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Jonathan R Moll

Publications and source records attributed to Jonathan R Moll.

4 recordsLinked to original sources
PubMed2002

A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I, V, L, N, A, and K.

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.2 kcal/mol per dimer more stable than the A-A interaction and 4.0 kcal/mol per dimer more stable than either the L-L or V-V interaction, and 7.0 kcal/mol per dimer more stable than the N-N interaction. Only lysine was less stable than alanine. An alanine-based double-mutant thermodynamic cycle calculated coupling energies between the a and a' positions in the heterodimer. The aliphatic amino acids L, V, and I prefer to form homotypic interactions with coupling energies of -0.6 to -0.9 kcal/mol per dimer, but the heterotypic aliphatic interactions have positive coupling energies of <1.0 kcal/mol per dimer. The asparagine homotypic interaction has a coupling energy of -0.5 kcal/mol per dimer, while heterotypic interactions with the aliphatic amino acids produce coupling energies ranging from 2.6 to 4.9 kcal/mol per dimer. The homotypic K-K interaction is 2.9 kcal/mol per dimer less stable than the A-A interaction, but the coupling energy is only 0.3 kcal/mol per dimer. Heterotypic interactions with lysine and either asparagine or aliphatic amino acids produce similar coupling energies ranging from -0.2 to -0.7 kcal/mol per dimer. Thus, of the amino acids that were examined, asparagine contributes the most to dimerization specificity because of the large positive coupling energies in heterotypic interactions with the aliphatic amino acids which results in the N-N homotypic interaction.

Alanine↗
PubMed2002

Magnesium is required for specific DNA binding of the CREB B-ZIP domain.

We have examined binding of the CREB B-ZIP protein domain to double-stranded DNA containing a consensus CRE sequence (5'-TGACGTCA-3'), the related PAR, C/EBP and AP-1 sequences and the unrelated SP1 sequence. DNA binding was assayed in the presence or absence of MgCl2 and/or KCl using two methods: circular dichroism (CD) spectroscopy and electrophoretic mobility shift assay (EMSA). The CD assay allows us to measure equilibrium binding in solution. Thermal denaturation in 150 mM KCl indicates that the CREB B-ZIP domain binds all the DNA sequences, with highest affinity for the CRE site, followed by the PAR (5'-TAACGTTA-3'), C/EBP (5'-TTGCGCAA-3') and AP-1 (5'-TGAGTCA-3') sites. The addition of 10 mM MgCl2 diminished DNA binding to the CRE and PAR DNA sequences and abolished binding to the C/EBP and AP-1 DNA sequences, resulting in more sequence-specific DNA binding. Using 'standard' EMSA conditions (0.25x TBE), CREB bound all the DNA sequences examined. The CREB-CRE complex had an apparent Kd of approximately 300 pM, PAR of approximately 1 nM, C/EBP and AP-1 of approximately 3 nM and SP1 of approximately 30 nM. The addition of 10 mM MgCl2 to the polyacrylamide gel dramatically altered sequence-specific DNA binding. CREB binding affinity for CRE DNA decreased 3-fold, but binding to the other DNA sequences decreased >1000-fold. In the EMSA, addition of 150 mM KCl to the gels had an effect similar to MgCl2. The magnesium concentration needed to prevent non-specific electrostatic interactions between CREB and DNA in solution is in the physiological range and thus changes in magnesium concentration may be a cellular signal that regulates gene expression.

Animals↗
PubMed2002

B-ZIP proteins encoded by the Drosophila genome: evaluation of potential dimerization partners.

The basic region-leucine zipper (B-ZIP) (bZIP) protein motif dimerizes to bind specific DNA sequences. We have identified 27 B-ZIP proteins in the recently sequenced Drosophila melanogaster genome. The dimerization specificity of these 27 B-ZIP proteins was evaluated using two structural criteria: (1) the presence of attractive or repulsive interhelical g<-->e' electrostatic interactions and (2) the presence of polar or charged amino acids in the 'a' and 'd' positions of the hydrophobic interface. None of the B-ZIP proteins contain only aliphatic amino acids in the'a' and 'd' position. Only six of the Drosophila B-ZIP proteins contain a "canonical" hydrophobic interface like the yeast GCN4, and the mammalian JUN, ATF2, CREB, C/EBP, and PAR leucine zippers, characterized by asparagine in the second 'a' position. Twelve leucine zippers contain polar amino acids in the first, third, and fourth 'a' positions. Circular dichroism spectroscopy, used to monitor thermal denaturations of a heterodimerizing leucine zipper system containing either valine (V) or asparagine (N) in the 'a' position, indicates that the V-N interaction is 2.3 kcal/mole less stable than an N-N interaction and 5.3 kcal/mole less stable than a V-V interaction. Thus, we propose that the presence of polar amino acids in novel positions of the 'a' position of Drosophila B-ZIP proteins has led to leucine zippers that homodimerize rather than heterodimerize.

Amino Acid Sequence↗