A considerable degree of variability exists in the way that 1H, 13C and 15N chemical shifts are reported and referenced for biomolecules. In this article we explore some of the reasons for this situation and propose guidelines for future chemical shift referencing and for conversion from many common 1H, 13C and 15N chemical shift standards, now used in biomolecular NMR, to those proposed here.

We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR. The structure consists of an eight-stranded beta-barrel connected by tight turns on the periplasmic side and larger mobile loops on the extracellular side. The solution structure of the barrel in DPC micelles is similar to that in n-octyltetraoxyethylene (C(8)E(4)) micelles determined by X-ray diffraction. Moreover, data from NMR dynamic experiments reveal a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein.

Subunit c is the H+-translocating component of the F1F0 ATP synthase complex. H+ transport is coupled to conformational changes that ultimately lead to ATP synthesis by the enzyme. The properties of the monomeric subunit in a single-phase solution of chloroform-methanol-water (4:4:1) have been shown to mimic those of the protein in the native complex. Triple resonance NMR experiments were used to determine the complete structure of monomeric subunit c in this solvent mixture. The structure of the protein was defined by >2000 interproton distances, 64 (3)JN alpha, and 43 hydrogen-bonding NMR-derived restraints. The root mean squared deviation for the backbone atoms of the two transmembrane helices was 0.63 A. The protein folds as a hairpin of two antiparallel helical segments, connected by a short structured loop. The conserved Arg41-Gln42-Pro43 form the top of this loop. The essential H+-transporting Asp61 residue is located at a slight break in the middle of the C-terminal helix, just prior to Pro64. The C-terminal helix changes direction by 30 +/- 5 degrees at the conserved Pro64. In its protonated form, the Asp61 lies in a cavity created by the absence of side chains at Gly23 and Gly27 in the N-terminal helix. The shape and charge distribution of the molecular surface of the monomeric protein suggest a packing arrangement for the oligomeric protein in the F0 complex, with the front face of one monomer packing favorably against the back face of a second monomer. The packing suggests that the proton (cation) binding site lies between packed pairs of adjacent subunit c.

S100B(beta beta), a member of the S100 protein family, is a Ca(2+)-binding protein with noncovalent interactions at its dimer interface. Each apo-S100 beta subunit (91 residues) has four alpha-helices and a small antiparallel beta-sheet, consistent with two predicted helix-loop-helix Ca(2+)-binding domains known as EF-hands [Amburgey et al. (1995) J. Biomol. NMR 6, 171-179]. The three-dimensional solution structure of apo-S100B(beta beta) from rat has been determined using 2672 distance (14.7 per residue) and 88 dihedral angle restraints derived from multidimensional nuclear magnetic resonance spectroscopy. Apo-S100B (beta beta) is found to be globular and compact with an extensive hydrophobic core and a highly charged surface, consistent with its high solubility. At the symmetric dimer interface, 172 intermolecular nuclear Overhauser effect correlations (NOEs) define the antiparallel alignment of helix I with I' and of helix IV with IV'. The perpendicular association of these pairs of antiparallel helices forms an X-type four-helical bundle at the dimer interface. Whereas, the four helices within each apo-S100 beta subunit adopt a unicornate-type four-helix bundle, with helix I protruding from the parallel bundle of helices II, III, and IV. Accordingly, the orientation of helix III relative to helices I, II, and IV in each subunit differs significantly from that known for other Ca(2+)-binding proteins. Indeed, the interhelical angle (omega) observed in the C-terminal EF-hand of apo-S100 beta is -142 degrees, whereas omega ranges from 118 degrees to 145 degrees in the apo state and from 84 degrees to 128 degrees in the Ca(2+)-bound state for the EF-hands of calbindin D9k, calcyclin, and calmodulin. Thus, a significant conformational change in the C-terminal EF-hand would be required for it to adopt a structure typical of the Ca(2+)-bound state, which could readily explain the dramatic spectral effects observed upon the addition of Ca2+ to apo-S100B(beta beta).

Wilson disease protein (ATP7B) is a copper-transporting P1B-type ATPase that regulates copper homeostasis and biosynthesis of copper-containing enzymes in human tissues. Inactivation of ATP7B or related ATP7A leads to severe neurodegenerative disorders, whereas their overexpression contributes to cancer cell resistance to chemotherapeutics. Copper-transporting ATPases differ from other P-type ATPases in their topology and the sequence of their nucleotide-binding domain (N-domain). To gain insight into the structural basis of ATP7B function, we have solved the structure of the ATP7B N-domain in the presence of ATP by using heteronuclear multidimensional NMR spectroscopy. The N-domain consists of a six-stranded ␤-sheet with two adjacent ␣-helical hairpins and, unexpectedly, shows higher similarity to the bacterial K ؉ -transporting ATPase KdpB than to the mammalian Ca 2؉ -ATPase or Na ؉ ,K ؉ -ATPase. The common core structure of P-type ATPases is retained in the 3D fold of the N-domain; however, the nucleotide coordination environment of ATP7B within this fold is different. The residues H1069, G1099, G1101, I1102, G1149, and N1150 conserved in the P1B-ATPase subfamily contribute to ATP binding. Analysis of the frequent disease mutation H1069Q demonstrates that this mutation does not significantly affect the structure of the N-domain but prevents tight binding of ATP. The structure of the N-domain accounts for the disruptive effects of >30 known Wilson disease mutations. The unique features of the N-domain provide a structural basis for the development of specific inhibitors and regulators of ATP7B.

The U6 RNA intramolecular stem-loop (ISL) structure is an essential component of the spliceosome and binds a metal ion required for pre-messenger RNA splicing. The metal binding internal loop region of the stem contains a partially protonated C67-(+)A79 base pair (pK(a) = 6.5) and an unpaired U80 nucleotide that is stacked within the helix at pH 7.0. Here, we determine that protonation occurs with an exchange lifetime of approximately 20 micros and report the solution structures of the U6 ISL at pH 5.7. The differences between pH 5.7 and 7.0 structures reveal that the pH change significantly alters the RNA conformation. At lower pH, U80 is flipped out into the major groove. Base flipping involves a purine stacking interaction of flanking nucleotides, inversion of the sugar pucker 5' to the flipped base, and phosphodiester backbone rearrangement. Analysis of residual dipolar couplings as a function of pH indicates that base flipping is not restricted to a local conformational change. Rather, base flipping alters the alignment of the upper and lower helices. The alternative conformations of the U6 ISL reveal striking structural similarities with both the NMR and crystal structures of domain 5 of self-splicing group II introns. These structures suggest that base flipping at an essential metal binding site is a conserved feature of the splicing machinery for both the spliceosome and group II self-splicing introns.

The fruit of Pentadiplandra brazzeana Baillon contains a small, sweet-tasting protein named brazzein. The structure of brazzein in solution was determined by proton nuclear magnetic resonance spectroscopy at pH 5.2 and 22 degrees C. The brazzein fold, which contains one alpha-helix and three strands of antiparallel beta-sheet, does not resemble that of either of the other two sweet-tasting proteins with known structures, monellin and thaumatin. Instead, the structure of brazzein resembles those of plant gamma-thionins and defensins and arthropod toxins. Sequence comparisons predict that members of a newly-identified family of serine proteinase inhibitors share the brazzein fold.

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