In order to quantify the interactions between molecules of biological interest, the determination of the dissociation constant (K d) is essential. Estimation of the binding affinity in this way is routinely performed in "favorable" conditions for macromolecules. Crucial data for ligand-protein binding elucidation is mainly derived from techniques (e.g., macromolecular crystallography) that require the addition of high concentration of salts and/or other additives. In this study we have evaluated the effect of temperature, ionic strength, viscosity, and hydrophobicity on the K d of three previously characterized protein-ligand systems, based on variation in their binding sites, in order to provide insight into how these often overlooked unconventional circumstances impact binding affinity. Our conclusions are as follows: (1) increasing solvent viscosity in general is detrimental to ligand binding, (2) moderate increases in temperature have marginal effects on the dissociation constant, and (3) the degree of hydrophobicity of the ligand and the binding site determines the extent of the influence of cosolvents and salt concentration on ligand binding affinity.
The cyclin groove is an important
recognition site for substrates
of the cell cycle cyclin dependent kinases and provides an opportunity
for highly selective inhibition of kinase activity through a non-ATP
competitive mechanism. The key peptide residues of the cyclin binding
motif have been studied in order to precisely define the structure–activity
relationship for CDK kinase inhibition. Through this information,
new insights into the interactions of peptide CDK inhibitors with
key subsites of the cyclin binding groove provide for the replacement
of binding determinants with more druglike functionality through REPLACE,
a strategy for the iterative conversion of peptidic blockers of protein–protein
interactions into pharmaceutically relevant compounds. As a result,
REPLACE is further exemplified in combining optimized peptidic sequences
with effective N-terminal capping groups to generate more stable compounds
possessing antitumor activity consistent with on-target inhibition
of cell cycle CDKs. The compounds described here represent prototypes
for a next generation of kinase therapeutics with high efficacy and
kinome selectivity, thus avoiding problems observed with first generation
CDK inhibitors.
BackgroundStudies on bacterial signal transduction systems have revealed complex networks of functional interactions, where the response regulators play a pivotal role. The AtoSC system of E. coli activates the expression of atoDAEB operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that atoSC is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the atoDAEB promoter as the single, cis-regulatory binding site of the AtoC response regulator. In this work, we used a computational approach to explore the presence of yet unidentified AtoC binding sites within other parts of the E. coli genome.ResultsThrough the implementation of a computational de novo motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the E. coli genome. In order to assess the biological relevance of the motifs and to select for experimental validation of those sequences related robustly with distinct cellular functions, we implemented a novel approach that applies Gene Ontology Term Analysis to the motif hits and selected those that were qualified through this procedure. The computational results were validated using Chromatin Immunoprecipitation assays to assess the in vivo binding of AtoC to the predicted sites. This process verified twenty-two additional AtoC binding sites, located not only within intergenic regions, but also within gene-encoding sequences.ConclusionsThis study, by tracing a number of putative AtoC binding sites, has indicated an AtoC-related cross-regulatory function. This highlights the significance of computational genome-wide approaches in elucidating complex patterns of bacterial cell regulation.
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