p27 controls cell proliferation by binding and regulating nuclear cyclin-dependent kinases (CDKs). In addition, p27 interacts with other nuclear and cytoplasmic targets and has diverse biological functions. We seek to understand how the structural and dynamic properties of p27 mediate its several functions. We show that, despite showing disorder before binding its targets, p27 has nascent secondary structure that may have a function in molecular recognition. Binding to Cdk2-cyclin A is accompanied by p27 folding, and kinetic data suggest a sequential mechanism that is initiated by binding to cyclin A. p27 regulates CDK-cyclin complexes involved directly in cell cycle control and does not interact with other closely related CDKs. We show that p27-cyclin interactions are an important determinant of this specificity and propose that the homologous cell cycle regulators p21 and p57 function by a similar sequential, folding-on-binding mechanism.

Until recently most scientific and patent documents dealing with chemistry have described molecular structures either with systematic names or with graphical images of Kekulé structures. The latter method poses inherent problems in the automated processing that is needed when the number of documents ranges in the hundreds of thousands or even millions since such graphical representations cannot be directly interpreted by a computer. To recover this structural information, which is otherwise all but lost, we have built an optical structure recognition application based on modern advances in image processing implemented in open source tools -OSRA. OSRA can read documents in over 90 graphical formats including GIF, JPEG, PNG, TIFF, PDF, and PS, automatically recognizes and extracts the graphical information representing chemical structures in such documents, and generates the SMILES or SD representation of the encountered molecular structure images.

The D1A mutant of recombinant NP2 has been prepared and shown to have the expression-initiation methionine-0 cleaved during expression in E. coli, as is the case for recombinant NP4, where Ala is the first amino acid for the recombinant protein, as well as for the mature native protein. The heme substituent 1 H NMR chemical shifts of NP2-D1A and those of its imidazole, N-methylimidazole and cyanide complexes are rather different from those of NP2-M0D1. This difference is likely due to the much smaller size of the N-terminal amino acid (A) of NP2-D1A, which allows formation of the closed loop form of this protein, as it does for NP4 (Weichsel, A.; Andersen, J. F.; Roberts, S. A.; Montfort, W. R Nature Struct. Biol. 2000, 7, 551-554). The ratio of the two hemin rotational isomers A and B is different for the two proteins, and the rate at which the A:B ratio reaches equilibrium is strikingly different (NP2-M0D1 t 1/2 for heme rotation ∼2 h, NP2-D1A t 1/2 ∼43 h). This difference is consistent with a high stability of the closed loop form of the NP2-D1A protein, and infrequent opening of the loops that could allow heme to at least partially exit the binding pocket in order to rotate about its α,γ-meso axis. Consistent with this, the rates of histamine binding and release to/from NP2-D1A are significantly slower than for NP2-M0D1 at pH 7.5. This work suggests that care must be taken in interpreting data obtained from proteins that carry the expression-initiation M0.The nitrophorins (nitro = NO, phorin = carrier) are a group of NO-carrying heme proteins found in the saliva of at least two species of blood-sucking insects, Rhodnius prolixus, the "kissing bug", which has four such proteins in the adult insect ( 1-5 ) and at least three additional nitrophorins in earlier stages of development (6 , 7 ), and Cimex lectularius, the bedbug, which has only one nitrophorin protein (8 , 9 ). These interesting heme proteins sequester nitric oxide that is produced by a nitric oxide synthase (NOS) present in the cells of the salivary glands that is similar to vertebrate constituitive NOS (10 -12 ). NO is kept stable for long periods of time by binding it as an axial ligand to a ferriheme Fe center (1 , 3 -5 ). Upon injection into the tissues of the victim, NO dissociates, diffuses through the tissues to the nearby capillaries to cause vasodilation and thereby allows more blood to be transported to the site of the wound. At the same time, histamine, whose role is to cause swelling, itching, and initiating the immune response, is released by mast cells and platelets of the victim. In the case of the Rhodnius proteins, this histamine binds to the heme Fe sites of the nitrophorins, hence preventing the insect's detection for a period of time, which allows it to obtain a sufficient blood meal (13). † This work was supported by National Institutes of Health grant HL54826.Address correspondence to: F. Ann Walker, Department of Chemistry, The University of Arizona, Tucson, Arizona 85721-0041, Tel. + +520 621-8645; Fax. ++520 626-93...

We report the preparation, structure, and reactions of a stable metallapyridine complex of tantalum prepared in the course of model studies of hydrodenitrogenation (HDN) reactions.Monomeric Ta(dNC t BudCHC t BudCH)(OAr) 2 (THF) (5‚THF) is isolated upon thermolyzing the η 2 (N,C)-pyridine complex [η 2 (N,C)-2,4,6-NC 5 t Bu 3 H 2 ]Ta(OAr) 2 Me (2) in the presence of THF, while the metallapyridine dimer [Ta(µ-NC t BudCHC t BudCH)(OAr) 2 ] 2 (6) is isolated when this reaction is carried out in benzene. Complete NMR characterization of 5‚THF is described, along with its conversion into 6. The bis(pyridine) adduct Ta(dNC t BudCHCt BudCH)(OAr) 2 (py) 2 (5‚py) is also described. Ta(dNC t BudCHC t BudCH)(OAr) 2 (THF) (5‚THF) is shown to react with t BuNCO and i PrNCN i Pr to afford the σ (η 1 ) and π (η 3 ) insertion products respectively Ta[dNC t BudCHC t BudCHC(dN t Bu)O](OAr) 2 (7) and Ta[dNC t BudCHC t BudCH-(η 3 -C(dN i Pr) 2 )](OAr) 2 (8). The molecular structure of the metallapyridine Ta(dNC t BudCHCt BudCH)(OAr) 2 (THF) (5‚THF) was detemined by X-ray crystallography and shown to adopt a trigonal-bipyramidal configuration with aryl oxide oxygens and the metallacyclic carbon occupying equatorial positions. The TaNC 4 metallacycle is very nearly planar, and discrete single and double bonds are evident around the ring. This π localization clearly favors the imido form Ta(dNC t BudCHC t BudCH)(OAr) 2 (THF) rather than a carbene structure. The relevance of these compounds to hydrodenitrogenation catalysis is described.

A Pseudo-Rotational Online Service and Interactive Tool (PROSIT) designed to perform complete pseudorotational analysis of nucleosides and nucleotides is described. This service is freely available at http://cactus.nci.nih.gov/prosit/. Files containing nucleosides/nucleotides or DNA/RNA segments, isolated or bound to other molecules (e.g., a protein) can be uploaded to be processed by PROSIT. The service outputs the pseudorotational phase angle P, puckering amplitude numax, and other related information for each nucleoside/nucleotide detected. The service was implemented using the chemoinformatics toolkit CACTVS. PROSIT was used for a survey of nucleosides contained in the Cambridge Structural Database and nucleotides in high-resolution crystal structures from the Nucleic Acid Database. Special cases discussed include nucleosides having constrained sugar moieties with extreme puckering amplitudes, and several specific DNA/RNA helices and protein-bound DNA oligonucleotides (Dickerson-Drew dodecamer, RNA/DNA hybrid viral polypurine tract, Z-DNA enantiomers, B-DNA containing (L)-alpha-threofuranosyl nucleotides, TATA-box binding protein/TATA-box complex, and DNA (cytosine C5)-methyltransferase complexed with an oligodeoxyribonucleotide containing transition state analogue 5,6-dihydro-5-azacytosine). When the puckering amplitude decreases to a small value, the sugar becomes increasingly planar, thus reducing the significance of the phase angle P. We introduce the term "central conformation" to describe this part of the pseudorotational hyperspace in contrast to the conventional north and south conformations.