Proof for a nonproteinaceous calcium-selective channel in Escherichia coli by total synthesis from ( R )-3-hydroxybutanoic acid and inorganic polyphosphate

Abstract

Traditionally, the structure and properties of natural products have been determined by total synthesis and comparison with authentic samples. We have now applied this procedure to the first nonproteinaceous ion channel, isolated from bacterial plasma membranes, and consisting of a complex of poly(3-hydroxybutyrate) and calcium polyphosphate. To this end, we have now synthesized the 128-mer of hydroxybutanoic acid and prepared a complex with inorganic calcium polyphosphate (average 65-mer), which was incorporated into a planar lipid bilayer of synthetic phospholipids. We herewith present data that demonstrate unambiguously that the completely synthetic complex forms channels that are indistinguishable in their voltage-dependent conductance, in their selectivity for divalent cations, and in their blocking behavior (by La 3؉ ) from channels isolated from Escherichia coli. The implications of our finding for prebiotic chemistry, biochemistry, and biology are discussed.Synthetic and naturally occurring ion channels are amphiphilic structures with an outer coat of nonpolar residues and a lining of polar and charged residues (1-7). In this report, we demonstrate the formation of synthetic nonproteinaceous ion channels from two structurally distinct polymers that share the above attributes in a cooperative fashion. The polymers in question, poly(3-hydroxybutyrate) (PHB) and inorganic polyphosphate (polyP), are ubiquitous constituents of biological cells (8)(9)(10)(11)(12). PHB is an amphiphilic homopolymer of (R)-3-hydroxybutanoic acid, which in bacteria is synthesized from acetyl-CoA in three steps: dimerization to form acetoacetyl-CoA, reduction by NADPH to form 3-hydroxybutyrylCoA, and polymerization to form PHB (13,14). Under growth-limiting conditions, some bacteria produce highmolecular-mass PHB (60,000 to Ͼ1,000,000 Da) in amounts of up to 90% of the cell dry weight. PolyP, a polyanion composed of phosphate residues linked by anhydride bonds, is formed by repetitive phosphoryl transfer from ATP or other high-energy phosphates (15-17).Both PHB and polyP have molecular characteristics that are consistent with a role in ion conduction. PHB has salt-solvating properties that derive from the recurrence of electrondonating ester carbonyl oxygens at regular and frequent intervals along its flexible backbone (18,19). The solvating abilities of PHB are illustrated by studies that demonstrate its capacity to transport cations across methylene chloride layers in U-tubes (20), form ion-conducting complexes with lithium perchlorate (21), or large-conductance nonselective ion channels in planar lipid bilayers (22). PolyP has a flexible backbone and high density of monovalent negative charges that create a large capacity for ion exchange and a stronger affinity for multivalent over monovalent cations (23).Plasma membrane vesicles of Escherichia coli contain complexes of PHB and polyP that function as calcium-selective channels in planar lipid bilayers (24). In vivo, such channels may control the influx of calcium to ma...

Obcells as Proto-Organisms: Membrane Heredity, Lithophosphorylation, and the Origins of the Genetic Code, the First Cells, and Photosynthesis

“…Thus, the periplasmic space would become an external "gut," enabling the protocell to consume the genomes and ribosomes of obcells until they became extinct (just as obcells had earlier consumed and caused a mass extinction of free replicators). Since polyP naturally combines with ␤-polyhydroxybutyrate and Ca ++ to form hydrophilic voltage-gated pores in the cytoplasmic membrane (Das et al 1997), it is possible that, initially, the pores might not have been of protein. More likely, however, they were protein precursors of porins.…”

How did cells get their size?

“…An intriguing possibility is that the liposomes had abiotic cation pores fashioned of polyphosphate and polyhydroxybutyrate (PHB), since these simple materials self-assemble as cation pores in lipid bilayers (Das et al, 1997).…”

“…Diverse small ␣-helical peptides (Table 2) interact strongly with bilayers, and by virtue of their ␣-helical dipole, dissolve into them at higher rates if the transmembrane voltage is nonzero. Prebiotic liposomes may have had nonzero potentials due to abiotic cation channels (Das et al, 1997) and/or asymmetrically disposed photo-or chemoreactive amphiphiles (Segre et al, 2001). In any case, it is reasonable to expect that in protocells manufacturing, say, 10 -20-mer peptides, peptide molecules sometimes found their way into the bilayer.…”

Biodegradable Polymers: Recent Developments and New Perspectives

“…Furthermore, oligo-PHB-Ca-polyphosphate complexes were found in eukaryotic membranes, suggesting their possible existence in all living cells [20,21]. Oligo-PHB-Ca-polyphosphate complexes play a role in not only the transport of inorganic cations [22][23][24] but also the transport of DNA molecules [25][26][27].…”