Poly-(R)-3-hydroxybutyrates (PHB) are Atherogenic Components of Lipoprotein Lp(a).

The hypothesis is that poly-(R)-3-hydroxybutyrates (PHB), linear polymers of the ketone body, R-3-hydroxybutyrate (R-3HB), are atherogenic components of lipoprotein Lp(a). PHB are universal constituents of biological cells and are thus components of all foods. Medium chain-length PHB (<200 residues) (mPHB) are located in membranes and organelles, and short-chain PHB (<15 residues) are covalently attached to certain proteins (cPHB). PHB are highly insoluble in water, but soluble in lipids in which they exhibit a high intrinsic viscosity. They have a higher density than other cellular lipids and they are very adhesive, i.e. they engage in multiple noncovalent interactions with other molecules and salts via hydrogen, hydrophobic and coordinate bonds, thus producing insoluble deposits. Following digestive processes, PHB enter the circulation in chylomicrons and very low density lipoproteins (VLDL). The majority of the PHB (>70%) are absorbed by albumin, which transports them to the liver for disposal. When the amount of PHB in the diet exceed the capacity of albumin to safely remove them from the circulation, the excess PHB remain in the lipid core of LDL particles that become constituents of lipoprotein Lp(a), and contribute to the formation of arterial deposits. In summary, the presence of PHB ­ water-insoluble, dense, viscous, adhesive polymers ­ in the lipid cores of the LDL moieties of Lp(a) particles supports the hypothesis that PHB are atherogenic components of Lp(a).

Molecular complementarity between simple, universal molecules and ions limited phenotype space in the precursors of cells.

BACKGROUND: Fundamental problems faced by the protocells and their modern descendants include how to go from one phenotypic state to another; escape from a basin of attraction in the space of phenotypes; reconcile conflicting growth and survival strategies (and thereby live on 'the scales of equilibria'); and create a coherent, reproducible phenotype from a multitude of constituents. PRESENTATION OF THE HYPOTHESIS: The solutions to these problems are likely to be found with the organic and inorganic molecules and inorganic ions that constituted protocells, which we term SUMIs for Simple Universal Molecules and Ions. These SUMIs probably included polyphosphate (PolyP) as a source of energy and of phosphate; poly-(R)-3-hydroxybutyrate (PHB) as a source of carbon and as a transporter in association with PolyP; polyamines as a source of nitrogen; lipids as precursors of membranes; as well as peptides, nucleic acids, and calcium. Here, we explore the hypothesis that the direct interactions between PHB, PolyP, polyamines and lipids - modulated by calcium - played a central role in solving the fundamental problems faced by early and modern cells. TESTING THE HYPOTHESIS: We review evidence that SUMIs (1) were abundant and available to protocells; (2) are widespread in modern cells; (3) interact with one another and other cellular constituents to create structures with new functions surprisingly similar to those of proteins and RNA; (4) are essential to creating coherent phenotypes in modern bacteria. SUMIs are therefore natural candidates for reducing the immensity of phenotype space and making the transition from a "primordial soup" to living cells. IMPLICATIONS OF THE HYPOTHESIS: We discuss the relevance of the SUMIs and their interactions to the ideas of molecular complementarity, composomes (molecular aggregates with hereditary properties based on molecular complementarity), and a prebiotic ecology of co-evolving populations of composomes. In particular, we propose that SUMIs might limit the initial phenotype space of composomes in a coherent way. As examples, we propose that acidocalcisomes arose from interactions and self-selection among SUMIs and that the phosphorylation of proteins in modern cells had its origin in the covalent modification of proteins by PHB.

The role of short-chain conjugated poly-(R)-3-hydroxybutyrate (cPHB) in protein folding.

Poly-(R)-3-hydroxybutyrate (PHB), a linear polymer of R-3-hydroxybutyrate (R-3HB), is a fundamental constituent of biological cells. Certain prokaryotes accumulate PHB of very high molecular weight (10,000 to >1,000,000 residues), which is segregated within granular deposits in the cytoplasm; however, all prokaryotes and all eukaryotes synthesize PHB of medium-chain length (~100-200 residues) which resides within lipid bilayers or lipid vesicles, and PHB of short-chain length (<12 residues) which is conjugated to proteins (cPHB), primarily proteins in membranes and organelles. The physical properties of cPHB indicate it plays important roles in the targeting and folding of cPHB-proteins. Here we review the occurrence, physical properties and molecular characteristics of cPHB, and discuss its influence on the folding and structure of outer membrane protein A (OmpA) of Escherichia coli.

Physiological importance of poly-(R)-3-hydroxybutyrates.

Poly-(R)-3-hydroxybutyrates (PHB), linear polymers of (R)-3-hydroxybutyrate, are components of all biological cells in which short polymers (<200 monomer residues) are covalently attached to certain proteins and/or noncovalently associated with polyphosphates - inorganic polyphosphate (polyP), RNA, and DNA. The low concentrations, lack of unusual atoms or functional groups, and flexible backbones of this complexed PHB, referred to as cPHB, make them invisible to many analytical procedures; whereas other physical properties - water-insolubility, high intrinsic viscosity, temperature sensitivity, multiple bonding interactions with other molecules - make them requisite participants in vital physiological processes as well as contributors to the development of certain diseases.

Insights into the structure and assembly of Escherichia coli outer membrane protein A.

Outer membrane protein A (OmpA) of Escherichia coli is a paradigm for the biogenesis of outer membrane proteins; however, the structure and assembly of OmpA have remained controversial. A review of studies to date supports the hypothesis that native OmpA is a single-domain large pore, while a two-domain narrow-pore structure is a folding intermediate or minor conformer. The in vitro refolding of OmpA to the large-pore conformation requires isolation of the protein from outer membranes with retention of an intact disulfide bond followed by sufficient incubation in lipids at temperatures of ≥ 26 °C to overcome the high energy of activation for refolding. The in vivo maturation of the protein involves covalent modification of serines in the eighth ß-barrel of the N-terminal domain by oligo-(R)-3-hydroxybutyrates as the protein is escorted across the cytoplasm by SecB for post-translational secretion across the secretory translocase in the inner membrane. After cleavage of the signal sequence, protein chaperones, such as Skp, DegP and SurA, guide OmpA across the periplasm to the ß-barrel assembly machinery (BAM) complex in the outer membrane. During this passage, a disulfide bond is formed between C290 and C302 by DsbA, and the hydrophobicity of segments of the C-terminal domain, which are destined for incorporation as ß-barrels in the outer membrane bilayer, is increased by covalent attachment of oligo-(R)-3-hydroxybutyrates. With the aid of the BAM complex, OmpA is then assembled into the outer membrane as a single-domain large pore.

Resolving the native conformation of Escherichia coli OmpA.

The native conformation of the 325-residue outer membrane protein A (OmpA) of Escherichia coli has been a matter of contention. A narrow-pore, two-domain structure has vied with a large-pore, single-domain structure. Our recent studies show that Ser163 and Ser167 of the N-terminal domain (1-170) are modified in the cytoplasm by covalent attachment of oligo-(R)-3-hydroxybutyrates (cOHBs), and further show that these modifications are essential for the N-terminal domain to be incorporated into planar lipid bilayers as narrow pores (≈ 80 pS, 1 m KCl, 22 °C). Here, we examined the potential effect(s) of periplasmic modifications on pore structure by comparing OmpA isolated from outer membranes (M-OmpA) with OmpA isolated from cytoplasmic inclusion bodies (I-OmpA). Chemical and Western blot analysis and 1H-NMR showed that segment 264-325 in M-OmpA, but not in I-OmpA, is modified by cOHBs. Moreover, a disulfide bond is formed between Cys290 and Cys302 by the periplasmic enzyme DsbA. Planar lipid bilayer studies indicated that narrow pores formed by M-OmpA undergo a temperature-induced transition into stable large pores (≈ 450 pS, 1 M KCl, 22 °C) [energy of activation (Ea) = 33.2 kcal·mol(-1)], but this transition does not occur with I-OmpA or with M-OmpA that has been exposed to disulfide bond-reducing agents. The results suggest that the narrow pore is a folding intermediate, and demonstrate the decisive roles of cOHB-modification, disulfide bond formation and temperature in folding OmpA into its native large-pore configuration.

Importance of oligo-R-3-hydroxybutyrates to S. lividans KcsA channel structure and function.

In the polyphosphate model of the Streptomyces lividans potassium channel KcsA, four polypeptides, each covalently modified by oligo-(R)-3-hydroxybutyrates (cOHB), surround a core molecule of inorganic polyphosphate (polyP). PolyP attracts, binds, and conducts K(+) in response to an electrochemical stimulus whilst the polypeptides govern access to polyP and regulate its selectivity. However, the role of cOHB has remained uncertain. Here we identify cOHB-conjugated residues in the ion pathway, S102 and S129, and mutate them to determine the influence of cOHB on channel properties. We find that the mutations have no discernible effect on tetramer formation or tetramer stability; however, cOHB influences polyP incorporation and/or retention, i.e. single mutants S102G and S129G contain ∼1/3 and double mutant S102G:S129G ≈ 1/2 as much polyP as wild-type. Moreover, planar lipid bilayer studies of wild-type and mutant proteins indicate that cOHB has a critical effect on channel function: at positive potentials, only ∼5% of S102G and S129G currents and <1% of S102G:S129G currents consist of well-structured channels; at negative potentials, S102G and S129G display only irregular conductance and S102G:S129G exhibits no conductance whatsoever. The results indicate that cOHB facilitates the incorporation and/or retention of polyP and plays a critical role in maintaining the flexible polyP molecule in an optimal transbilayer orientation for efficient K(+) transport.

Hypothesis: poly-(R)-3-hydroxybutyrate is a major factor in intraocular pressure.

Short chain poly-(R)-3-hydroxybutyrate (cPHB) is a ubiquitous molecule that readily adheres to others, that is covalently added to proteins and that forms, with polyphosphate, ion channels. High levels of cPHB have been implicated in atherosclerosis and in diabetes. Here, we propose a hypothesis in which cPHB adheres to filaments in the extracellular matrix and this raises intraocular pressure. In a corollary, we propose that a positive feedback relationship between cPHB adherence to filaments, raised IOP and filament stretching constitutes a switch.

Role of polyphosphate in regulation of the Streptomyces lividans KcsA channel.

We examine the hypotheses that the Streptomyces lividans potassium channel KcsA is gated at neutral pH by the electrochemical potential, and that its selectivity and conductance are governed at the cytoplasmic face by interactions between the KcsA polypeptides and a core molecule of inorganic polyphosphate (polyP). The four polypeptides of KcsA are postulated to surround the end unit of the polyP molecule with a collar of eight arginines, thereby modulating the negative charge of the polyP end unit and increasing its preference for binding monovalent cations. Here we show that KcsA channels can be activated in planar lipid bilayers at pH 7.4 by the chemical potential alone. Moreover, one or both of the C-terminal arginines are replaced with residues of progressively lower basicity-lysine, histidine, valine, asparagine-and the effects of these mutations on conductance and selectivity for K(+) over Mg(2+) is tested in planar bilayers as a function of Mg(2+) concentration and pH. As the basicity of the C-terminal residues decreases, Mg(2+) block increases, and Mg(2+) becomes permeant when medium pH is greater than the pI of the C-terminal residues. The results uphold the premise that polyP and the C-terminal arginines are decisive elements in KcsA channel regulation.

Poly-3-hydroxybutyrate synthase from the periplasm of Escherichia coli.

This is the first report of a poly-3-hydroxybutyrate (PHB) synthase in Escherichia coli. The enzyme was isolated from the periplasm using ammonium sulfate fractionation, hydrophobic, and size-exclusion chromatography and identified by LC/MS/MS as YdcS, a component of a putative ABC transporter. Green Fluorescent Protein-tagged ydcS, purified by 2D native gel electrophoresis, also exhibited PHB synthase activity. Optimal conditions for enzyme activity were 37 degrees C, pH 6.8-7.5, 100 mM KCl. K(m) was 0.14 mM and V(max) was 18.7 nmol/mg protein/min. The periplasms of deletion mutants displayed <25% of the activity of the parent strain. Deletion mutants exhibited approximately 25% less growth in M9 medium, glucose, and contained approximately 30% less PHB complexed to proteins (cPHB) in the outer membranes, but the same concentration of chloroform-extractable PHB as wild-type cells. The primary sequence of YdcS suggests it may belong to the alpha-/beta-hydrolase superfamily which includes polyhydroxybutyrate (PHB) synthases, lipases, and esterases.

Sorting signal of Escherichia coli OmpA is modified by oligo-(R)-3-hydroxybutyrate.

Escherichia coli outer membrane protein A (OmpA) is a well-established model for the study of membrane assembly. Previous studies have shown that the essential sequence for outer membrane localization, known as the sorting signal, is contained in a segment of the eighth beta-strand, residues 163-171. Sequential digestion of OmpA, purified from outer membranes or inclusion bodies with cyanogen bromide and Staphylococcus aureus GluC, yielded peptides 162-174(LSLGVSYRFGQGE). Western blot and chemical assays indicated that the peptide was covalently modified by oligo-(R)-3-hydroxybutyrate (cOHB), a flexible, amphipathic oligoester. MALDI/MS was consistent with modification of peptides 162-174 by up to ten R-3-hydroxybutyrate (HB) residues. Western blot analysis of mutants of the peptide, using anti-OHB IgG, indicated that cOHB modification was not inhibited by the single mutations S163G, S167G, Y168F, R169N or R169D; however, cOHB was not detected on peptides containing the double mutations S163G:S167G S163G:V166G, L162G:S167G, and L164G:S167G. MALDI/MS/MS of double mutant S163G:S167G confirmed the absence of cOHB-modification. The results suggest that cOHB may be attached to one or both serines, and point to the importance of the flanking hydrophobic residues. Modification by cOHB may play a role in outer membrane targeting and assembly of OmpA.

Insight into the selectivity and gating functions of Streptomyces lividans KcsA.

Streptomyces lividans KcsA is a 160-aa polypeptide that oligomerizes to form a tetrameric potassium channel. The three-dimensional structure of the polypeptides has been established, but the selectivity and gating functions of the channel remain unclear. It has been shown that the polypeptides copurify with two homopolymers, poly[(R)-3-hydroxybutyrate] (PHB) and inorganic polyphosphate (polyP), which have intrinsic capacities for cation selection and transport. PHB/polyP complexes are highly selective for divalent cations when pH is greater than the pK(2) of polyP ( approximately 6.8), but this preference is lost when pH is < or =pK(2). It is postulated that KcsA polypeptides attenuate the divalent negative charge of the polyP end unit at physiological pH by strategic positioning of two C-terminal arginines. Here we mutate one or both of the C-terminal arginines and observe the effects on channel selectivity in planar lipid bilayers. We find that channels formed by KcsA polypeptides that retain a single C-terminal arginine remain highly selective for K(+) over Mg(2+), independent of medium pH; however, channels formed by KcsA polypeptides in which both C-terminal arginines have been replaced with neutral residues are selective for Mg(2+) when pH is >7 and for K(+) when pH is <7. Channel gating may be triggered by changes in the balance between the K(+) polyP(-) binding energy, the membrane potential, and the gradient force. The results reveal the importance of the C-terminal arginines to K(+) selectivity and argue for a supramolecular structure for KcsA in which the host polypeptides modify the cation preference of a guest PHB/polyP complex.

A large, voltage-dependent channel, isolated from mitochondria by water-free chloroform extraction.

We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria. Reconstituted channels showed multiple subconductance levels and were voltage-dependent, showing an increased probability of higher conductance states at voltages near zero. In symmetric 150 mM KCl, the maximal conductance of the channel ranged from 350 pS to 750 pS. For voltages >+/-60 mV, conductance fluctuated in the range of approximately 50- approximately 200 pS. In the presence of a 1:3 gradient of KCl, at pH = 7.4, selectivity periodically switched between different states ranging from weakly anion-selective (V(rev) approximately -15 mV) to ideally cation-selective (V(rev) approximately +29 mV), without a significant change in its conductance. Overall, the diverse, but highly reproducible, channel activity most closely resembled the behavior of the permeability transition pore channel seen in patch-clamp experiments on native mitoplasts. We suggest that the isolated complex may represent the ion-conducting module from the permeability transition pore.

Polyhydroxybutyrate-enhanced transformation of log-phase Escherichia coli.

Transformation of Escherichia coli plays an important role in recombinant DNA technology. Most current transformation protocols require that the cells be treated to attain a particular physiological state known as "competence," and this makes transformation procedures lengthy and arduous. Here we describe a protocol for transforming log-phase E. coli using dimethyl sulfoxide (DMSO) solutions of poly-(R)-3-hydroxybutyrate (PHB) to facilitate the transfer of plasmid DNA into cells, and certain reagents and temperature shocks to promote DNA uptake. The protocol was optimized using factorial design techniques across variables that included PHB molecular weight and concentration, DMSO concentration, monovalent and divalent salts, glucose, cold and heat shocks, cell density, and pH. Using 10 ng DNA, the optimized protocol produces approximately 1000 colony-forming units (CFUs) from 100 microL early log-phase cell culture or approximately 300 CFU from a 21-24 h single colony, sufficient for many applications. The total volume of the transformation reaction mixture is only 150 microL suggesting that the procedure may be adapted for use in microplates or automated transformation technologies.

Posttranslational modification of E. coli histone-like protein H-NS and bovine histones by short-chain poly-(R)-3-hydroxybutyrate (cPHB).

Short-chain poly-(R)-3-hydroxybutyrate (cPHB), a highly flexible, amphiphilic molecule with salt-solvating properties, is a ubiquitous constituent of prokaryotic and eukaryotic cells, wherein it is mainly conjugated to proteins. The solvating properties and cellular distribution of cPHB suggest it may be associated with proteins that bind and/or transfer DNA. Here we examine Escherichia coli protein H-NS and calf thymus histones, H1, H2A, H2B, H3, and H4, for the presence of cPHB. The proteins are related in that all bind to DNA and are implicated in the compact organization of the chromosome. The presence of cPHB in E. coli H-NS was first detected in Western blots of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of total cell proteins, probed with anti-cPHB IgG, and then by Western blot analysis of the purified protein. Western blot analysis of the calf thymus histones indicated that each contained cPHB. The presence of cPHB in H-NS and histones was confirmed by chemical assay. The in vivo size of conjugated cPHB could not be established due to the lack of standards and degradation of cPHB during protein purification and storage. The molecular characteristics of cPHB and its presence in histone-like and histone proteins of diverse organisms suggest it may play a role in DNA binding and/or DNA organization.