ARTICLE Quantitative one- and two-dimensional 13 C spectra of microcrystalline proteins with enhanced intensity Rudra N. Purusottam • Geoffrey Bodenhausen • Piotr Tekely Received: 13 May 2013 / Accepted: 23 June 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract We recorded quantitative, uniformly enhanced one- and two-dimensional 13 C spectra of labelled micro- crystalline proteins. The approach takes advantage of efficient equilibration of magnetization by low-power proton irradiation using Phase Alternated Recoupling Irradiation Schemes and benefits simultaneously from uniform sensitivity enhancement due to efficient spin exchange that can overcome T 1 ( 13 C) constraints and the presence of heteronuclear Overhauser effects. Keywords Solid-state NMR Á PARIS rf-irradiation Á Microcrystalline proteins Á Quantitative 13 C spectra Á Spin diffusion Á Heteronuclear Overhauser enhancement Introduction Recording sensitive and quantitative spectra of low-gamma nuclei such as 13 C constitutes a long-standing challenge for solid-state NMR spectroscopy. Most 13 C spectra are inherently non-quantitative since cross-polarization (CP) from protons to carbons is not uniform, and single-pulse experiments are not quantitative when the delays between successive acquisitions are too short to allow a uniform recovery of the longitudinal 13 C magnetization of all sites. The most pronounced distortions of intensities in CP spectra are observed between proton-carrying carbons on the one hand, and carbonyl, carboxyl, or quaternary car- bons on the other, since the latters’ magnetization suffers from a slow build-up during cross polarization. Additional deviations from quantitative peak intensities can also arise in CP spectra because local variations in internal mobility lead to variations in CP efficiency and/or rotating-frame relaxation rates R 1q . We have recently discussed the requirements for recording quasi-quantitative single pulse or CP spectra of labelled amino acids (Herbert-Pucheta et al. 2012a). We have demonstrated that recoupling schemes such as Phase Alternated Recoupling Irradiation Schemes (PARIS) (Weingarth et al. 2009b, c) and PARIS-xy (Weingarth et al. 2010) permit one to reach similar peak amplitudes (±10 %) for chemically different sites on time scales as short as a few hundreds of milliseconds. We have previ- ously used these pulse schemes to record sensitive 2D correlation spectra of microcrystalline proteins (Weingarth et al. 2009c; Herbert-Pucheta et al. 2012b), amyloid fibrils (Weingarth et al. 2011b) and mixtures of crystallographic forms (Herbert-Pucheta et al. 2011), and to restore the symmetry in 2D homonuclear correlation experiments of simple amino acids (Herbert-Pucheta et al. 2012a). In this work we show quantitative one- and two-dimen- sional 13 C spectra of microcrystalline proteins recorded after equilibration of magnetization by low-power PARIS or PARIS-xy irradiation. These spectra benefit simultaneously from a uniform sensitivity enhancement (1) due to efficient spin diffusion that allows one to overcome T 1 ( 13 C) con- straints and (2) the presence of heteronuclear Overhauser effects. Such NOE effects have first been observed in solids R. N. Purusottam Á G. Bodenhausen Á P. Tekely (&) De ´partement de Chimie, Ecole Normale Supe ´rieure, 24 rue Lhomond, 75005 Paris, France e-mail: piotr.tekely@ens.fr R. N. Purusottam Á G. Bodenhausen Á P. Tekely Universite ´ Pierre-et-Marie Curie, Place Jussieu, 75005 Paris, France R. N. Purusottam Á G. Bodenhausen Á P. Tekely Laboratoire des Biomole ´cules, CNRS, UMR 7203, 24 rue Lhomond, 75005 Paris, France 123 J Biomol NMR DOI 10.1007/s10858-013-9759-1