ABSTRACT
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.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Ubiquitin/chemistry , Carbon Isotopes , Crystallography, X-Ray , Diffusion , Models, Molecular , Protein Conformation , Protein Structure, TertiaryABSTRACT
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)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.
Subject(s)
Antiporters/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/metabolism , Glutamic Acid/chemistry , Biological Transport , Cell-Free System , Dimerization , Freeze Fracturing , Lipids/chemistry , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Molecular Conformation , Mutation , Protein Conformation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The application of a spin-state-selective coherence transfer experiment (INADEQUATE-SSS) to solid-state NMR spectroscopy is described. Two-dimensional (13)C double-quantum/single-quantum spectra without J splittings in both dimensions lead to enhanced spectral resolution. The method is demonstrated to significantly improve the spectral resolution of the crowded C'-C(alpha) region of two proteins.
Subject(s)
Algorithms , Carbon Isotopes/chemistry , Magnetic Resonance Spectroscopy/methods , Models, Chemical , Computer Simulation , Sensitivity and Specificity , Spin LabelsABSTRACT
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.
