Polyamine binding

About: Polyamine binding is a research topic. Over the lifetime, 188 publications have been published within this topic receiving 9206 citations.

Characterization of the NspS-MbaA signaling system controlling biofilm formation from polyamine input to phenotypic output

Abstract: Biofilm formation plays a critical role in the infectious cycle of the bacterial pathogen, Vibrio cholerae. The polyamine, norspermidine, has a positive influence on biofilm formation while spermidine has the opposite effect. These influences occur through a signaling system comprised of the periplasmic polyamine binding protein, NspS, and the transmembrane phosphodiesterase, MbaA. In the absence of NspS, MbaA acts as a phosphodiesterase cleaving the pro-biofilm second messenger, cyclic di-GMP, indicating that NspS may interact with and inhibit phosphodiesterase activity and biofilm repression by MbaA. In this study, I have shown that several nspS ligand-binding pocket mutants have altered sensitivity to norspermidine and spermidine as indicated by vps transcription and biofilm assays. I have also shown using thermal shift assays that these mutants are capable of binding both polyamines. LC-MS/MS analyses indicate that the NspS-MbaA signaling pathway is a high specificity c-di-GMP signaling system. Additionally, a system for reproducible demonstration of the NspS-MbaA interaction was developed which may subsequently be used in future experiments to study the NspS and MbaA interaction. Altogether, this study provides a thorough investigation of the steps involved in polyamine signal input to phenotypic output for a novel polyamine responsive signaling system controlling V. cholerae biofilm formation.

Recombinant anti-polyamine antibodies: identification of a conserved binding site motif

Abstract: Polyamines are small linear polycations found ubiquitously in eukaryotic cells. They are involved in nucleic acid and protein synthesis and rises in cellular polyamine levels have been correlated with cell proliferation. Antibodies to these molecules have potential as prognostic indicators of disease conditions and indicators of treatment efficacy. Antipolyamine monoclonal antibodies of differing but defined specificities have been generated in our laboratory using polyamine ovalbumin conjugates as immunogens. These antibodies show small but significant cross reactivities with other polyamine species; IAG-1 cross reacts with spermidine (8%), JAC-1 with spermine (6%) and JSJ-1 with both putrescine (11%) and spermine (6%). We have rescued and sequenced the heavy and light chain variable regions of all three of these antibodies. While the light chains of two antibodies, IAG-1 and JSJ-1, were 93% homologous at the amino acid level, none of the heavy chains displayed any significant sequence homology. However, computer-generated models of all three antibody binding sites revealed a three-dimensionally conserved polyamine binding site motif. The polyamine appears to bind into a negatively charged cleft lined with acidic and polar residues. The cleft is partially or completely closed at one end and the specificity of the interaction is determined by placement of acidic residues in the cleft. Aromatic residues contribute to polyamine binding interacting with the carbon backbone. The polyamine-binding motif we have identified is very similar to that observed in the crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli.

Designed DNA Interactions

Abstract: Using a fluorescence-detected ethidium displacement array, the complexation properties of several tri- and tetracationic polyamines with natural and synthetic polynucleotides were determined. The compounds exhibited different binding affinities which were dependent upon the structure and charge of the polyamine compound. The GC/AT DNA binding selectivities were measured for several of the polyamines, and two of the tricationic derivatives (compounds 3 and 4) were observed to exhibit significant GC-selectivity. Computer modelling results were in agreement with a major groove spanning binding mode which possessed site specific interactions between the polyamines and DNA hydrogen bonding sites.