Oligo-(R)-3-hydroxybutyrate modification of sorting signal enables pore formation by Escherichia coli OmpA.

The outer membrane protein A (OmpA) of Escherichia coli is a well-known model for protein targeting and protein folding. Wild-type OmpA, isolated either from cytoplasmic inclusion bodies or from outer membranes, forms narrow pores of approximately 80 pS in planar lipid bilayers at room temperature. The pores are well structured with narrow conductance range when OmpA is isolated using lithium dodecyl sulfate (LDS) or RapiGest surfactant but display irregular conductance when OmpA is isolated with urea or guanidine hydrochloride. Previous studies have shown that serine residues S163 and S167 of the sorting signal of OmpA (residues 163-169), i.e., the essential sequence for outer membrane incorporation, are covalently modified by oligomers of (R)-3-hydroxybutyrate (cOHB). Here we find that single-mutants S163 and S167 of OmpA, which still contain cOHB on one serine of the sorting signal, form narrow pores in planar lipid bilayers at room temperature with lower and more irregular conductance than wild-type OmpA, whereas double mutants S163:S167 and S163:V166 of OmpA, with no cOHB on the sorting signal, are unable to form stable pores in planar lipid bilayers. Our results indicate that modification of serines in the sorting signal of OmpA by cOHB in the cytoplasm enables OmpA to incorporate into lipid bilayers at room temperature as a narrow pore. They further suggest that cOHB modification may be an important factor in protein targeting and protein folding.

[Seasonal dependence of the parameters of quantitative ultrasonic measurements on the peripheral skeleton]. / Saisonale Abhängigkeit der Parameter des quantitativen Ultraschalls am peripheren Skelett.

PURPOSE: To investigate the dependence of parameters of quantitative ultrasound (QUS) on seasonable temperature variation. MATERIAL AND METHODS: 10 adolescents were examined by QUS at the heel (Sahara, Hologic), at the tibia, at the radius and at the proximal phalanges (Omnisense 7000 P, Sunlight). Measuring took place 0 (T1), 30 (T2), 60 (T3), and 120 (T4) minutes after entering the building at constant room temperature. The skin temperature was measured. The speed of sound (SOS; m/s) and the broadband ultrasound attenuation (BUA; dB/MHz) were determined as QUS parameters. Investigations took place every two weeks from January to July. RESULTS: Between skin temperature at the heel after entering the building and the measured SOS at the point of time (T1), a significantly negative correlation was stated (R = -0.47; p < 0.01); after 30 minutes (T2) the correlation was R = -0.21 (p < 0.01); after 60 minutes (T3) and after 120 minutes (T4) the correlation was not significantly anymore (R = -0,1). Minor, significant correlation was stated at the proximal phalanx at point of time T 1 (R = -0.2; p < 0.01). There was no significant correlation between temperature and SOS at the radius and tibia. CONCLUSION: The speed of sound at the heel depends inversely on outside temperature. This effect decreases with the longer duration of stay in the building. The measuring error is not relevant in relation to the population variability, but should be considered for individual process controls. There is no temperature dependence for the broadband ultrasonic attenuation. The SOS on the radius and tibia does not depend on temperature.

Haemophilus influenzae outer membrane protein P5 is associated with inorganic polyphosphate and polyhydroxybutyrate.

Outer membrane protein P5 of nontypeable (acapsulate) Haemophilus influenzae (NTHi P5) forms large pores in planar lipid bilayers between symmetric solutions that unpredictably display a nonzero reversal potential. Moreover, NTHi P5 has a high theoretical isoelectric point, calculated as 9.58, which is not in agreement with the experimental isoelectric point, determined as 6.3-6.8, or with its preference for cations, disproportionately strong at one side. These anomalous results intimate that NTHi P5 is associated with a polyanion. Chemical and immunological analyses revealed the presence of inorganic polyphosphate (polyP), and the amphiphilic, solvating polyester, poly-(R)-3-hydroxybutyrate, frequently associated with polyP. A sharp reduction in cation selectivity was observed after addition of Saccharomyces cerevisiae exopolyphosphatase X to the bilayer, providing functional evidence for the involvement of polyP in selectivity. The results suggest that NTHi P5 associates with polyP and poly-(R)-3-hydroxybutyrate to create large, cation-selective pores in the outer membrane of H. influenzae.

Pore characteristics of nontypeable Haemophilus influenzae outer membrane protein P5 in planar lipid bilayers.

The structure of outer membrane protein P5 of NTHi, a homolog of Escherichia coli OmpA, was investigated by observing its pore characteristics in planar lipid bilayers. Recombinant NTHi P5 was overexpressed in E. coli and purified using ionic detergent, LDS-P5, or nonionic detergent, OG-P5. LDS-P5 and OG-P5 could not be distinguished by their migration on SDS-PAGE gels; however, when incorporated into planar bilayers of DPhPC between symmetric aqueous solutions of 1 M KCl at 22 degrees C, LDS-P5 formed narrow pores (58 +/- 6 pS) with low open probability, whereas OG-P5 formed large pores (1.1 +/- 0.1 nS) with high open probability (0.99). LDS-P5 narrow pores were gradually and irreversibly transformed into large pores, indistinguishable from those formed by OG-P5, at temperatures >or=40 degrees C; the process took 4-6 h at 40 degrees C or 35-45 min at 42 degrees C. Large pores were stable to changes in temperatures; however, large pores were rapidly converted to narrow pores when exposed to LDS at room temperatures, indicating acute sensitivity of this conformer to ionic detergent. These studies suggest that narrow pores are partially denatured forms and support the premise that the native conformation of NTHi P5 is that of a large monomeric pore.

Kinetics of folding of Escherichia coli OmpA from narrow to large pore conformation in a planar bilayer.

The outer membrane protein of Escherichia coli, OmpA, is currently alleged to adopt two native conformations: a major two-domain conformer in which 171 N-terminal residues form a narrow eight beta-barrel pore and 154 C-terminal residues are in the periplasm and a minor one-domain conformer in which all 325 residues create a large pore. However, recent studies in planar bilayers indicate the conformation of OmpA is temperature-sensitive and that increasing temperature converts narrow pores to large pores. Here we examine the reversibility and kinetics of this transition for single OmpA molecules in planar bilayers of diphytanoylphosphatidylcholine (DPhPC). We find that the transition is irreversible. When temperatures are decreased, large pores close down, and when temperatures are stabilized they reopen in the large pore conformation, with gradually increasing open time. Large pores are converted to narrow pores only by denaturing agents. The transition from narrow to large pores requires temperatures >or= 26 degrees C and is a biphasic process with rates that rise steeply with temperature. The first phase, a flickering stepwise transition from a low-conductance to a high-conductance state requires approximately 7 h at 26 degrees C but only approximately 13 min at 42 degrees C, signifying an activation energy of 139 +/- 12 kJ/mol. This is followed by a gradual increase in conductance and open probability, interpreted as optimization of the large pore structure. The results indicate that the two-domain structure is a partially folded intermediate that is kinetically stable at lower temperatures and that mature fully folded OmpA is a large pore.

Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA.

Here we present functional evidence for involvement of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP) in ion conduction and selection at the intracellular side of the Streptomyces lividans potassium channel, KcsA. At < or = 25 degrees C, KcsA forms channels in planar bilayers that display signal characteristics of PHB/polyP channels at the intracellular side; i.e., a preference for divalent Mg(2+) cations at pH 7.2, and a preference for monovalent K+ cations at pH 6.8. Between 25 and 26 degrees C, KcsA undergoes a transition to a new conformation in which the channel exhibits high selectivity for K+, regardless of solution pH. This suggests that basic residues of the C-terminal polypeptides have moved closer to the polyP end unit, reducing its negative charge. The data support a supramolecular structure for KcsA in which influx of ions is prevented by the selectivity pore, whereas efflux of K+ is governed by a conductive core of PHB/polyP in partnership with the C-terminal polypeptide strands.

Streptomyces lividans potassium channel KcsA is regulated by the potassium electrochemical gradient.

It is currently held that the Streptomyces lividans potassium channel, KcsA, requires an intracellular pH<5 to exhibit activity in planar lipid bilayers. Here, we show that KcsA functions well at normal physiological pH in the presence of a potassium electrochemical gradient. Single-channel conductance and open probability increased directly as the extracellular potassium concentration was decreased. Channel activity was sensitive to both the membrane potential and the size of the gradient, thus indicating that gating of the channel depends on both components of the electrochemical potential. When [K(+)(in)]/[K(+)(ex)] was 200 mM/10 mM, chord conductance was 24pS with subconductance 15pS; open probability was 0.9. The permeability series was K(+) > Rb(+) >>> Cs(+); K(+) selectivity over Rb(+) was 1.2-fold and selectivity over Na(+) was 12-fold. The channels were disrupted by intracellular Na(+) and blocked by intracellular Ba(2+). A hypothetical supramolecular model for the channel is presented.

Outer membrane protein A of Escherichia coli forms temperature-sensitive channels in planar lipid bilayers.

The temperature dependence of single-channel conductance and open probability for outer membrane protein A (OmpA) of Escherichia coli were examined in planar lipid bilayers. OmpA formed two interconvertible conductance states, small channels, 36-140 pS, between 15 and 37 degrees C, and large channels, 115-373 pS, between 21 and 39 degrees C. Increasing temperatures had strong effects on open probabilities and on the ratio of large to small channels, particularly between 22 and 34 degrees C, which effected sharp increases in average conductance. The data infer that OmpA is a flexible temperature-sensitive protein that exists as a small pore structure at lower temperatures, but refolds into a large pore at higher temperatures.

Increased poly-(R)-3-hydroxybutyrate concentrations in streptozotocin (STZ) diabetic rats.

Poly-(R)-3-hydroxybutyrate, a linear polymer of the ketone body, R-3-hydroxybutyric acid, is an amphiphilic, water-insoluble, salt-solvating polymer. In humans, shortchain, complexed polyhydroxybutyrate has been found in a wide variety of tissues and in atherosclerotic plaques. In the circulation, plasma polyhydroxybutyrate concentrations correlate strongly with atherogenic lipid profiles. We compared polyhydroxybutyrate levels in plasma, kidney, eye, sciatic nerve, aorta, and brain of streptozotocin-diabetic and healthy control Sprague-Dawley rats, three weeks after injection. With the exception of brain, which showed only a marginal increase (1.3-fold), polyhydroxybutyrate levels were 3- to 8-fold greater in tissues from diabetic vs. control rats. Increases in polyhydroxybutyrate levels between normal and diabetic rat tissues were in order: sciatic nerve (9.0-fold), kidney (7.2-fold), plasma (6.0-fold), aorta (4.4-fold), and whole eye (2.9-fold). These data indicate a significant increase in polyhydroxybutyrate levels in organs affected by complications of diabetes, and further suggest that plasma polyhydroxybutyrate levels may serve as a marker for the disease.

Differential effects of temperature on E. coli and synthetic polyhydroxybutyrate/polyphosphate channels.

Complexes of poly-(R)-3-hydroxybutyrate and inorganic polyphosphate (PHB/polyP), isolated from the plasma membranes of Escherichia coli or prepared synthetically (HB(128)/polyP(65)), form Ca(2+)-selective ion channels in planar lipid bilayers that exhibit indistinguishable gating and conductance characteristics at 22 degrees C. Here we examine the gating and conductance of E. coli and synthetic PHB/polyP complexes in planar lipid bilayers as a function of temperature from 15 to 45 degrees C. E. coli PHB/polyP channels remained effectively open throughout this range, with brief closures that became more rare at higher temperatures. Conversely, as temperatures were gradually increased, the open probability of HB(128)/polyP(65) channels progressively decreased. The effect was fully reversible. Channel conductance exhibited three distinct phases. Below 25 degrees C, as PHB approached its glass temperature (ca. 10 degrees C), the conductance of both E. coli and synthetic channels remained at about the same level (95-105 pS). Between 25 degrees C and ca. 40 degrees C, the conductance of E. coli and synthetic channels increased gradually with temperature coefficients (Q(10)) of 1.45 and 1.42, respectively. Above 40 degrees C, E. coli channel conductance increased sharply, whereas the conductance of HB(128)/polyP(65) channels leveled off. The discontinuities in the temperature curves for E. coli channels coincide with discontinuities in thermotropic fluorescence spectra and specific growth rates of E. coli cells. It is postulated that E. coli PHB/polyP complexes are associated with membrane components that inhibit their closure at elevated temperatures.

pH regulates cation selectivity of poly-(R)-3-hydroxybutyrate/polyphosphate channels from E. coli in planar lipid bilayers.

Poly-(R)-3-hydroxybutyrate/polyphosphate (PHB/polyP) complexes, whether isolated from the plasma membranes of bacteria or prepared from the synthetic polymers, form ion channels in planar lipid bilayers that are highly selective for Ca(2+) over Na(+) at physiological pH. This preference for divalent over monovalent cations is attributed to a high density of negative charge along the polyP backbone and the higher binding energies of divalent cations. Here we modify the charge density of polyP by varying the pH, and observe the effect on cation selectivity. PHB/polyP complexes, isolated from E. coli, were incorporated into planar lipid bilayers, and unitary current-voltage relations were determined as a function of pH. When Ca(2+) was the sole permeant cation, conductance diminished steadily from 97 +/- 6 pS at pH 7.4 to 47 +/- 3 pS at pH 5.5. However, in asymmetric solutions of Ca(2+) and Na(+), there was a moderate increase in conductance from 98 +/- 4 at pH 7.4 to 129 +/- 4 pS at pH 6.5, and a substantially larger increase to 178 +/- 6 pS at pH 5.6, signifying an increase in Na(+) permeability or disorganization of channel structure. Reversal potentials point to a sharp decrease in preference for Ca(2+) over Na(+) over a relatively small decrease in pH. Ca(2+) was strongly favored over Na(+) at physiological pH, but the channels became nonselective near the pK(2) of phosphate (approximately 6.8), and displayed weak selectivity for Na(+) over Ca(2+) at acidic pH. Evidently, PHB/polyP complexes are versatile ion carriers whose selectivity may be modulated by small adjustments of the local pH. The results may be relevant to the physiological function of PHB/polyP channels in bacteria and the role of PHB and polyP in the Streptomyces lividans potassium channel.

Transmembrane ion transport by polyphosphate/poly-(R)-3-hydroxybutyrate complexes.

Transmembrane ion transport, a critical process in providing energy for cell functions, is carried out by pore-forming macromolecules capable of discriminating among very similar ions and responding to changes in membrane potential. It is widely regarded that ion channels are exclusively proteins, relatively late arrivals in cell evolution. Here we discuss the formation of ion-selective, voltage-activated channels by complexes of two simple homopolymers, namely, inorganic polyphosphates (polyPs) and poly-(R)-3-hydroxybutyrates (PHBs), derived from phosphate and acetate, respectively. Each has unique molecular characteristics that facilitate ion selection, solvation, and transport. Complexes of the two polymers, isolated from bacterial plasma membranes or prepared from the synthetic polymers, form voltage-dependent, Ca2+-selective channels in planar lipid bilayers that are selective for divalent over monovalent cations, permeant to Ca2+, Sr2+, and Ba2+, and blocked by transition metal cations in a concentration-dependent manner. Recently, both polyP and PHB have been found to be components of ion-conducting proteins: namely, the human erythrocyte Ca2+-ATPase pump and the Streptomyces lividans potassium channel. The contribution of polyP and PHB to ion selection and/or transport in these proteins is yet unknown, but their presence gives rise to the hypothesis that these and other ion transporters are supramolecular structures in which proteins, polyP, and PHB cooperate in forming well-regulated and specific cation transfer systems.

Frequent expression of the B-cell-specific activator protein in Reed-Sternberg cells of classical Hodgkin's disease provides further evidence for its B-cell origin.

The neoplastic cells of classical Hodgkin's disease (cHD), ie, Hodgkin and Reed-Sternberg cells (HRS cells), contain clonally rearranged Ig genes, but are dissimilar to normal B cells in that they mostly do not display B-cell antigens such as CD20 or CD19. The transcription factor B-cell-specific activator protein (BSAP) influences numerous B-cell functions such as B-cell antigen expression, Ig expression, and class switch. We analyzed the expression of BSAP in cHD and control tissues by isotopic in situ hybridization and immunohistochemistry to determine whether BSAP is expressed in HRS cells and, if so, whether it may be involved in the genesis of the abnormal phenotype of these cells. Both in normal lymphoid tissue and non-Hodgkin lymphomas, BSAP transcripts and protein were almost exclusively found in B cells and B-cell lymphomas (40 cases), but were absent from the tumor cells of T-cell neoplasms (41 cases), including 19 cases of anaplastic large cell lymphoma of T- and null-cell type. Among cHD, variable numbers of HRS cells exhibited BSAP transcripts (22 of 25 cases) and protein (28 of 31 cases). Our findings show that BSAP is sufficiently specific to serve as B-cell marker. BSAP expression in HRS cells provides further strong evidence for a frequent B-cell origin of cHD and helps distinguish this disease from anaplastic large cell lymphoma of T- and null-cell type. Because BSAP is much more frequently expressed in HRS cells than the conventional B-cell antigens, the abnormal immunophenotype of HRS cells with frequent absence of B-cell antigens does not appear to be due to absent BSAP expression.

Heterologous gene expression in a membrane-protein-specific system.

We have constructed an expression system for heterologous proteins which uses the molecular machinery responsible for the high level production of bacteriorhodopsin in Halobacterium salinarum. Cloning vectors were assembled that fused sequences of the bacterio-opsin gene (bop) to coding sequences of heterologous genes and generated DNA fragments with cloning sites that permitted transfer of fused genes into H. salinarum expression vectors. Gene fusions include: (i) carboxyl-terminal-tagged bacterio-opsin; (ii) a carboxyl-terminal fusion with the catalytic subunit of the Escherichia coli aspartate transcarbamylase; (iii) the human muscarinic receptor, subtype M1; (iv) the human serotonin receptor, type 5HT2c; and (v) the yeast alpha mating factor receptor, Ste2. Characterization of the expression of these fusions revealed that the bop gene coding region contains previously undescribed molecular determinants which are critical for high level expression. For example, introduction of immunogenic and purification tag sequences into the C-terminal coding region significantly decreased bop gene mRNA and protein accumulation. The bacteriorhodopsin-aspartate transcarbamylase fusion protein was expressed at 7 mg per liter of culture, demonstrating that E. coli codon usage bias did not limit the system's potential for high level expression. The work presented describes initial efforts in the development of a novel heterologous protein expression system, which may have unique advantages for producing multiple milligram quantities of membrane-associated proteins.

Streptomyces lividans potassium channel contains poly-(R)-3-hydroxybutyrate and inorganic polyphosphate.

The Streptomyces lividans KcsA potassium channel, a homotetramer of 17.6 kDa subunits, was found to contain two nonproteinaceous polymers, namely, poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP). PHB and polyP are ubiquitous cellular constituents with a demonstrated capacity for cation selection and transport. PHB was detected in both tetramer and monomer species of KcsA by reaction to anti-PHB IgG on Western blots, and estimated as 28 monomer units of PHB per KcsA tetramer by a chemical assay in which PHB is converted to its unique degradation product, crotonic acid. PolyP was detected in KcsA tetramers, but not in monomers, by metachromatic reaction to o-toluidine blue stain on SDS-PAGE gels. A band of free polyP was also visible, suggesting that polyP is released when tetramers dissociate. The exopolyphosphatase of Saccharomyces cerevisiae degraded the free polyP, but tetramer-associated polyP was not affected, indicating it was inaccessible to the enzyme. PolyP in KcsA was estimated as 15 monomer units per tetramer by an enzymatic assay in which polyphosphate kinase is used to transfer phosphates from polyP to [(14)C]ADP, yielding [(14)C]ATP. The experimentally determined isoelectric point of KcsA tetramer was 6.5-7.5, substantially more acidic than the theoretical pI of 10.3, and consistent with the inclusion of a polyanion. The results suggest that PHB is covalently bound to KcsA subunits while polyP is held within tetramers by ionic forces. It is posited that KcsA protein creates an environment in which PHB/polyP is selective for K(+). The basic amino acids attenuate the negative charge density of polyP, thereby transforming the cation binding preference from multivalent to monovalent, and discrimination between K(+) and Na(+) is accomplished by adjusting the ligand geometry in cation binding cavities formed by PHB and polyP.

Gating kinetics of E. coli poly-3-hydroxybutyrate/polyphosphate channels in planar bilayer membranes.

Nonproteinaceous calcium channel complexes from Escherichia coli, composed of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP), exhibit two distinct gating modes (modes 1 and 2) in planar lipid bilayers. Here we report the kinetic characterization of the channel in mode 2, a mode characterized by two well-defined conductance levels, a fully open state (87 +/- 3 pS), and a major subconductance state (56 +/- 2 pS). Other subconductance states and full closures are rare (<0.5% of total time). Several kinetic properties of the channel showed asymmetric voltage-dependence indicating an asymmetry in the channel structure. Accordingly, single channels responded to potential change in one of two mirror-image patterns, postulated to arise from opposite orientations of the asymmetrical channel complex in the bilayer. The fraction of time spent in each conductance level was strongly voltage-sensitive. For channels reported in this study, presumably all oriented in the same direction, residence time in the fully open state increased as clamping potentials became more positive whereas residence time in the major subconductance state increased at more negative potentials. Analysis of open time distributions revealed existence of two kinetically distinct states for each level. The shorter time constants for both conductance states exhibited weak voltage-sensitivity; however, the longer time constants were strongly voltage-sensitive. A kinetic scheme, consistent with the complex voltage dependence of the channel, is proposed.

Mapping and use of a sequence that targets DNA ligase I to sites of DNA replication in vivo.

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed "functional organization" of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1-28 and 111-179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins. We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.

Novel components and enzymatic activities of the human erythrocyte plasma membrane calcium pump.

The plasma membrane Ca2+ pump is essential for the maintenance of cystolic calcium ion concentration levels in eukaryotes. Here we show that the Ca2+-ATPase, purified from human erythrocytes, contains two homopolymers, poly(3-hydroxybutyrate) (PHB) and inorganic polyphosphate (polyP), which form voltage-activated calcium channels in the plasma membranes of Escherichia coli and other bacteria. Furthermore, we demonstrate that the plasma membrane Ca2+-ATPase may function as a polyphosphate kinase, i.e. it exhibits ATP-polyphosphate transferase and polyphosphate-ADP transferase activities. These findings suggest a novel supramolecular structure for the functional Ca2+-ATPase, and a new mechanism of uphill Ca2+ extrusion coupled to ATP hydrolysis.

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

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 La3+) from channels isolated from Escherichia coli. The implications of our finding for prebiotic chemistry, biochemistry, and biology are discussed.