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Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.

The function of the ATR (ataxia-telangiectasia mutated and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is central to the cellular response to replication stress and DNA damage. In order to better understand the function of this complex, we have studied its interaction with DNA. We find that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro. Although replication protein A (RPA) is sufficient for the recruitment of ATRIP to ssDNA, we show that a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA. In this latter complex, we show that neither ATR nor ATRIP are able to bind DNA individually, nor do they bind DNA in a cooperative manner. However, the addition of HeLa nuclear extract is able to reconstitute the DNA binding of both ATR and ATRIP, suggesting the requirement for an additional protein activity. We also show that ATR is necessary for ATRIP to bind DNA in this low affinity mode and to form a large DNA binding complex. These observations suggest that there are at least two in vitro ATR-ATRIP DNA binding complexes, one which binds DNA with high affinity in an RPA-dependent manner and a second, which binds DNA with lower affinity in an RPA-independent manner but which requires an as of yet unidentified protein.

The role of helix 0 of the alpha chain (TrpA) of the tryptophan synthetase alpha2beta2 multi-functional enzyme complex of Escherichia coli was examined by deleting amino-terminal residues 2-6, 2-11, or 2-19 of TrpA. Selected substitutions were also introduced at TrpA positions 2-6. The altered genes encoding these polypeptides were overexpressed from a foreign promoter on a multicopy plasmid and following insertion at their normal chromosomal location. Each deletion polypeptide was functional in vivo. However all appeared to be somewhat more labile and insoluble and less active enzymatically than wild type TrpA. The deletion polypeptides were overproduced and solubilized from cell debris by denaturation and refolding. Several were partially purified and assayed in various reactions in the presence of tryptophan synthetase beta2 (TrpB). The purified TrpADelta2-6 and TrpADelta2-11 deletion polypeptides had low activity in both the indole + serine --> tryptophan reaction and the indoleglycerol phosphate + serine --> tryptophan reaction. Poor activity in each reaction was partly due to reduced association of TrpA with TrpB. The addition of the TrpA ligands, alpha-glycerophosphate or indoleglycerol phosphate, during catalysis of the indole + serine --> tryptophan reaction increased association and activity. These findings suggest that removal of helix 0 of TrpA decreases TrpA-TrpB association as well as the activity of the TrpA active site. Alignment of the TrpA sequences from different species indicates that several lack part or all of helix 0. In some of these polypeptides, extra residues at the carboxyl end may substitute for helix 0.

Residue Asp60 of the tryptophan synthetase alpha chain of Escherichia coli is though to interact with the pyrrole NH of substrate indole-3-glycerol phosphate and facilitate its cleavage to indole and glyceraldehyde 3-phosphate. Two distinguishable partial revertants of DN60 tryptophan synthetase alpha mutant trpA34 were analyzed. The slower growing partial revertant, PR1, had the second-site change, YD102. The other partial revertant, PR2, lacked three consecutive base pairs, resulting in replacement of Ala59 and Asn60 of the DN60 mutant alpha polypeptide by Asp. Inspection of the three-dimensional structure of the enzyme-substrate analog complex revealed that Tyr102 is in the vicinity of the pyrrole NH of the substrate. The PR1 alpha chain has a near normal Km for substrate, whereas the PR2 polypeptide has greatly reduced substrate affinity. The PR2 polypeptide is more active than the PR1 polypeptide in the alpha beta reaction in vitro and appears to be more active than the PR1 polypeptide in vivo. Attempts to obtain repeat occurrences of the PR2 deletion mutation were unsuccessful. A third type of trpA34 partial revertant, PR3, that grows very poorly in minimal medium, also has a Tyr102 replacement: YF102. These findings demonstrate that each of the second-site mutations affects a residue located in the vicinity of the active site residue altered by the primary mutation. Slightly leaky mutant trpA89, genetically altered near the site of the trpA34 mutation, was found to have a GS61 substitution.