We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized alpha/beta-barrel protein. The design was elaborated in two steps. First, the idealized backbone was defined with geometric parameters representing our target fold: a central eight parallel-stranded beta-sheet surrounded by eight parallel alpha-helices, connected together with short structural turns on both sides of the barrel. An automated sequence selection algorithm, based on the dead-end elimination theorem, was used to find the optimal amino acid sequence fitting the target structure. A synthetic gene coding for the designed sequence was constructed and the recombinant artificial protein was expressed in bacteria, purified and characterized. Far-UV CD spectra with prominent bands at 222nm and 208nm revealed the presence of alpha-helix secondary structures (50%) in fairly good agreement with the model. A pronounced absorption band in the near-UV CD region, arising from immobilized aromatic side-chains, showed that the artificial protein is folded in solution. Chemical unfolding monitored by tryptophan fluorescence revealed a conformational stability (DeltaG(H2O)) of 35kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T(m) of 65 degrees C. Moreover, the artificial protein did not exhibit any affinity for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS), providing additional evidence that the artificial barrel is not in the molten globule state, contrary to previously designed artificial alpha/beta-barrels. Finally, 1H NMR spectra of the folded and unfolded proteins provided evidence for specific interactions in the folded protein. Taken together, the results indicate that the de novo designed alpha/beta-barrel protein adopts a stable three-dimensional structure in solution. These encouraging results show that de novo design of an idealized protein structure of more than 200 amino acid residues is now possible, from construction of a particular backbone conformation to determination of an amino acid sequence with an automated sequence selection algorithm.

A reinvestigation of Strychnos icaja roots has resulted in the isolation of two tertiary quasi-symmetric bisindole alkaloids named strychnogucines A (1) and B (2). Their structures were identified by means of spectroscopic data interpretation. Compound 2 was highly active in vitro and compound 1 moderately active against four strains of Plasmodium falciparum. Strychnogucine B (2) was more active against a chloroquine-resistant strain than against a chloroquine-sensitive one (best CI 50 , 80 nM against the W2 strain). In addition, this compound showed a selective antiplasmodial activity with 25-180 times greater toxicity toward P. falciparum, relative to cultured human cancer cells (KB) or human fibroblasts (WI38).

Cadmium is often used as a useful NMR 1 probe to study zincor calcium-containing proteins; 2 we present here a method for identification of cadmium-coordinated histidine via 1 H-15 N HMQC, which does not require any Cd excitation pulse. The cross-peaks show an E-COSY 3 type of pattern which allows the measurement of the 3 J H-Cd and 1 J N-Cd coupling constants.In many metalloproteins, notably those containing zinc or calcium, the natural metal is not conveniently detectable by NMR. To overcome this problem, substitution of the metal by cadmium has often been used. In particular, cadmium-113 NMR has often been used to study zinc-containing proteins; 2 the 113 Cd chemical shift is very sensitive to the nature, number, and geometric arrangement of the ligands within the coordination sphere. In many cases, the substitution of Zn 2+ by Cd 2+ has been shown to have only a modest effect on the catalytic activity of metalloenzymes. 4 113 Cd resonances are commonly detected by direct observation ( 113 Cd has spin 1 / 2 and a sensitivity 63% that of 13 C) or by inverse detection of 113 Cd scalar-coupled to 1 H. Cysteine, methionine, and histidine residues have been successfully identified as the coordinating ligands using 1 H-113 Cd HMQC, 113 Cdedited 1 H-1 H COSY or 1 H-113 Cd heteroTOCSY experiments. [5][6][7][8] These inverse experiments require a time-delay for transfer of magnetization between 1 H and 113 Cd spin, and also a knowledge of the 113 Cd chemical shift, in view of the very large chemical shift range of 113 Cd, which can also make direct detection difficult. These prerequisites make the experiments difficult in many cases.It would therefore be desirable to have a method by which the ligands of 113 Cd can be identified which does not rely on the polarization transfer between 1 H and 113 Cd spins. We describe here a method for identifying cadmium-coordinated histidines using a two-dimensional 1 H-15 N HMQC experiment which does not require Cd excitation pulses and which permits the identification of the histidine imidazole proton and nitrogen resonances of 113 Cd-bound imidazoles and the determination of the 3 J H-Cd and 1 J N-Cd coupling constants.We demonstrate the method using the zinc -lactamase from Bacillus cereus strain 569/H/9 (BCII). Zinc -lactamases 9 in pathogenic bacteria, which can be plasmid-encoded, confer resistance toward all the -lactam antibiotics and represent a real threat to antibiotic therapy because no inhibitors are presently available for clinical purposes. 10 The crystal structure 11 of -lactamase BCII shows two zinc cations in the active site, one (site I) coordinated by three histidines and one water molecule, and the other (site II) coordinated by a histidine, a cysteine, an aspartate, and an unknown molecule, likely to be a carbonate ion. These two zinc ions can be replaced by 113 Cd with minimal change in the kinetic parameters of the enzyme. The 113 Cd spectrum of the enzyme showed two signals, at 143 and 266 ppm relative to 0.1 M Cd-(ClO 4 ) 2 ; comparison with previously rep...

The reinvestigation of Strychnos nux-vomica resulted in the isolation of a colored monoquaternary bisindole alkaloid from the roots. The structure of this new orange substance, strychnochrysine (1), was defined by detailed spectroscopic methods.