Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination.

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H]-transverse relaxation-optimized spectroscopy (TROSY) (3-5) of scalar couplings across the Watson-Crick base pairs in isotope-labeled DNA, which affords direct observation of the hydrogen bonds in these structures. Scalar couplings across hydrogen bonds have been previously reported for organicsynthetic compounds (6, 7), RNA fragments (8), and a metalloprotein (9, 10). The variability of such couplings observed so far indicates that they may become sensitive new parameters for detection of hydrogen bond formation and associated subtle conformational changes. Furthermore, in conjunction with quantum-chemical calculations, precise measurements of scalar couplings across hydrogen bonds can be expected to provide novel insights into the nature of hydrogen bonds in chemicals and in biological macromolecules. MATERIALS AND METHODSFully and partially 13 C, 15 N-doubly labeled DNA oligomers were synthesized on a DNA synthesizer (Applied Biosystems model 392-28) by the solid-phase phosphoroamidite method, by using isotope-labeled monomer units that had been synthesized according to a previously described strategy (11). Approximately 1 mol of oligomer was obtained from 5 mol of nucleoside bound to the resin. NMR samples of the DNA duplex at a concentration of Ϸ2 mM were prepared in 90% H 2 O͞10% D 2 O containing 50 mM potassium phosphate and 20 mM KCl at pH 6.0. NMR measurements were performed at 15°C on Bruker DRX500 and DRX750 spectrometers equipped with H bond length, the solid-state NMR value of 0.11 nm for G and T in a hydrated DNA duplex (19) was used. Relaxation of the imino proton due to dipole-dipole (DD) coupling with remote protons in the DNA duplex was represented as follows (2): in the Watson-Crick AAT pair by an adenosine amino proton at a distance of 0.24 nm and the adenosine C2 proton at 0.3 nm; in G'C by a guanosine amino proton at 0.22 nm and a cytosine amino proton at 0.25 nm. For both base pairs, two imino protons in sequentially stacked bases at 0.4 nm also were considered. Following the calculations outlined in refs. 3-5, the use of TROSY at a polarizing magnetic field B o ϭ 17.6 T is expected to yield 65% and 30% reductions of the 15 N and 1 H linewidth, respectively, for AAT base pairs and 55% and 20% reductions for G'C base pairs. If the contributions from dipolar interactions with remote protons are neglected, the calculations predict reductions of 85% and 75% for 15 N and 1

The solution structure of the DNA dodecamer d(CGCGAATTCGCG)2 has been studied in an aqueous liquid crystalline medium containing 5% w/v bicelles. These phospholipid particles impose a small degree of orientation on the DNA duplex molecules with respect to the magnetic field and permit the measurement of dipolar interactions. Experiments were carried out on several samples with different isotopic labeling patterns, including two complementary samples, in which half of the nucleotides were uniformly enriched with 13C and deuterated at the H2‘ ‘and H5‘ positions. From this, 198 13C−1H and 10 15N−1H one-bond dipolar coupling restraints were derived, in addition to 200 approximate 1H−1H dipolar coupling and 162 structurally meaningful NOE restraints. Although loose empirical restraints for the phosphodiester backbone torsion angles were essential for obtaining structures that satisfy all experimental data, they do not contribute to the energetic penalty function of the final minimized structures. Except for additional regular Watson−Crick hydrogen bond restraints and standard van der Waals and electrostatic terms used in the molecular dynamics-based structure calculation, the structure is determined primarily by the dipolar couplings. The final structure is highly regular, without any significant bending or kinks, and with C2‘-endo/C1‘-exo sugar puckers corresponding to regular B-form DNA. Most local parameters, including sugar puckers, glycosyl torsion angles, and propeller twists, are also tightly determined by the NMR data. The precision of the determined structures is limited primarily by the uncertainty in the exact magnitude and rhombicity of the alignment tensor. This causes considerable spread in parameters such as the degree of base-pair opening and the width of the minor groove, which are relatively sensitive to the alignment tensor values used.