Multiplet-filtered and gradient-selected zero-quantum TROSY experiments for 13 C 1 H 3 methyl groups in proteins

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1 J iomol NMR (011) 51:5 51 DOI /s RILE Multiplet-filtered and gradient-selected zero-quantum ROSY eperiments for 3 methyl groups in proteins Michelle L. Gill rthur G. Palmer III Received: 5 May 011 / ccepted: 11 July 011 / Published online: 15 September 011 Ó Springer Science+usiness Media.V. 011 bstract Multiplet-filtered and gradient-selected heteronuclear zero-quantum coherence (gshzq) ROSY eperiments are described for measuring correlations for H 3 methyl groups in proteins. hese eperiments provide improved suppression of undesirable, broad outer components of the heteronuclear zero-quantum multiplet in medium-sized proteins, or in fleible sites of larger proteins, compared to previously described HZQ sequences (ugarinov et al. in J m hem Soc 16:91 95, 00; Ollerenshaw et al. in J iomol NMR 33:5 1, 005). Hahn-echo versions of the gshzq eperiment also are described for measuring zero- and double-quantum transverse relaation rate constants for identification of chemical echange broadening. pplication of the proposed pulse sequences to Escherichia coli ribonuclease HI, with a molecular mass of 18 kd, indicates that improved multiplet suppression is obtained without substantial loss of sensitivity. Keywords Methyl ROSY Zero-quantum HZQ HMQ RNase H Introduction Increased sensitivity of the HMQ pulse sequence, relative to the HSQ eperiment, for the study of Electronic supplementary material he online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. M. L. Gill. G. Palmer III (&) Department of iochemistry and Molecular iophysics, olumbia University, 630 Wes68th Street, New York, NY 1003, US agp6@columbia.edu correlations in fully protonated methyl groups, termed the methyl-rosy effect, has been demonstrated by Kay and coworkers (ugarinov et al. 003; Ollerenshaw et al. 003; Korzhnev et al. 00). dditional line narrowing is obtained by selective recording of heteronuclear zero-quantum coherence (HZQ), rather than miing zero- and doublequantum signals, as in the HMQ eperiment (ugarinov et al. 00; ugarinov and Kay 00; Ollerenshaw et al. 005). urrent HZQ sequences rely upon differential relaation during the evolution period to suppress the outer components, represented by the spin operators H? i - ( aa [\ aa ) j,k and H? i - ( bb [\ bb ) j,k, where i, j, k [ {1,, 3} and i = j = k, relative to the desired central component, H? i - ( ab [\ ab? ba [\ ba ) j,k, of the zero-quantum multiplet. onsequently, suppression is imperfect for methyl groups in medium sized proteins and in fleible regions of large proteins. o address this issue, gradient-selected HZQ pulse sequences have been developed in which the signals from the respective outer multiplet components are removed via a methyl multiplet filter. he sequences are validated using U-[ H, 15 N], Ile d1-[ 3 ], Leu d-[ 3 ], Val c-[ 3 ] ribonuclease HI (RNase H) from E. coli. Methods Protein epression and purification Escherichia coli strain L1(DE3) was transformed with a plasmid containing E. coli ribonuclease HI (RNase H) under control of a 7lac promoter (Hollien and Marqusee 1999; Mandel et al. 1995). he cells were grown in 1 L of M9 minimal media containing 99% H O, 15 N-ammonium chloride, and 98% H 7 -glucose to OD 600 = 0.7. Selective 13

2 6 J iomol NMR (011) 51:5 51 [ 3 ]-labeling of Ile-d1 and stereospecific labeling of Val/Leu-c were achieved by supplementing the growth media with 80 mg/l of -keto-3-methyl- H 3-3- H-- butyric acid (99%, 98% H, ambridge Isotopes) and 50 mg/l of -keto-3- H -- -butyric acid (99%, 98% H, Sigma ldrich) an hour before induction (Goto et al. 1999; Gardner and Kay 1997). Induction of protein epression and purification proceeded as described (Yang et al. 1990; Mandel et al. 1995; Kroenke et al. 1998). NMR spectroscopy NMR samples were 50 lm protein in 100 mm H 3 - sodium acetate, ph 5.5 in 99% H O. Spectra were acquired a8.8 and 1.1 on ruker vance spectrometers equipped with triple resonance z-ais gradient cryoprobes. Sample temperature was calibrated to 83 K using 98% H -methanol as previously described (Findeisen et al. 007). Spectra were recorded with transients per free-induction decay (16 transients per point) and, ,536 (t 9 ) points. Spectral widths were khz or khz (t 9 ) for 18.8 and 1.1, respectively. he and radiofrequency carriers were set to.0 ppm (after presaturation of the water resonance) and 19.5 ppm. Multipart eperiments were acquired with identical points interleaved. Relaation data were acquired a.1 on ruker DRX600 console equipped with a triple resonance z-ais gradient cryoprobe. Spectra were recorded using 8 scans per increment and, points for t 9. he spectral width was khz. Relaation delays () were set to n/(j H ) where n = {1, 10, 5, 0}. Relaation rates were determined for duplicate data sets and subsequently averaged. Data processing D E G 1 ζ ζ +ζ ζ G G 3 G G φ +ζ φ φ G G +ζ G ζ ζ ζ Spectra were processed using NMRPipe (Delaglio et al. 1995). o preserve lineshape features, data used for analysis were processed without further apodization in either dimension. Peak assignments were made using known chemical shifts (Yamazaki et al. 1993; utterwick et al. 00). Peak fitting was performed in NMRPipe. Relaation rates were determined by fitting the parameters of a monoeponential decay to peak heights using in-house Python scripts. Results and discussion hree versions of the gradient-selected HZQ (gshzq) eperiment are shown in Fig. 1a c. he basic pulse sequence shown in Fig. 1a utilizes only magnetization originating on the spins and actively suppresses magnetization from the natural polarization. Inclusion of the methyl multiplet filter in this sequence adds an additional inversion pulse (Fig. 1a, gray) and two delays of length s/ = 1/(8J H ) (&.0 ms, total), relative to the original HZQ eperiment (ugarinov et al. 00) (Fig. 1d). he versions of the eperiment shown in Fig. 1b and c illustrate two methods for constructive utilization of the natural polarization. he sequence depicted in Fig. 1b is similar to the scheme proposed by Ollerenshaw and coworkers (Ollerenshaw et al. 005) (Fig. 1e), but contains one fewer inversion pulse. he alternative approach depicted in Fig. 1c contains one fewer and 13

3 J iomol NMR (011) 51: b Fig. 1 omparison of gradient selected pulse sequences for measuring heteronuclear zero-quantum coherences (gshzq) to previously proposed HZQ schemes (ugarinov et al. 00; Ollerenshaw et al. 005). a simple implementation of the gshzq eperiment. he dashed bo denotes four distinct parts of the pulse sequence: two for inversion of the outer multiplet components and two for echo/antiecho frequency discrimination during. hese alterations are achieved by a pulse sequence element in which the positions of the composite ( y 90 ) and 180 pulses flanking (gray bars) are shifted (see Fig. ). he phases u 1 and u are given in Fig. for the first step of the phase cycle. he 90 pulse labeled u 1 and the receiver are inverted on alternate scans for isotope filtration. ll other pulse phases are {}. Weak presaturation was used to suppress the water resonance. WLZ-16 (Shaka et al. 1983) was used for decoupling during t. G 1 = (1 ms, 7 G/cm), = (500 ls, -.5 G/cm), = (500 ls, 30 G/cm), = (500 ls, 18 G/cm), and s = 1/(J H ) & 3.91 ms. b and c gshzq pulse sequences with signal enhancement using natural polarization. he pulse train for begins on {-} and inverts with the isotope filter. G = (500 ls, 5 G/cm), G 3 = (500 ls, -5 G/cm) and f = 1.5 ms (corresponding to sin(pj H f) = 3-1/ ). For (), = (500 ls,.5 G/cm). Pulse sequences and parameter sets suitable for ruker spectrometers are provided for (a c) in supplementary material. d Previous HZQ pulse sequence by ugarinov and coworkers (ugarinov et al. 00) that utilizes magnetization and differential relaation rates to eliminate signal from outer multiplet components. e n unfiltered HZQ eperiment by Ollerenshaw and coworkers (Ollerenshaw et al. 005) that includes polarization enhancement. he delays, s and f, are the same as in (a) and (b), respectively. Gradient strengths and pulse phases for (d) and (e) are as described previously (ugarinov et al. 00; Ollerenshaw et al. 005) one fewer inversion pulse compared to Fig. 1b; however magnetization is placed in the transverse plane for a time period s-f(&.1 ms) longer and magnetization is inverted for a time period s/-f(&1. ms) compared to Fig. 1b. he relative sensitivity of these two enhancement methods is discussed below. he methyl multiplet filter has no effect (other than relaation losses) when applied to the spin operators representing the center band of the zero-quantum multiplet, H i? - ( ab [\ ab? ba [\ ba ) j,k. In contrast, the outer multiplet components, H i? - ( aa [\ aa ) j,k and H i? - ( bb [\ bb ) j,k, acquire phase factors of ep(-ip/) and ep(ip/), respectively, from evolution under the 1 J H scalar coupling Hamiltonian during the filter. ccordingly, the outer components are dispersive relative to the center component and 180 out of phase with respect to each other. Dividing the filter around the delay allows the composite inversion pulse (Fig. 1a c, gray) to both refocus chemical shift evolution during the filter and control echo/ antiecho frequency discrimination (see below), thereby reducing the number of required inversion pulses by one. Elimination of the undesirable components is achieved by addition of successive data sets in which the phases of the outer components are inverted by alternating the position of the inversion pulse (Fig. 1a c, gray). ollectively, the Fig. he four parts of the gshzq eperiment are depicted as a 9 grid in which rows denote inversion of the outer multiplet components and columns generate echo/ antiecho data. Frequency discrimination is controlled by placing the composite 180 pulse before or after the evolution period, while the phase of the outer multiplet components is controlled by placement of the 180 pulse. Due to variation in the position of the and 180 pulses, the encode gradient must select either zero- or double-quantum coherence (ZQ or DQ), as shown in the coherence level diagrams. For both and, the coherence pathways selected by the encode and decode gradients are shown as solid lines. he relative signs of the encode and decode gradients depict the coherence selection of the pulse sequences in Fig. 1a, c. he dashed boes are consistent with those of Fig. 1 13

4 8 J iomol NMR (011) 51:5 51 ZQ DQ D ZQ DQ G 1 G 1 + filter and echo/antiecho frequency discrimination require the generation of four free-induction decays for each point, which can be understood by considering a 9 matri (Fig. ) in which the columns generate echo/antiecho hypercomple pairs and the rows generate outer multiplet components with alternating phases. he data + Δ 1 Δ Δ 1 Δ G φ Frequency Discrimination Echo ntiecho G + Frequency Discrimination ntiecho Echo b Fig. 3 Hahn-echo gshzq pulse sequences for measurement of multiple-quantum transverse relaation rates. a In the first half of the methyl multiplet filter, ZQ or DQ coherence is selected during the relaation period () using a pulse sequence element shown in part and similar to Fig.. b In the second half of the multiplet filter, the phase of the outer multiplet components is inverted using a pulse sequence element shown in part D. c grid similar to that in Fig. describes the evolution of magnetization through the pulse sequence in (a). In this case, the rows correspond to the multiple quantum coherence (ZQ or DQ) present during the relaation delay and only one phase of the outer multiplet components is considered. he coherence selection (ZQ or DQ) during the relaation delay is controlled by alternating the position of the 180 before and after. Frequency discrimination is obtained by shifting the position of composite ( y 90 ) pulse. d Inversion of the outer multiplet component in the pulse sequence in part is achieved by shifting the two 180 pulses between the relaation delay (/) and the multiplet filter delay (s/). he relaation delay () is equal to n/ (J H ) where n is any integer 1. Gradient strengths, phase cycles, and delays are as described in Fig. 1, ecept that the second 90 pulse is used for the isotope filter. = (500 ls,.5 G/cm) in (a) and (b) corresponding to the rows in Fig. are added to suppress the multiplet components, after which the resulting two data sets are processed according to the Rance-Kay protocol (avanagh et al. 1991; Palmer et al. 1991; Palmer et al. 199; Kay et al. 199) to produce a frequency-discriminated two-dimensional spectrum. Gradient coherence selection is incorporated during the second half of the methyl multiplet filter (Figs. 1, ). he coherence pathway diagrams shown below the pulse sequence elements in Fig. illustrate the gradient selection scheme. When the and inversion pulses occur on the same side of the interval (Fig., top row) the first gradient must encode the zero-quantum (ZQ) frequency. When the two inversion pulses are on opposite sides of (Fig., bottom row), inversion of either the or coherence occurs after and the first gradient must encode the double-quantum frequency (DQ). he decode gradient selects for single-quantum magnetization in all cases. he multiplet filtered sequences are longer by a time period s/ (&.0 ms), compared to the unfiltered versions. he sensitivity of the filtered sequences is reduced by (I unf -I fil )/I unf = 1-[ep(-R ZQ s/)? ep(-r DQ s/)]/ & R MQ s/, in which I unf and I fil are the peak intensities in the unfiltered and filtered spectra, respectively, and R MQ = (R DQ? R ZQ )/. he sensitivity loss is epected to be\7% for proteins with rotational diffusion times\70 ns (ugarinov et al. 00). Differential relaation of the zeroand double-quantum coherence orders (ZQ vs. DQ) also creates a slight imbalance between the two halves of the filter, which could result in incomplete cancellation of the outer components. ecause the length of the filter is short, differential relaation is unlikely to be an issue with molecules for which the filter is necessary.

5 J iomol NMR (011) 51: Hahn-echo relaation period (Rance and yrd 1983; Davis 1989) for measurement of zero- (R ZQ ) and double- (R DQ ) quantum relaation rate constants is incorporated into the basic gshzq eperiment in Fig. 3a, b. he pulse sequence in Fig. 3a utilizes an element similar to Fig. ecept that alternating the position of the inversion pulse selects either ZQ or DQ relaation during the relaation delay () (Fig. 3c). second pulse sequence (Fig. 3b) is necessary to invert the phase of the outer multiplet components. In this sequence, the order of the delays for relaation and multiplet filtration are alternated to select the desired coherence during (Fig. 3d). o ensure the outer multiplet components have the correct relative phase during the two halves of the filter, = n/ (J H ), where n is any integer 1. he polarization enhancement schemes of Fig. 1b, c can be incorporated into the Hahn-echo sequences in a straightforward fashion. he pulse sequences depicted in Fig. 3a, b measure pure ZQ or DQ relaation rate constants. he desired coherence is always ZQ during, which maimizes sensitivity and resolution and ensures the peaks are located at the same frequency in the indirect dimension of each eperiment. s before, the two halves of the multiplet filter are slightly imbalanced due to the differential relaation rates for ZQ and DQ magnetization. When R ZQ is measured, the desired coherence is ZQ during the two s/ delays for the first eperiment (Fig. 3a, c) and DQ for the second eperiment (Fig. 3b, d). he opposite is true for the eperiment that measures R DQ. his imbalance does not affect the measured relaation rate constants. he methyl region of the HZQ spectrum of RNase H is greatly simplified when outer multiplet components are actively suppressed (Fig. a) using the pulse sequence of Fig. 1a, compared to the unfiltered spectrum (Fig. b) acquired using the sequence of Fig. 1d. races through the resonances of selected peaks are shown in Fig. c to illustrate the degree of multiplet suppression. Incorporation of the methyl multiplet filter in a non- polarization enhanced version of the gshzq results in slight sensitivity loss compared to the analogous HZQ by ugarinov et al. (00). Using the measured values of R ZQ and R DQ (vide infra), the new eperiment is epected to be 3% less sensitive than the original unfiltered version, while an empirical value (.0 ± 0.)% (average of data acquired a8.8 and 1.1 ) was determined for the spectra shown in Fig.. he enhanced eperiments in Fig. 1b, c were 9 and 13% more sensitive, respectively, than the unenhanced gshzq (Fig. b) when applied to RNase H. he increased sensitivity of the pulse sequence depicted in Fig. 1c reflects the detrimental effect of the additional inversion pulses in the sequence of Fig. 1b. s protein size increases, relaation losses during the longer polarization transfer period in Fig. 1c will become more significant, and the eperiment of Fig. 1b may become more sensitive. Multiple-quantum relaation decay curves (R DQ and R ZQ ) measured using the Hahn-echo pulse sequences in Fig. 3a, b L6 δ L6 δ1 L136 δ1 L1 δ1 L δ1 L6 δ L6 δ1 L136 δ1 L1 δ1 L δ1 I116 δ1 ZQ (ω H ω, Hz) L107 δ1 L δ L1 δ L136 δ L16 δ V7 γ1 V153 γ1 V153 γ V11 γ1 L59 δ L59 δ L67 δ V5 γ L103 δ V7 γ1 V5 γ1 V5 δ 00 V153 γ1 V5 γ1 V153 γ I53 δ1 I5 δ1 I7 δ1 L107 δ1 L δ L1 δ L136 δ L16 δ V153 γ (ppm) ZQ (Hz) L67 δ V5 γ L103 δ V5 γ1 V5 δ V5 γ1 I53 δ1 I5 δ1 I7 δ1 600 I116 δ1 I116 δ1 800 I66 δ1 V11 γ1 I66 δ D Fig. omparison of gshzq and unfiltered HZQ pulse sequences. a gshzq (Fig. 1a) spectrum of selected Leu d1 and d and Val c1 and c resonances in the methyl region of RNase H. region containing Ile d1 resonances are shown in the inset. Unassigned peaks are marked with an asterisk. b n unfiltered HZQ (Fig. 1d, ugarinov et al. 00) spectrum of the same region. Dashed lines connect the outer multiplet components to the central component. oth (a) and (b) were collected a.1 and contour levels have been normalized to their respective noise floors. c and d omparison of traces along the dimension of HZQ pulse sequences for I116 d1(c) and V153 c1(d). olors are as in (a) and (b) 13

6 50 J iomol NMR (011) 51:5 51 Normalized Intensity 1.0 I8 δ L107 δ L111 δ1 L111 δ quantities are.0 ± 1.0 and 10.9 ± 3.3, respectively. he rate constant g MQ depends upon cross-correlation effects between the methyl group and eternal and H spins and chemical echange broadening of zero- and double-quantum coherences (ugarinov et al. 00). he magnitude of g MQ is consistent with previous studies indicating RNase H is not subject to significant line broadening due to chemical echange at 83 K (Mandel et al. 1996). onclusion Occurrence Occurrence Relaation Delay, (ms) η = (R R )/ (s 1 ) MQ DQ ZQ R = (R + R )/ (s 1 ) MQ DQ ZQ Fig. 5 Measurement of multiple-quantum transverse relaation rates in RNase H. a Zero- and double-quantum relaation profiles, R ZQ (filled squares) and R DQ (open squares), are shown for selected residues. Dashed lines are the best fits of a monoeponential decay to the respective profiles. Peak intensities were normalized and averaged for display. Error bars are smaller than the plotted symbols and represent the error associated with the averaged data sets. he mean ± one standard deviation of R DQ and R ZQ for two data sets are 13.3 ± 0.1 s -1 and 6.9 ± 0.1 s -1 for I8 d1, 19.9 ± 0.1 s -1 and 13.0 ± 0. s -1 for L107 d1, 17.7 ± 0. s -1 and 13.0 ± 0. s -1 for L111 d1, and 18.9 ± 0. s -1 and 1.0 ± 0.1 s -1 for L111 d, respectively. b and c Histograms of g MQ = (R DQ -R ZQ )/ and R MQ = (R DQ? R ZQ )/ for RNase H. he mean ± one standard deviation is g MQ =.0 ± 1.0 s -1 for (b) and R MQ = 10.9 ±. s -1 for (c) are shown for representative methyl groups in Fig. 5a. Histograms of g MQ = (R DQ -R ZQ )/ (Fig. 5b) and R MQ = (R DQ? R ZQ )/ (Fig. 5c) are shown. he average values of the Multiplet-filtered and gradient-selected heteronuclear zeroquantum coherence (gshzq) eperiments have been presented for recording coherences in H 3 spin systems. he gshzq sequences provide improved resolution for medium-sized proteins or larger ones with fleible regions by active suppression of outer components of the HZQ multiplet using a methyl multiplet filter. Sensitivity of the eperiments also is improved by incorporation of polarization enhancement while minimizing the number of inversion pulses. ddition of a Hahn-echo element to the gshzq eperiment enables measurement of relaation rate constants for zero- and double-quantum coherences while maintaining zero-quantum coherence during. he new gshzq eperiments etend the applicability of methyl-rosy eperiments for investigations of structure, interactions, and dynamics of proteins. cknowledgments.g.p. and M.L.G. acknowledge support from National Institute of Health grants GM5091 and GM08907, respectively..g.p. is a member of the New York Structural iology enter (NYS). he NYS is a SR center supported by the New York State Office of Science, echnology and cademic Research. Data acquired a8.8 and 1.1 were collected at the NYS. he 900 MHz (1.1 ) spectrometers were purchased with funds from the National Institute of Health (US), the Keck Foundation (New York State), and the NY Economic Development orporation..g.p. and M.L.G. thank Mark Rance (University of incinnati) and Daròn Freedberg (ER/FD) for helpful scientific discussions. References utterwick J, Patrick Loria J, strof NS, Kroenke D, ole R, Rance M, Palmer G (00) Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes. J Mol iol 339: avanagh J, Palmer G, Wright PE, Rance M (1991) Sensitivity improvement in proton-detected two-dimensional heteronuclear relay spectroscopy. J Magn Reson (1969) 91:9 36 Davis DG (1989) Elimination of baseline distortions and minimization of artifacts from phased D NMR spectra. J Magn Reson (1969) 81: Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, a (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J iomol NMR 6:

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