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Start Over Topic biochemistry Publication Year Range Last 50 years Journal biochemistry
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1. Determination of cyclic nucleotide-dependent protein kinase substrate specificity by the use of peptide libraries on cellulose paper

Author

Wolfgang R. Dostmann, Werner Tegge, Ronald Frank, and Franz Hofmann

Subjects
Paper, Molecular Sequence Data, Peptide, Biochemistry, chemistry.chemical_compound, Cyclic nucleotide, Structure-Activity Relationship, Adenosine Triphosphate, Escherichia coli, Histone octamer, Amino Acid Sequence, Cellulose, Phosphorylation, Protein kinase A, Cyclic GMP, chemistry.chemical_classification, Kinase, Cyclic AMP-Dependent Protein Kinases, Recombinant Proteins, Amino acid, Kinetics, chemistry, Oligopeptides, Protein Kinases
Abstract

An iterative approach to the a priori determination of the substrate specificity of cAMP- and cGMP-dependent protein kinases (PKA and PKG) by the use of peptide libraries on cellulose paper is described. The starting point of the investigation was an octamer library with the general structure Ac-XXX12XXX, where X represents mixtures of all 20 natural amino acids and 1 and 2 represent individual amino acid residues. The library thus contained all possible 2.56 x 10(10) octamers, divided into 400 sublibraries with defined amino acids 1 and 2 each consisting of 6.4 x 10(7) sequences. After phosphorylation with the kinases in the presence of [gamma-32P]ATP, the sublibrarys Ac-XXXRRXXX and Ac-XXXRKXXX were identified as the best substrates for PKA and PKG, respectively. The second-generation libraries had the structures Ac-XXXRR12X and Ac-XXXRK12X for PKA and PKG and resulted in the most active sequence pools Ac-XXXRRASX and Ac-XXXRKKSX. After delineation of every position in the octameric sequence and extension of the investigation to decameric peptides, the best sequences, Ac-KRAERKASIY and Ac-TQKARKKSNA, were obtained for PKA and PKG, respectively. Promising octameric and decameric peptides were assembled 5 or 10 times each and assayed in order to determine the experimental scatter inherent in the approach. The kinetic data of several octameric and decameric sequences were determined in solution and compared to data for known substrates. The recognition motif of PKA was confirmed by this approach, and a novel substrate sequence for PKG was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Published
1995

2. On the determination of deoxyribonucleic acid-protein interaction parameters using the nitrocellulose filter-binding assay

Author

von Hippel Ph and Charles P. Woodbury

Subjects
Paper, Chromatography, Ligand binding assay, Proteins, DNA, Biochemistry, Models, Biological, chemistry.chemical_compound, Kinetics, chemistry, Filter (video), Molecule, Binding site, Nitrocellulose, Nitrocellulose filter, Mathematics, Protein Binding
Abstract

We examine the effects of filter efficiency on DNA-protein binding data obtained by the popular nitrocellulose filter-binding assay. Graphical procedures for determining the efficiency parameter epsilon (for the efficiency of retention of DNA on the filter, per bound protein molecule) are established. Filter efficiency modified formulas for determining thermodynamic binding parameters are derived for four simple prototypes of DNA-protein binding systems. The effects of experimental error on discrimination between models are considered. Finally, we discuss conceptual errors often made in calculating binding stoichiometry from filter-binding experiments and suggest a general protocol for the analysis of filter-binding data.

Published
1983