Proton-driven 13C spin diffusion (PDSD) is a simple and robust two-dimensional NMR experiment. It leads to spectra with a high signal-to-noise ratio in which cross-peaks contain information about internuclear distances. We show that the total information content is sufficient to determine the atomic-resolution structure of a small protein from a single, uniformly 13C-, 15N-labeled microcrystalline sample. For the example of ubiquitin, the structure was determined by a manual procedure followed by an automatic optimization of the manual structure as well as by a fully automated structure determination approach. The relationship between internuclear distances and cross-peak intensities in the spectra is investigated.

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We describe a low-power approach for heteronuclear cross-polarization (CP) at high magic-angle spinning (MAS) frequencies. It is based on second-order CP at the n = 0 Hartmann-Hahn condition. The mechanism for the polarization transfer in the low-power CP experiment relies on second-order cross terms between homonuclear and heteronuclear dipolar couplings. At a MAS frequency of 65 kHz, rf-field amplitudes below 10 kHz are sufficient to efficiently transfer polarization from 1 H to 13 C. The low-rf field requirements of this approach make it well suited for the investigation of proteins and other temperature-sensitive samples without the risk of sample heating and degradation.

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report 13 Multidrug drug resistance, in particular bacterial resistance to clinical antibiotics, is a widely known phenomenon. Basic defense mechanisms of bacteria include permeability barriers, inactivation of antimicrobials, modification of antibiotic targets, and active drug efflux (1). Active efflux is conducted by primary and secondary active transport proteins and the latter are found in almost all transporter families (2). The molecular mechanism of the broad substrate specificity of multidrug efflux pumps is not yet fully understood. To this end structural studies are desirable, but currently only 13 three-dimensional structures of different transport proteins are known and not every transport family is represented. The only multidrug transporter with known three-dimensional structure is AcrB (4, 5). A three-dimensional structure of the ABC transporter Sav1866 has also been reported (6), which is assumed to function as a multidrug efflux pump.Here, we are especially interested in Escherichia coli EmrE, a member of the medically relevant SMR 2 protein family (TC number 2.A.7.1 (8, 9)). Due to its small size (12 kDa), EmrE was originally proposed as ideal structure-function paradigm (10). It has attracted significant interest due to its controversial topological organization (11-15), oligomerization state (16 -19), transport cycle steps (12, 20 -23), and unknown three-dimensional structure (24). EmrE transports a diverse array of aromatic, positively charged substrates in exchange for protons (21) via at least one occluded transport cycle intermediate state (25). Other SMR proteins have overlapping but significantly different substrate specificities with measured affinities in the nanomolar to millimolar range (20, 26). All SMR proteins are of similar size (ϳ11-12 kDa), have a 4-transmembrane helix topology and a highly conserved key residue Glu 14 (20,27). It has been shown that Glu 14 is an essential residue and directly involved in drug and proton binding (28 -30). It can reasonably be assumed, that Glu 14 of both protomers in a dimer form a shared binding pocket (31,32).Whether EmrE forms a symmetric or an asymmetric dimer should be reflected in the chemical shifts of residues such as Glu 14 , which are likely to be found at the dimerization interface. We therefore 13 C-labeled Glu 14 in EmrE by utilizing a cell-free expression system. To allow an unambiguous NMR analysis, the nonessential residue Glu 25 was replaced with alanine (EmrE E25A) to create a single glutamate mutant. The protein was reconstituted into E. coli lipids allowing the most native environment possible. The sample was maintained at pH 8.0. Under these conditions, withou...

We describe the simplification of 13C-13C correlation spectra obtained from a microcrystalline protein sample expressed on a growth medium of 10% fully 13C labeled glucose diluted in 90% natural abundance glucose as compared to a fully labeled sample. Such a labeling scheme facilitates the backbone and side-chain resonance assignment of Phe, Tyr, His, Asp, Asn, Ile, Lys and Pro and yields an unambiguous stereospecific assignment of the valine Cgamma1, Cgamma2 13C resonances and of Leucine Cdelta2.