HNHC a triple resonance experiment for correlatin

Authors: Jörg Rinnenthal Harald Schwalbe

Publish Date: 2009/05/08

Volume: 44, Issue: 2, Pages: 101-105

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Abstract

A novel NMR pulse sequence has been developed that correlates the H2 resonances with the C2 and the N1 N3 resonances in adenine nucleobases of 13C 15N labeled oligonucleotides The pulse scheme of the new 3DHNHC experiment is composed of a 2J15NHSQC and a 1J13CHSQC and utilizes large 2JH2 N1N3 and 1JH2 C2 couplings The experiment was applied to a mediumsize 13C 15Nlabeled 36mer RNA It is useful to resolve assignment ambiguities occurring especially in larger RNA molecules due to resonance overlap in the 1Hdimension Therefore the missing link in correlating the imino H3 resonances of the uracils across the AU base pair to the H8 resonances of the adenines via the novel pulse sequence and the TROSY relayed HCCHCOSY Simon et al in J Biomol NMR 20173–176 2001 is providedThe availability of isotope labeled RNA oligonucleotides Batey et al 1992 Nikonowicz et al 1992 Quant et al 1994 Batey et al 1995 turned NMR spectroscopy into a powerful tool to determine structure and dynamics of RNAs up to a size of 100 nucleotides Varani and Tinoco 1991 Varani et al 1996 Wijmenga 1998 Cromsigt et al 2001 Fürtig et al 2003 Heteronuclear NMR experiments allow resonance assignment of the NMR active 1H 13C 15N and 31P nuclei which is a prerequisite for further structural and dynamical studies on RNA molecules In general most of the experiments use large heteronuclear couplings in order to create a throughbond correlation of distinct nuclei However in some cases these couplings are too small and transverse relaxation especially in the nucleobases is too fast so that NOEbased strategies complement the set of suitable resonance assignment experiments Usually resonance assignment starts with an NOEbased correlation of the imino resonances since imino resonances show high dispersion especially in the 1H dimension The number of peaks is comparably small because only those imino groups give rise to an NMR signal that participate in a hydrogen bridge From the imino signal the assignment is extended to the nonexchangeable aromatic protons and then to the H1′ resonances of the ribose moiety Varani et al 1996 Wijmenga 1998 Fürtig et al 2003The assignment of the adenine and cytosine bases which do not possess imino groups is more difficult It relies on correlations across the base pair starting from the imino resonances of guanine and uracil nucleobases For the adenine nucleobases the assignment of H2 protons in 13C 15Nlabeled RNA is usually obtained from the strong NOE between the uracil H3 and the adenine H2 in an AU base pair and assignment of the adenine N1 is usually obtained via the HNNCOSY experiment in which the uracil H3N3 signal is correlated to the N1 resonance of the adenine Dingley and Grzesiek 1998 Then it is possible to assign the H2N1 resonance pair in the adenine nucleobase in the 2J15NHSQC unambiguously The adenine throughbase connectivity is most suitably obtained via the TROSY relayed HCCHCOSY experiment Simon et al 2001 which allows the assignment of the H2C2 and the H8C8 signals to the same nucleobase via the correlation to the C4 C5 and C6 resonances Base connectivity can also be obtained using a 2D 1H 13CHMBC experiment van Dongen et al 1996 which is recommended for smaller RNA or DNA molecules where spectral overlap in the proton dimension only rarely occursSchematic representation of the scalar couplings involved in the coherence transfer pathway of the 3DHNHC experiment in adenine Fiala et al 2004 The 1JC N couplings that are used for the HCN experiment are shown in gray Fiala et al 2004 The schematic coherence transfer pathways of the 3DHNHC pulse sequence are also indicatedPulse sequence of the 3DHNHC experiment in the soft Watergate water suppression Piotto et al 1992 implementation Narrow and wide filled bars correspond to rectangular 90° and 180° pulses applied with RF field strengths of 255 kHz 1H 195 kHz 13C 695 kHz 15N respectively Gradients G 1–7 are indicated as black filled semiellipses Bipolar gradients BG1 BG2 Sklenar 1995 are indicated as unfilled black rectangles The default pulse phase is x The pulse sequence was optimized on a Bruker spectrometer with the Bruker typical phase settings Roehrl et al 2005 The wide filled bar indicated with cw is an optional presaturation pulse on the water resonance Fixed delays are adjusted as follows Updelta = 125textmsleft 1/left 21 textJ textH2C2 right right tau = 16textmsleft 1/left 42 textJ textH2N13 right right η = 204 μs length of the 180° pulse on the 1H channel + t 10 = 264 μs The proton carrier frequency is centered at the water frequency 47 ppm The carbon carrier frequency is set to 144 ppm middle between C2 and C8 and the nitrogen carrier frequency is set to 221 ppm middle between N1 N3 and N7 At 700 MHz band selective pulses are set as follows 90° square pulse small gray semiellipse water flipback 15 ms 90° square pulse small black semiellipse soft Watergate water suppression 085 ms Asynchronous GARP decoupling Shaka et al 1985 on the carbon and the nitrogen channel is used to suppress heteronuclear scalar couplings during acquisition The pulse field gradients G 1–G 7 have sine bell shaped amplitudes G 1–G 6 are 1 ms and G 7 is 200 μs in length All gradients are applied along the zaxis and have the following strengths G 1 41 G 2 7 G 3 11 G 4 53 G 5 23 G 6 29 G 7 85 100 of gradient strength corresponds to 55 Gauss/cm The bipolar pulse field gradients BG1 and BG2 Sklenar 1995 are applied along the zaxis have rectangular shaped amplitude and are t 1/2 BG1 and t 2/2 BG2 in length The bipolar gradients have the following strengths BG1 2 BG2 3 Phase cycling φ1 = x φ2 = x −x φ3 = 2x 2−x φ4 = x φ5 = 4x 4−x φ6 = x φrec = x −x x −x −x x −x x φ1 and φ2 are incremented in a StatesTPPI Marion et al 1989 manner to achieve quadrature detection in the 15N ω1 dimension and φ4 and φ5 are incremented according to StatesTPPI Marion et al 1989 to achieve quadrature detection in the 13C ω2 dimensionIn the 3D15N 13C 1HHNHC pulse sequence Fig 2 the coherence 2H2 z N13 z at time point a is created via an INEPT step utilizing the strong 2JH2 N1N3 coupling 15 Hz Between a and b the coherence is labeled with the 15N chemical shift In the refocusing INEPT evolution of the 2JH2 N1N3 coupling is concatenated with the evolution of the 1JH2 C2 coupling 200 Hz leading to the coherence 2H2 z C2 z expiωN13 t 1 at time point c C2 chemical shift evolution occurs between c and d Finally the 1JH2 C2 coupling 200 Hz is refocused resulting in H2 x expiωN13 t 1expiωC2 t 2 inphase magnetization in e Neglecting relaxation effects optimal sensitivity is achieved by setting the delay τ to 16 ms 1/42JH2N13 However for larger RNA molecules showing high R2 rates of the H2 protons the delay τ has to be optimized towards shorter delays in order to obtain optimal sensitivity The pulse sequence illustrated in Fig 2 contains a soft Watergate water suppression element Piotto et al 1992 which makes the pulse sequence applicable to H2Osamples Although the pulse sequence can be applied to a D2O sample we optimized gradients and water flip back pulses on an H2O sample so that the important experiments such as 2D NOESY HNNCOSY HNHC and TROSY relayed HCCHCOSY can be performed on the same sample Additionally to this implementation an echo/antiecho sensitivity enhanced Fig S1 Kay et al 1992 and an echo/antiecho TROSY Fig S2 version Nietlispach 2005 of the 3D15N 13C 1HHNHC experiment have been developed The pulse sequences are part of the supplementary material We found that the soft Watergate version has a slightly higher sensitivity than the echo/antiecho sensitivity enhanced version For the 36mer test RNA the TROSY implementation is less sensitive by a factor of 2 but the signals have smaller line widths especially in the 13C dimension All the soft pulses applied on the water frequency small gray semiellipses Fig 2 are optimized to reduce the intensity of the water signal They are applied in a way that the water magnetization is stored along z Gradients are applied to eliminate artifacts and suppress radiation damping of the water signal during the time periods in which the water magnetization is transverse or along −z During the chemical shift evolution periods the water magnetization is along −z for time periods of t 1/2 and t 2/2 respectively Therefore bipolar gradients Sklenar 1995 are used to suppress radiation damping during t 1 and t 2We also investigated the alternative option to obtain the required correlations by exciting double and zeroquantum DQ/ZQ coherence such as 4H2 z N13+/−C2+/− and 4H2 z N13+/−C2−/+ and evolve sums and differences of the chemical shifts of the N13 and C2 nuclei in a concatenated manner Such an experiment would be shorter for one CHINEPT transfer period than the SQ version we propose However the DQ/ZQ experiment requires postacquisition processing and a more elaborate phase cycle to separate DQ and ZQ coherences

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