Abstract
Using a dimeric bZIP protein, we have designed a leucine zipper that becomes more stable after a serine in the e position is phosphorylated by protein kinase A (delta delta GP = -1.4 kcal mol-1 dimer-1 or -0.7 kcal mol-1 residue-1). Mutagenesis studies indicate that three arginines form a network of inter-helical (i,i' + 5; i, i' + 2) and intra-helical (i, i + 4) attractive interactions with the phosphorylated serine. When the arginines are replaced with lysines, the stabilizing effect of serine phosphorylation is reduced (delta delta GP = -0.5 kcal mol-1 dimer-1). The hydrophobic interface of the leucine zipper needs a glycine in the d position to obtain an increase in stability after phosphorylation. The phosphorylated protein binds DNA with a 15-fold higher affinity. Using a transient transfection assay, we document a PKA dependent four-fold activation of a reporter gene. Phosphorylation of a threonine in the same e position decreases the stability by delta delta GP = +1.2 kcal mol-1 dimer-1. We present circular dichroism (CD) thermal denaturations of 15 bZIP proteins before and after phosphorylation. These data provide insights into the structural determinants that result in stabilization of a coiled coil by phosphorylation.
MeSH terms
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Amino Acid Sequence
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Arginine / chemistry
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Avian Proteins*
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Basic-Leucine Zipper Transcription Factors
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Centrifugation, Isopycnic
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Circular Dichroism
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Cyclic AMP-Dependent Protein Kinases / metabolism
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DNA Probes / metabolism
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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G-Box Binding Factors
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Genes, Reporter
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Glutamic Acid
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Glycine / chemistry
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Glycine / genetics
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Glycine / metabolism
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Hot Temperature
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Leucine Zippers* / genetics
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Phosphoproteins / metabolism
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Phosphorylation
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Protein Binding
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Protein Denaturation
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Protein Engineering
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Protein Structure, Secondary*
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Serine / chemistry
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Serine / genetics
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Serine / metabolism*
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Threonine / chemistry
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Threonine / genetics
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Threonine / metabolism
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Transcription Factors / chemistry
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Transcriptional Activation
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Transfection
Substances
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Avian Proteins
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Basic-Leucine Zipper Transcription Factors
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Carrier Proteins
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DNA Probes
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DNA-Binding Proteins
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G-Box Binding Factors
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Phosphoproteins
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Recombinant Proteins
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Transcription Factors
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Threonine
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Glutamic Acid
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Serine
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Arginine
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Cyclic AMP-Dependent Protein Kinases
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Glycine