Protein NMR chemical shifts are highly sensitive to local structure. A robust protocol is described that exploits this relation for de novo protein structure generation, using as input experimental parameters the 13 C ␣ , 13 C ␤ , 13 C , 15 N, 1 H ␣ and 1 H N NMR chemical shifts. These shifts are generally available at the early stage of the traditional NMR structure determination process, before the collection and analysis of structural restraints. The chemical shift based structure determination protocol uses an empirically optimized procedure to select protein fragments from the Protein Data Bank, in conjunction with the standard ROSETTA Monte Carlo assembly and relaxation methods. Evaluation of 16 proteins, varying in size from 56 to 129 residues, yielded full-atom models that have 0.7-1.8 Å root mean square deviations for the backbone atoms relative to the experimentally determined x-ray or NMR structures. The strategy also has been successfully applied in a blind manner to nine protein targets with molecular masses up to 15.4 kDa, whose conventional NMR structure determination was conducted in parallel by the Northeast Structural Genomics Consortium. This protocol potentially provides a new direction for high-throughput NMR structure determination. molecular fragment replacement ͉ protein structure prediction ͉ ROSETTA ͉ structural genomics

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

De novo proteins provide a unique opportunity for investigating the structure-function relationships of metalloproteins in a minimal, well-defined, and controlled scaffold. Herein, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the due ferri family. Originally created to catalyze O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyze the selective N-hydroxylation of arylamines by remodeling the substrate access cavity and introducing a critical third His ligand to the metal binding cavity. Additional second-and third-shell modifications were required to stabilize the His ligand in the core of the protein. These changes resulted in at least a 106 –fold increase in the relative rates of the two reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of geometric and electronic factors that influence the catalytic tuning of di-iron active sites.

DnaJ from Escheichia coli is a 376-amino acid protein that f in coRjunction with DnaK and GrpE as a chaperone machine. The N-terminal frmt of residues 2-108, DnaJ-(2-108), retains many of the activities of the full-length protein and conta astructural motif, the J domain of residues 2-72, which Is highly conserved in a superfamily of proteins. In this paper, NMR spectroscopy was used to determine the secondary structure and the three-dimensional polypeptide backbone fold of DnaJ-(2-108). By using 13C/l5N doubly labeled DnaJ-(2-108), nearly complete sequence-specific assignments were obtained for 1H, 5N, 13ca, and 13CP, and about 40% of the peripheral aliphatic carbon resonances were also asie Four a-helices in polypeptide segments ofresidues 6-11, 18-31, 41-55, and 61-68 in the J domain were identified by sequential and medium-range nuclear Overhauser effects. For the J domain, the three-dimensional structure was calculated with the program DANA from an input of 536 nuclear Overhauser effect upper-distance constraints and 52 spin coupling constants. The polypeptide backbone fold Is characterized by the formation of an antiparale bundle of two long helices, residues 18-31 and 41-55, which is stabilized by a hydrophobic core of side chains that are highly conserved in homologous J domain sequences. The Gly/Phe-rich region from residues 77 to 108 is flexibly disordered in solution.The Escherichia coli dnaJ, dnaK, and grpE heat shock genes encode a molecular chaperone machine that participates in a variety ofbiological processes. For example, they function in protein folding and transport, survival at high temperatures, negative autoregulation of the heat shock response, modulation of in vivo proteolysis rates, and the replication of bacteriophage A and certain plasmids (for reviews, see refs. 1 and 2). The J domain, which is the N-terminal 71 residues of DnaJ (3, 4), is an evolutionarily highly conserved motif found in a superfamily ofproteins that includes >50 members from prokaryotic and eukaryotic organisms (refs. 3-6 MATERIALS AND METHODS Preparation of 13C/15N Doubly Labeled DnaJ-(2-108).DnaJ-(2-108) was purified to >95% purity from a strain carrying the expression vector pDW19dnaJ(2-108) as described (8). To doubly label DnaJ-(2-108), a 100-ml culture grown in Isogro medium (Isotec, Miamisburg, OH) was used to inoculate 7 liters of M9 minimal medium (9) supplemented with ampicillin (100 Mg/ml), thiamine (2 pg/ml), MgSO4 (1 mM), MgCl2 (3 mM), CaCl2 (0.1 mM), FeCl3 (0.3 .M),[13Cd-D-glucose (0.3%, Isotec), and 5NH4Cl (1 g/liter, Isotec). The culture was induced with isopropyl 3-D-thiogalactopyranoside (1 mM) at an A595 of 0.7 for 4 h before harvest. Protein sequence analysis revealed that Ala-2 represents the N-terminal residue.NMR Spectroscopy. All NMR spectra were recorded on a Bruker AMX 600 spectrometer equipped with four channels, using a single sample of15N/'3C doubly labeled DnaJ-(2-108).The protein concentration was =1 mM in 90% H2O/10%o 2H20 at pH 6.2 and 280C. The spectra that were recorded to...