0 and the Mascot program. The fol lowing parameters were used for database searches, monoisotopic mass accuracy up to 0. 2 Da for internally calibrated spectra, up to one missed cleavage site, carba midomethylation of cysteine as fixed chemical Enzastaurin Phase 3 modifica tion, and oxidation of methionine as variable chemical modification. The protein was identified as a leucyl ami nopeptidase. Phylogenetic relationship of LAPTc with other LAPs Twenty nine sequences were selected from the nonre duntant protein database of NCBI after a search for M17 family members from different organisms under the following accession numbers, Sequence alignments were conducted with the ClustalX software package. Phylogenetic analysis and statisti cal neighbor joining bootstrap tests of the phylogenies were performed with the Mega package.

The PCR product was cloned into the pCR2. 1 TOPO vector. The clone was digested with NdeI and XhoI and the 1563 bp full length fragment was cloned into the pET 19b expression vector. Gene cloning was confirmed by DNA sequencing. The N terminal His tagged rLAPTc was produced in E. coli BL21 through 1. 0 mM IPTG induction at 20 C over 5 h. Cells were harvested by centrifugation, resuspended in lysis buffer, sub mitted to sonication on ice and centrifuged at 15,000 �� g for 10 min at 4 C. Then, the supernatant was sub mitted to affinity chromatography on a nickel column and rLAPTc was eluted with 400 mM imidazole and further purified by size exclusion chromatography on a Superose 6 HR 10 30 column as described above.

rLAPTc, the main peak of activity obtained after the last purification step, was used for enzymatic assays and analyzed by 8% PAGE in the presence of 0. 1 or 0. 01% SDS, followed by Coomassie staining of the gel. Molecular organization assay, analytical ultracentrifugation and light scattering Sedimentation velocity experiments were performed using a Beckman XL I analytical ultracentrifuge and an AN 60 TI rotor. Experiments were carried out at 10 C for rLAPTc, obtained after affinity chromatography, at 170, 56 and 10 uM in 25 mM Tris pH 8. 0, 150 mM NaCl, corresponding to absorbancies at 280 nm of 3. 5, 1. 2 and 0. 2, respectively. A volume of 110 ul or 420 ul was loaded into 0. 3 or 1. 2 cm path cells and centrifuged at 42,000 rpm. Scans were recorded every 6 min, over night, at 295 and 285 nm and by interference.

We used the Sednterp software to estimate the partial specific volume of the polypeptide chain, v, the solvent density, r 1. 00667 g ml, and the solvent viscosity, h 1. 335 mPa. s, at 10 C. Sedimentation profiles were analyzed by the size distribution Batimastat analysis of Sedfit. In Sedfit, finite element solutions of the Lamm equation for a large number of discrete, independent species, for which a relationship between mass, sedimentation and diffusion coefficients, s and D, is assumed, are combined with a maximum entropy regularization to represent a continuous size distribution.