Dissecting the Engrailed homeodomain-DNA interaction by phage-displayed shotgun scanning

Phage-displayed alanine shotgun scanning was used to dissect contributions by engrailed homedomain (En-HD) residues 17 through 46, which indirectly influence recognition of DNA. The relative contributions of such indirect contacts, quantified by shotgun scanning, highlight previously unexplored En-HD residues. Two motifs dominate En-HD function in this region. First, two surface-exposed aromatic residues (F20 and Y25) bracket the hydrophobic core. Second, two sets of turn-forming residues are highlighted, including carboxamide-requiring residues E22/N23 and a leucine/isoleucine splint. The En-HD hydrophobic core exhibits a surprising degree of malleability, as demonstrated by homolog shotgun scanning. Most selectants from in vitro shotgun scanning mirror the consensus human homeodomain sequence. Thus, natural evolution and in vitro selection use similar selection criteria: affinity, specificity, and stability. However, homolog shotgun scanning identified mutations capable of improving the affinity and specificity of En-HD.     

The mass spectra of some 1H-imidazo[1,2-a]pyrrolo[3,2-e]pyridines

We report here the mass spectral fragmentations of some derivatives of a new heterocyclic system 1H-imidazo[1,2-a]pyrrolo[3,2-e]pyridine. These compounds combine structural features of 1H-pyrrolo[2,3-b]pyridines and imidazo[1,2-a]pyridines, but follow fragmentation pathways similar to the former.     

A semi-continuum model for the solvated electron in methanol

The semi-continuum model for solvated electrons has been applied to methanol at 300°K. The configurational stability of the ground state was established and various physical properties of the solvated electron have been calculated and compared with experiment.     

On the formation and stability of O− and O−2 radicals in type a zeolites and demonstration of cation interactions

Electron spin resonance studies show tha O^? is formed as the major paramagnetic oxygen species in γ-irradiated Ca₆-A zeolite followed by oxygen adsorption. This is a new method to generate this highly reactive catalytic intermediate. O^?₂ is formed in addition to O^? if oxygen is adsorbed prior to irradiation. In Na₁₂-A zeolite O^? is also seen but it transforms to O^?₂ in several hours. Thus O^? appears to be more stable in divalent exchanged zeolites. By electron spin echo modulation spectrometry interactions fo O^?₂ with Li⁺ have been detected which suggests that oxygen species locations in zeolites can be delineated.     

Matrix endor linewidths of trapped silver atoms and aromatic nitroxide radicals in polycrystalline and glassy matrices at 77°K: correlation with matrix polarity

Matrix ENDOR linewidths are shown to serve as effective probes of weak interactions between radicals and their molecular environment in solid disordered matrices. The linewidths of aromatic nitroxides and silver atoms are analyzed in terms of an effective radius of the unpaired electron distribution which increases monotonically with increasing matrix polarity.     

Stabilization and electron spin resonance characterization of Ru(bpy)3 and Ru(bpy)3 by radiolysis of Ru(bpy)3 adsorbed on silica gel

Ru(bpy)₃³⁺, which is important in artificial photosynthetic systems due to its high reduction potential, is stabilized together with its counter anion, Ru(bpy)₃⁺, by radiolysis of Ru(bpy)₃²⁺ adsorbed on silica gel at 77 K. Both species are characterized by electron spin resonance.     

Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure and phospho-acceptor specificity of the wild-type enzyme

The direct substrates of one protein kinase in a cell can be identified by mutation of the ATP binding pocket to allow an unnatural ATP analog to be accepted exclusively by the engineered kinase. Here, we present structural and functional assessment of peptide specificity of mutant protein kinases with unnatural ATP analogs. The crystal structure (2.8 A resolution) of c-Src (T338G) with N(6)-(benzyl) ADP bound shows that the creation of a unique nucleotide binding pocket does not alter the phospho-acceptor binding site of the kinase. A panel of optimal peptide substrates of defined sequence, as well as a degenerate peptide library, was utilized to assess the phospho-acceptor specificity of the engineered "traceable" kinases. The specificity profiles for the mutant kinases were found to be identical to those of their wild-type counterparts.     

Selective kinase inhibition by exploiting differential pathway sensitivity

Protein kinase inhibitors are optimized to have high affinity for their intended target(s) to elicit the desired cellular effects. Here, we asked whether differences in inhibitory sensitivity between two kinase signaling pathways, controlled by the cyclin-dependent kinases Cdk1 and Pho85, can be sufficient to allow for selective targeting of one pathway over the other. We show the oxindole inhibitor GW297361 elicits a Pho85-selective response in cells despite having a 20-fold greater biochemical potency for Cdk1 in vitro. We provide evidence that partial inhibition of Pho85 is sufficient to activate Pho85-dependent signaling, but partial inhibition of Cdk1 is not sufficient to block Cdk1-dependent cell proliferation. Identification of highly sensitive kinases may provide a means to achieve selective perturbation of kinase signaling pathways complementary to efforts to achieve maximal differences between in vitro IC50 values.