The first application of protein deuteration was demonstrated in solution-state NMR spectros-copy in the late 1960s, while the approach has been highly extended over the past decades [Crespi et al.,1968,Markley et al.,1968,Crespi and Katz,1969,Kalbitzer et al.,1985,Lemaster and Richards,1988,Lemaster,1994,Yamazaki et al.,1994a,Grzesiek et al.,1995,Shan et al., 1996,Garrett et al.,1997,Gardner and Kay,1998]. Nowadays, deuteration, as well as TROSY techniques, are routinely applied to improve spectral sensitivity and resolution, as1H,1H dipole mediated line broadening is chemically suppressed [Pervushin et al.,1997,Fiaux et al., 2002, Tugarinov et al.,2002,Fernandez and Wider,2003].
In the solid-state, peptide and protein deuteration was introduced about thirty years later by Zilm, Reif and co-workers [Reif et al.,2001,Reif and Griffin,2003,Reif et al.,2003,Morcombe et al.,2005,Chevelkov et al.,2006,Hologne et al.,2006,Linser et al.,2011b,Reif,2012]. As molecular tumbling is absent in the solid-state, anisotropic interactions are not averaged (cf.
Figure 1.1, page 5). Therefore, the strong1H,1H dipolar coupling network induces severe line broadening, particularly to 1H resonances, as 1H possesses the largest gyromagnetic ratio. In
this manner,1H-detection is strongly impeded in the solid-state.
Employing1H,1H homonuclear decoupling sequences, proton line widths of about 100-500 Hz can be achieved [Bielecki et al., 1989, Levitt et al., 1993, Vinogradov et al., 1999, Sakellar-iou et al., 2000, Bosman et al., 2004], which are still two orders of magnitude larger than solution-state line widths. However, it was shown, that in the solid-state, the proton line width of exchangeable protons can amount to 20-40 Hz, in case heavily deuterated samples are em-ployed [Chevelkov et al., 2006, Schanda et al.,2009, Akbey et al.,2010,Knight et al., 2011, Lewandowski et al.,2011a], since the1H,1H dipolar network is diluted. To enable1H-detected
1H,15N correlation experiments in the solid-state, the protein sample is expressed, using uni-formly deuterated glucose, as well as15N-labeled ammonium chloride [Chevelkov et al.,2006].
To further dilute the1H,1H dipolar coupled spin bath, the purified protein is back-exchanged in a buffer containing 10-20% H2O and 90-80% D2O, respectively.
Methyl protons are accessible by making use of specific precursors for amino acid biosyn-thesis [Agarwal et al.,2006,Agarwal and Reif,2008,Huber et al.,2011,Schanda et al.,2011a, Huber et al., 2012], or by exploiting the fact that commercially available precursors are typi-cally not 100% enriched in2H [Agarwal and Reif, 2008]. The deuteration scheme is not only applicable to microcrystalline proteins, but is also successfully implemented in amyloid fibrils, membrane proteins [Linser et al.,2011b] and, as shown in the present thesis, protein-RNA com-plexes (section 3.7, page 137).
Deuteration not only improves the resolution by increasing the effectiveT2 times, it also en-ables1H-detection in the solid-state [Reif and Griffin, 2003,Chevelkov et al., 2006]. Proton detection can, in principle, provide a gain in sensitivity by a factor of 8 and 31, compared to the13C- or15N-detected version of the experiment, respectively, according to(γH/γX)3/2(X =
13C,15N) [Cavanagh et al.,1996]. Therefore, proton is preferred over heteronuclear detection.
However, in practice, to determine a sensitivity enhancement factor also the quality factor and the geometry of the rf coil has to be considered [Ishii et al., 2001], yielding a slightly smaller enhancement [Reif and Griffin,2003].
In biomolecular solid-state NMR spectroscopy, routinely cross polarization (CP) is employed, in order to improve the experimental sensitivity of low-abundant spins (Figure 1.2A), such as
C and N, which are coupled to a proton spin bath [Hartmann and Hahn,1962,Pines et al., 1973,Schaefer and Stejskal,1976,Stejskal et al.,1977].Sspin (13C,15N) detection is common in the solid-state, as directI spin (1H) detection is hampered by large1H,1H dipolar couplings.
However, S spins usually possess low gyromagnetic ratios, compromising the achievable sen-sitivity per unit time. To obtainISCP in rotating solids and increase theSpolarization, the Hartmann-Hahn matching condition has to be fulfilled (zero-quantum condition) [Hartmann and Hahn,1962,Marks and Vega,1996,Rovnyak,2008]
ω1Iω1S=kωr, (1.2)
in which ω1I andω1S are the spin-lock fields applied to the I andS channels, ωr is the MAS rotation frequency andk=±1,±2 [Marks and Vega,1996], respectively. Matching the sideband atk=±1 yields optimal sensitivity for short spin-lock contact times. At very long contact times thek=±2 sidebands are about equally populated [Meier,1992]. To broaden the CP matching profile, an amplitude ramp is employed [Metz et al.,1994]. An intensity maximum is obtained by centering the ramp on a sideband, matching the Hartmann-Hahn condition, and by adjusting the ramp size to cover the full-width of the sideband. The ramp can be implemented by linearly changing the rf field strength on theIorSchannel, respectively.
Figure 1.2:Sensitivity enhancement ofSspins with a typically low gyromagnetic ratio and abundance by employing (A) a cross polarization (CP) (“SL” means spin-lock) or (B) an Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) sequence, respectively. Including the bracketed part, yields a refocussed INEPT sequence. TheIspin is an abundant, high-γnucleus, usually1H.
In solution-state NMR spectroscopy, the most established heteronuclear polarization transfer
technique, Insensitive Nuclei Enhanced by Polarization Transfer (INEPT), is used for improving the sensitivity of low-γnuclei [Becker et al.,1969,Bodenhausen and Freeman,1977,Morris and Freeman,1979]. As opposed to CP, INEPT solely relies on through-bond scalar transfers. The typical INEPT building block is depicted in Figure 1.2B. Immediately before the first bracket, the magnetization has evolved to an anti-phase operator [Sorensen et al.,1983,Cavanagh et al., 1996], 2IzSx, while setting theτ delay to 1/4JIS. Here,JIS is the heteronuclear scalar coupling constant between spinI andS, respectively. Applying a decoupling field to theI spins at this point of the pulse sequence would eliminate theS spin magnetization due to destructive inter-ference, as the positive and negative component of the anti-phase doublet would cancel each other. Extending the sequence by another so-called spin echo element (sequence in brackets), yields the in-phase operator,Sx. The experiment including the spin echo is known as refocussed INEPT [Burum and Ernst,1980,Morris,1980].
In principle, the enhancement factor for both CP and INEPT is given by the ratioγI/γS. How-ever, a further sensitivity gain per unit time is achieved by excitation ofI spins as compared to Sspins. Most commonly, theI spin is1H and theSspin13C or15N, respectively. As the1HT1 relaxation times are about an order of magnitude shorter than13C and15NT1 times,I excited experiments require a significantly shorter recycle delay for recovery of Boltzmann magnetiza-tion.