are a 181.0 and b 187.6 ppm. The 1H-1Hdistances corresponding to NOESY cross-peaks are shown for Glu35and Glu65 using the first model of the structural ensemble (PDB code

1b1 g). The prochiral hydrogen atoms of the CcH2 groups are shownin yellow. Oxygen and nitrogen atoms are shown in red and blue. In(a), grey dashed lines show short distances that have been used toobtain stereospecific assignments for Glu35. In (b), the distances withGlu65 are not compatible, suggesting an improper orientation ofGlu65 side-chain

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shown as supporting information. Overall, the signal of a

proton in the CHD-filtered spectrum is similar or higherthan in the CH2-filtered spectrum. A significant exception

occurs for amino acids produced by biosynthesic pathways

where the b methylene group stems from the C6 methylene

group of glucose. Thus, CbHb2 sites of serines and aromatic

residues show lower populations of CHpro-RDpro-S andCDpro-RHpro-S isotopomers compared to CH2.

Figure 3 illustrates the dramatically different cross-re-

laxation effects observed for two pairs of diastereotopicprotons in 3D H(C)CO-based experiment. When the three-

dimensional structure of a protein is known, a large number

of distance restraints can be derived to make stereospecificassignments of prochiral proton signals reliable (Fig. 3a).

In a few instances, such as the CcH2 group of Glu65

(Fig. 3b), the cross-relaxation networks are not compatiblewith the previously reported structure, indicating incon-

sistencies in side-chain orientations. In order to compare

the cross-relaxation networks derived from CHD-filteredexperiments with those obtained in non-filtered experi-

ments, we recorded two interleaved 3D NOESY–H(C)CO

experiments with opposite values for the phase /2 (seeFig. 1). Proton decoupling was avoided to preserve the

signals of CH2 groups. The sum of these two experiments

gave a CHD-filtered spectrum while the difference yieldeda CH2-filtered experiment. Figure 4 shows signals of the

Hb,pro-R and Hb,pro-S protons of Asn21 in calbindin D9k. The

patterns observed in the CHD-filtered experiment (Fig. 4a)are identical to those obtained with the 3D CHD–NOESY–

H(C)CO experiment and signal intensities differ signifi-

cantly for both prochiral protons. On the other hand, thesignals in the CH2–NOESY–H(C)CO experiment (Fig. 4b)

are almost identical, which makes stereospecific assign-

ment unreliable.The 4D versions of H(C)CO- and HSQC-based experi-

ments make stereospecific assignments of all methyleneresonances straightforward. Figure 5 shows two examples

taken from a 4D CHD–HMQC–NOESY–HSQC. Since the

CHD filtration occurs before the NOESY mixing time, we

display only x313Cð Þ=x4

1Hð Þ planes (the latter dimension

is detected directly), which can easily be assigned to dis-

tinct diastereotopic protons. These planes exhibit excellentspectral resolution and provide structural information that

is in agreement with the crystal structure (PDB code 4icb).

Like in Figs. 3 and 4, the efficiency of CHD-filters isconfirmed by the differentiation between cross-relaxation

rates involving diastereotopic protons.

In conventional protein structure determination, stere-ospecific assignments are performed in a refinement phase

at the end of the procedure, through the analysis of pre-

liminary structures. We have explored the possibility ofobtaining stereospecific assignments directly during the

process of automated NMR structure determination. Both

3D CHD-filtered spectra are used as input for a modifiedversion of the UNIO–ATNOS/CANDID procedure that

iteratively identifies NOESY cross-peaks, makes NOE

assignments and structure calculations. The 3D structure ofthe nth iteration is used to obtain an increasingly reliable

and complete interpretation of NOESY signals in the

(n ? 1)th iteration. Diastereotopic atoms that have notbeen assigned stereospecifically are systematically swap-

ped between pro-R and pro-S assignments. At the outset of

the structure calculation, all prochiral atoms are subjectedto this atom-swapping procedure. In subsequent iterations,

a preliminary 3D structure is used to calculate a score

based on distance restraints for the two possible assign-ments of each prochiral group. If this score shows good

agreement with the experimental restraints for only one of

the two configurations, then a new stereospecific assign-ment is made and used in the next iteration of the NOE

assignment and structure calculation.

In order to assess the advantages of stereospecificassignments, two separate UNIO–ATNOS/CANDID cal-

culations were performed. Without stereospecific assign-

ments, we obtained 1130 meaningful distance restraintsthat led to a pairwise backbone RMSD of 0.687 A and an

average backbone RMSD of 1.583 A for residues 1–41 and

45–76 with respect to the crystal structure (PDB code

Fig. 4 Strip-plots extracted from 3D CHD-filtered (a) and CH2-filtered (b) NOESY-H(C)CO spectra of the CbHD respectively CbH2

methylene groups of Asn21. a Intra-methylene spin diffusion issuppressed by deuteration in CbHD groups. b Spin diffusion in CH2

groups results in altered NOESY cross-peaks intensities that areinconsistent with structural data. For both strip plots, the 13C chemicalshift in the x2 dimension is 177.86 ppm. The insert shows theconformation of Asn21 in the structure of PDB code 1b1 g

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4icb). With our automated stereospecific assignment rou-tine, UNIO–ATNOS/CANDID yielded 1078 meaningful

distance restraints, a pairwise backbone RMSD of 0.695 A,

and an average RMSD of 1.293 A with respect to the X-raystructure, i.e., a significant improvement of * 0.3 A. No

less than 71 of the 110 diastereotopic side-chain methylene

groups were automatically assigned, in good agreementwith manual assignments based on 3D CHD–NOESY–

H(C)CO spectra (see Table 1). While the number of

meaningful distance restraints and the precision of theresulting structure bundles were comparable, a significant

increase of the accuracy of atomic coordinates (* 0.3 A)

was observed for the average coordinates of the NMRstructure bundle calculated with automatically determined

stereospecific assignments.

In order to compare the approaches described here,stereospecific assignments of methylene groups adjacent

to side-chain carboxyl and carbonyl groups have been

made with four different methods, as summarized inTable 1. Assignments were obtained by comparing the

analysis of 3D CHD–NOESY–H(C)CO spectra with (1)

the NMR structure of PDB code 1b1 g, (2) the crystalstructure PDB code 4icb, and (3) the new NMR structure

described above. Automated stereospecific assignments

(4) were also compared to manual assignments. Auto-mated and manual assignments from the new NMR

structure were identical, showing that the automated

process worked well. The consensus assignment for themethylene protons was defined as the one obtained in at

least two of the three structures (1, 2, 3). Wrong stere-

ospecific assignments were obtained for only onemethylene group in the crystal structure PDB code 4icb

and in the NMR structure PDB code 1b1 g, as well as for

only two methylene groups in the new NMR structure. Inat least one case, i.e., for Asp58, the error stems from the

improper description of the interaction with a Ca2? ion,

which is always a difficult task in NMR.Differences in populations of isotopomers (for instance

for CbHb2 sites of serines and aromatic residues) have a

minor impact on the resulting global and local NMR

structure, as documented by the RMSD values given above

and the large number of stereo-specific assignmentsobtained for these residues (we obtained automatic stereo-

specific assignments for 3 of 5 serines and for all 5

phenylalanines). UNIO–ATNOS/CANDID uses an r-6

relationship between NOESY cross peak volumes and

upper distance bounds. The lower bounds are set to the van

der Waals radii of the involved atoms. The calibrationconstant is automatically set so that the structure of the

preceding iteration does not violate more than a predeter-

mined percentage of all upper distance bounds. When thecross-peak intensities are scaled down, the upper bound

will be too large, resulting in a certain loss of information.

The cooperative effect of many NOE distance constraints

Fig. 5 Cross-sections extractedfrom 4D C,C-edited CHD–HMQC–NOESY–HSQC spectrawith 13C shifts in x3 and

1Hshifts in x4. The signals of twoCHD groups are shown forLeu23 (a, b) and Leu28 (e, f).The corresponding fragments ofthe X-ray structure of calbindinD9k (PDB code 4icb) are shownin (c) and (d). The difference inintensity of observed intra- andinter-residual cross-peaks(arrows) reflects the localenvironments of thediastereotopic b protons

J Biomol NMR (2016) 64:27–37 33

123


usually compensates at least in part for this loss of infor-

mation. When the cross-peak intensities are scaled up, thecorresponding upper distance bound might lead to a con-

sistent constraint violation and hence will be eliminated

from the structure calculation. In general, the appliedautomated structure-based calibration method is suffi-

ciently robust to handle slightly ‘incorrect’ cross peak

intensities caused by isotopomer effects or common sour-ces of errors such as spin diffusion. Note that differences in

populations of CHpro-RDpro-S and CDpro-RHpro-S iso-topomers could be used to guide stereospecific assign-

ments, as demonstrated for carbon isotopomers in solid-

state NMR of proteins (Castellani et al. 2002).

Conclusions

We have presented a set of novel NOESY experimentsaimed at distinguishing the cross-relaxation patterns of

prochiral methylene CHD protons in partially deuterated

proteins. They can be carried out in a three- or in four-dimensional manner if resolution needs to be boosted. The

robustness of the stereospecific assignments benefits from

the differences between cross-relaxation rates involvingdiastereotopic protons. Stereospecific assignments were

also obtained by conventional means, by inspecting

hypothetical three-dimensional structures. A large numberof consistent stereo-specific assignments can be obtained

Table 1 Stereospecific assignments of proton resonances in methylene groups adjacent to side-chain carboxyl groups in calbindin D9k

Residuenumber

Consensusassignment

d(1H)(ppm)

Manual assignment fromprevious NMR structure(PDB code 1b1 g)

Comparison withcrystal structure(PDB code 4icb)

Comparisonwith newNMR structure

Automatedassignment

Comments

Glu 4 Hc2

Hc3

2.291

2.424

Correct Correct Correct Correct

Glu 5 Hc2

Hc3

2.452

2.300


Glu 11 Hc2

Hc3

2.314

2.695

Correct Correct Wrong Wrong New NMR structureincompatible withNOESY spectra

Glu 17 Hc2

Hc3

1.960

2.219


Asn 21 Hb2

Hb3

2.686

3.023

Wrong Correct Correct Correct New NMR structuresimilar with crystalstructure

Gln 22 Hc2

Hc3

2.008

2.254

Correct Wrong Correct Correct Orientation of side chainis different in crystalstructure

Gln 33 Hc2

Hc3

2.364

2.540


Glu 35 Hc2

Hc3

2.321

2.181


Asp 47 Hb2

Hb3

2.543

2.698


Glu 52 Hc2

Hc3

1.992

2.243


Asp 54 Hb2

Hb3

1.597

2.520


Asn 56 Hb2

Hb3

3.308

2.859


Asp 58 Hb2

Hb3

3.154

2.461

Correct Correct Wrong Wrong Artefacts due to ill-defined description ofinteraction with Ca2?

Gln 67 Hc2

Hc3

2.322

2.136


The ‘consensus assignment’ is defined as the assignment of the majority of structures

34 J Biomol NMR (2016) 64:27–37

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by automated NMR structure determination. The use of

stereospecific assignments leads to a remarkable improve-ment of the atomic coordinates.

Experimental section

A 100 lL sample of the calcium-loaded P43G mutant of

calbindin D9k with uniform 15N and 13C labeling and 50 %random deuteration was used (protein concentration 4 mM,

pH 6). Three-dimensional experiments were performed ona 600 MHz Bruker Avance spectrometer equipped with a

TXI room-temperature probe and triple-axis gradients.

Four-dimensional experiments were run on a Varian700 MHz spectrometer equipped with a room-temperature

triple resonance probe. The two 4D data sets were pro-

cessed using a signal separation algorithm (SSA) (Staneket al. 2012) leading to a reduction of the effective noise to

the thermal noise level from 1.6 to 1.0, and from 7.4 to 1.1

in 4D HMQC–NOESY–H(C)CO and CC-edited NOESY,respectively. Noise levels in the 4D spectra were calculated

by averaging over 38 and 363 points in the x4(1H)

dimension at the coordinates of auto-correlation peaks. Thedurations of the experiments were: 70 h for the 3D CHD–

NOESY–H(C)CO, 111 h for the CHD–NOESY–CT-

HSQC, 148 h for the 4D CHD–HMQC–NOESY–H(C)CO,and 149 h for the 4D CHD–HMQC–NOESY–HSQC. Such

durations are similar or slightly longer than what is typi-

cally used to record conventional NOESY spectra.The UNIO–ATNOS/CANDID algorithm comprises

seven iterations that differ in increasing threshold values

for the acceptance of NOE assignments.(Herrmann et al.2002a, b) In the standard UNIO–ATNOS/CANDID pro-

tocol, 80 conformers are calculated, and the 20 conformers

with the lowest target function values are selected. Theseconformers are then used to guide the NOESY analysis of

the following iteration. For each prochiral group

j (j = 1…M), three different average target functions arecalculated for each bundle of N conformers.

Tswap ¼1

N

XN

i¼1

Tiswap; T

jproR ¼ 1

N

XN

i¼1

Ti;jproR;

T jpros ¼

1

N

XN

i¼1

Ti;jproS

ð1Þ

The first target function is calculated by ‘atom swapping’

of all prochiral groups. The second target function is cal-culated by assuming the pro-R configuration for the

prochiral group j and atom swapping for all other prochiral

groups. The third target function is calculated like thesecond one but assuming the pro-S configuration for the

prochiral group j. A prochiral group j is definitely assigned

to the pro-R configuration if j exhibits at least 2 distance

restraints, provided the following three conditions are

simultaneously fulfilled:

(a) 1.1 TproRj < TproS

j

(b) TproRj <1.1 Tswap

(c) T jproR þ 0:2A

2\T jproS

The first condition assures that the energy difference

between the two configurations is at least 10 %. The sec-ond condition checks if a consistent stereo-assignment can

be achieved for all N conformers. The last criterion guar-

antees safe stereo-assignments in cases where the set ofdistance restraints is in good agreement with the bundle of

conformers for both configurations. Note that in early

iterations, the 3D structure is usually slightly distorted dueto erroneous NOE assignments and distance restraints, so

that the average target function value will be higher in

early runs than in later iterations. Therefore stereo-specificassignments can lead to an increase of the target function

compared to the atom-swapping technique, but this

increase should be below 10 % in early iterations (criterionb). In later iterations, the target function will always be

close to zero, so that criterion c will gain in importance.

Acknowledgments We thank Mikael Akke (Lund University) for asample of partially deuterated calbindin D9k, as well as DominiqueFrueh (Johns Hopkins University) and Lewis Kay (University ofToronto) for fruitful suggestions. This research was supported by thePolish budget funds for science in 2013–2014 (Project IP2012 057872awarded to J.S.) Access to the Research Infrastructure at University ofWarsaw was financed by the European Commission’s FP7 (Contract228461, EAST-NMR). Financial support of the Bio-NMR Project No.261863 is gratefully acknowledged.

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Nuclear overhauser spectroscopy of chiral CHD methylene groups

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