Adenylate kinases (AKs) play important roles in nucleotide metabolism in all organisms and in cellular energetics by means of phosphotransfer networks in eukaryotes. The crystal structure of a human AK named AK6 was determined by in-house sulfur single-wavelength anomalous dispersion phasing methods and refined to 2.0-Å resolution with a free R factor of 21.8%. Sequence analyses revealed that human AK6 belongs to a distinct subfamily of AKs present in all eukaryotic organisms sequenced so far. Enzymatic assays show that human AK6 has properties similar with other AKs, particularly with AK5. Fluorescence microscopy showed that human AK6 is localized predominantly to the nucleus of HeLa cells. The identification of a nuclear-localized AK sheds light on nucleotide metabolism in the nucleus and the energetic communication between mitochondria and nucleus by means of phosphotransfer networks.

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies.

Crystal structures of Thermoanaerobacter tengcongensis hypoxanthine-guanine phosphoribosyltransferase (HGPRT) apoenzyme and the enzyme–inosine monophosphate (IMP) complex have been determined to 2.5A ° and 2.2A ° resolution, respectively. The active form of the enzyme was identified as a tetramer in solution and the Ki value of IMP was measured to be 45 mM for a-D-phosphoribosyl-1-pyrophosphate (PRPP). Conformation of the flexible loop in T. tengcongensis HGPRT, which is involved in substrate PRPP binding, is different from that observed in phosphoribosyltransferases (PRTs). It contains a 3-10 helix, and a unique double serine repeat. This loop is ordered even in the apoenzyme and assumes a half-closed conformation. The primary magnesium ion is directly coordinated by side-chains of Glu101 and Asp102, and water molecules in the apoenzyme, suggesting a possible prerequisite role for substrate PRPP binding. Most interestingly, an alternative IMP binding mode is found in the structure of T. tengcongensis HGPRT-IMP complex. The 50-phosphate of IMP occupies the PPi position usually seen in PRT-PRPP complexes. This new observation is consistent with the lower Ki value of IMP and may suggest a mechanism involving multiple modes of interactions between IMP and T. tengcongensis HGPRT in product release and feedback inhibition. The structure of T. tengcongensis HGPRT is compared with those of mesophilic HPRTs, and several possible features contributing to its thermostability are elucidated. Overall, T. tengcongensis HGPRT appears to be more diverged from other PRTs.

We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson-Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert (<20 bp) containing the modification or non-Watson-Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields (<60%overall yield) and ease of use. Thismethod has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of nonsequence-specific nucleic acid-protein interactions.

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