As structural genomics and proteomics research has become popular, the importance of cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using cell-free protein synthesis. Among the many procedures for cell-free protein synthesis, the preparation of the cell extract is a crucial step to establish a highly efficient and reproducible workflow. In this article, we describe a detailed protocol for E. coli cell extract preparation for cell-free protein synthesis, which we have developed and routinely use. The cell extract prepared according to this protocol is used for many of our cell-free synthesis applications, including high-throughput protein expression using PCR-amplified templates and large-scale protein production for structure determinations.

A new carbon-carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki-Heck and Sonogashira reactions). Reaction conditions were screened by using a model peptide, and the stability of the Ras protein under the reaction conditions was examined by using the wild-type Ras protein. Finally, the iF-Ras proteins containing a 4-iodo-L-phenylalanine residue were subjected to organopalladium reactions with vinylated or propargylated biotin. Site-specific biotinylations of the Ras protein were confirmed by Western blot and LC-MS/MS.

Palladium-catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki-Heck reaction for site-specific carbon-carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His6-fused Ras protein containing 4-iodo-L-phenylalanine at position 32 (iF32-Ras-His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32-Ras-His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography-mass spectrometry (LC-MS). The regioselectivity of the Mizoroki-Heck reaction was furthermore confirmed by LC-MS/MS analysis. However, in addition to the biotinylated product (bF32-Ras-His), a dehalogenated product (F32-Ras-His) was detected by LC-MS/MS. This dehalogenation resulted from the undesired termination of the Mizoroki-Heck reaction due to steric and electrostatic hindrance around residue 32. The biotinylated Ras showed binding activity for the Ras-binding domain as its downstream target, Raf-1, with no sign of decomposition. This study is the first report of an application of organometallic chemistry in protein chemistry.

GCN2 is the ␣-subunit of the only translation initiation factor (eIF2␣) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an ␣ + ␤ sandwich fold consisting of two layers: a four-stranded antiparallel ␤-sheet, and three side-by-side ␣-helices, with an ␣␤␤␤␤␣␣ topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive ␤-turns that overlap with each other by two residues (triple ␤-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in ␣-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting ␤-strand 4 and ␣-helix 3. The structural architecture, including the triple ␤-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWDcontaining proteins.

The BolA-like proteins are widely conserved from prokaryotes to eukaryotes. The BolA-like proteins seem to be involved in cell proliferation or cell-cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA-like protein. The overall topology is alphabetabetaalphaalphabetaalpha, in which beta(1) and beta(2) are antiparallel, and beta(3) is parallel to beta(2). This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA-like proteins are assembled on the one side of the protein.