Helical structure of phospholamban in membrane bilayers.

The regulation of calcium levels across the membrane of the sarcoplasmic reticulum involves the complex interplay of several membrane proteins. Phospholamban is a 52 residue integral membrane protein that is involved in reversibly inhibiting the Ca(2+) pump and regulating the flow of Ca ions across the sarcoplasmic reticulum membrane during muscle contraction and relaxation. The structure of phospholamban is central to its regulatory role. Using homonuclear rotational resonance NMR methods, we show that the internuclear distances between [1-(13)C]Leu7 and [3-(13)C]Ala11 in the cytoplasmic region, between [1-(13)C]Pro21 and [3-(13)C]Ala24 in the juxtamembrane region and between [1-(13)C]Leu42 and [3-(13)C]Cys46 in the transmembrane domain of phospholamban are consistent with alpha-helical secondary structure. Additional heteronuclear rotational-echo double-resonance NMR measurements confirm that the secondary structure is helical in the region of Pro21 and that there are no large conformational changes upon phosphorylation. These results support the model of the phospholamban pentamer as a bundle of five long alpha-helices. The long extended helices provide a mechanism by which the cytoplasmic region of phospholamban interacts with residues in the cytoplasmic domain of the Ca(2+) pump.

Structure of the transmembrane dimer interface of glycophorin A in membrane bilayers.

The hydrophobic transmembrane domain of glycophorin A contains a sequence motif that mediates dimerization in membrane environments. Long-range interhelical distance measurements using magic angle spinning NMR spectroscopy provide high-resolution structural constraints on the packing of the dimer interface in membrane bilayers. We show that direct packing contacts occur between glycine residues at positions 79 and 83 in the transmembrane sequence. Additional interhelical constraints between Ile76 and Gly79 and between Val80 and Gly83 restrict the rotational orientation and crossing angle of the interacting helices. These results refine our previously proposed structure of the glycophorin A dimer [Smith, S. O., and Bormann, B. J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 488-491] which revealed that the methyl groups of Val80 and Val84 are packed against Gly79 and Gly83, respectively.

Magnetic field gradients in solid state magic angle spinning NMR.

Magnetic field gradients have proven useful in NMR for coherence pathway selection, diffusion studies, and imaging. Recently they have been combined with magic angle spinning to permit high-resolution measurements of semi-solids, where magic angle spinning averages any residual dipolar couplings and local variations in the bulk magnetic susceptibility. Here we show the first examples of coherence pathway selection by gradients in dipolar coupled solids. When the gradient evolution competes with dipolar evolution the experiment design must take into account both the strength of the dipolar couplings and the means to refocus it. Examples of both homonuclear and heteronuclear experiments are shown in which gradients have been used to eliminate the need for phase cycling.

Towards quantitative CP-MAS NMR.

Many of the combined cross polarization-magic angle spinning (CP-MAS) experiments that have been developed over the past ten years for structural studies of chemical and biological systems rely on the measurement of quantitative signal intensities. However, one of the drawbacks of the CP technique is that the efficiency of polarization transfer depends on the strength of the heteronuclear dipolar interaction which is generally different for different chemical groups. In this paper, we propose the use of a linear ramp of CP amplitudes in combination with high speed MAS to improve signal quantitation. The linear ramp is centered on one of the sidebands of the Hartmann-Hahn matching profile and the contact time is chosen to maximize the total signal in the CP-MAS spectrum. The approach is illustrated on N-t-Boc-alanine whose six carbons exhibit a range of heteronuclear dipolar couplings and molecular motions.