Irregular activity oscillations of a rotary molecular motor: A simple kinetic model of F<Subscript>1</Subscript>-ATPase

F₁-ATPase is a catalytic part of the F₁F_o-ATP synthase molecular motor. The cooperative hydrolysis of ATP at three catalytic sites of F₁-ATPase is accompanied by the rotation of the central γ-subunit inside a cylinder formed by three α-subunits and three β-subunits. Experimental works of different authors have shown that the γ-subunit rotates with irregular dwells. A simple kinetic model suggested in this article provides an explanation as to why dwells occur during the rotation of F₁-ATPase. According to this model, rotation dwells happen as a result of deterministic chaos, which in turn occurs at rate constants that are close to those demonstrated experimentally. The time duration of dwells in the model is in agreement with that observed experimentally. Our model explains the known irregular occupancy of catalytic sites of F₁-ATPase by nucleotides.     

Structure and Mechanism of Vacuolar Na<Superscript>+</Superscript>-Translocating ATPase From <Emphasis Type="Italic">Enterococcus hirae</Emphasis>

V-type Na⁺-ATPase from Entercoccus hirae consists of nine kinds of subunits (NtpA₃, B₃, C₁, D₁, E₁₋₃, F₁₋₃, G₁, I₁, and K₁₀) which are encoded by the ntp operon. The amino acid sequences of the major subunits, A, B, and K (proteolipid), were highly similar to those of A, B, and c subunits of eukaryotic V-ATPases, and those of β, α, and c subunits of F-ATPases. We modeled the A and B subunits by homology modeling using the structure of β and α subunits of F-ATPase, and obtained an atomic structure of NtpK ring by X-ray crystallography. Here we briefly summarize our current models of the whole structure and mechanism of the E. hirae V-ATPase.     

Evidence for centrophenoxine as a protective drug in aluminium induced behavioral and biochemical alteration in rat brain

The aim of the present work was to study the effects of an unilateral ischaemic-reperfusion injury on Na⁺, K⁺-ATPase activity, α₁ and β₁ subunits protein and mRNA abundance and ATP content in cortical and medullary tissues from postischaemic and contralateral kidneys. Right renal artery was clamped for 40 min followed by 24 and 48 h of reperfusion. Postischaemic and contralateral renal function was studied cannulating the ureter of each kidney. Postischaemic kidneys after 24 (IR24) and 48 (IR48) hours of reperfusion presented a significant dysfunction. Na⁺, K⁺-ATPase α₁ subunit abundance increased in IR24 and IR48 cortical tissue and β₁ subunit decreased in IR48. In IR24 medullary tissue, α₁ abundance increased and returned to control values in IR48 while β₁ abundance was decreased in both periods. Forty minutes of ischaemia without reperfusion (I40) promoted an increment in α₁ mRNA in cortex and medulla that normalised after 24 h of reperfusion. β₁ mRNA was decreased in IR24 medullas. No changes were observed in contralateral kidneys. This work provides evidences that after an ischaemic insult α₁ and β₁ protein subunit abundance and mRNA levels are independently regulated. After ischaemic-reperfusion injury, cortical and medullary tissue showed a different pattern of response. Although ATP and Na⁺, K⁺-ATPase activity returned to control values, postischemic kidney showed an abnormal function after 48 h of reflow.     

Structural elements of the C-terminal domain of subunit E (E<Subscript>133-222</Subscript>) from the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> V<Subscript>1</Subscript>V<Subscript>O</Subscript> ATPase determined by solution NMR spectroscopy

Subunit E of the vacuolar ATPase (V-ATPase) contains an N-terminal extended α helix (Rishikesan et al. J Bioenerg Biomembr 43:187–193, 2011) and a globular C-terminal part that is predicted to consist of a mixture of α-helices and β-sheets (Grüber et al. Biochem Biophys Res Comm 298:383–391, 2002). Here we describe the production, purification and 2D structure of the C-terminal segment E_133-222 of subunit E from Saccharamyces cerevisiae V-ATPase in solution based on the secondary structure calculation from NMR spectroscopy studies. E_133-222 consists of four β-strands, formed by the amino acids from K136-V139, E170-V173, G186-V189, D195-E198 and two α-helices, composed of the residues from R144-A164 and T202-I218. The sheets and helices are arranged as β1:α1:β2:β3:β4:α2, which are connected by flexible loop regions. These new structural details of subunit E are discussed in the light of the structural arrangements of this subunit inside the V₁- and V₁V_O ATPase.     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Analysis of the Open and Closed Conformations of the β Subunits in Thermophilic F1-ATPase by Solution NMR

F₁-ATPase, composed of α, β, γ, δ, and ? subunits, is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this function is the α₃β₃γ complex. We have investigated the α₃β₃γ?^ΔC (?^ΔC, truncated ?) complex from thermophilic Bacillus PS3 (TF₁′, 360 kDa) in the solution state by using the combination of extensive deuteration, segmental-labeling, and CRINEPT (cross-correlated relaxation-enhanced polarization transfer) NMR. Well-resolved CRINEPT-HMQC (heteronuclear multiple-quantum correlation) spectra of partially ¹⁵N-labeled TF₁′ were obtained for this huge and asymmetric protein complex. The spectrum of the C-terminal domain of the β subunit revealed that the open form of the β subunit in the TF₁′ complex is similar to that of the free β monomer. The open β subunit in the TF₁′ complex does not exhibit high affinity for nucleotides unlike the monomer, but this is in agreement with the results of single-molecule analysis of TF₁α₃β₃γ. On the other hand, the closed form of the β subunit in the TF₁′ complex was shown to be distinct from that of the nucleotide-bound β monomer. This is consistent with a previous report that the closed form of the TF₁β monomer could be a catalytically activated state. The loop between the N-terminal β-barrel and the central domain is highly flexible in the TF₁′ complex, in contrast to that in the α₃β₃ hexamer, suggesting that it is affected by the presence of the γ subunit in this area.     

γ<Subscript>1</Subscript>-Dependent Down-regulation of Recombinant Voltage-gated Ca<Superscript>2+</Superscript> Channels

(1) Voltage-gated Ca²⁺ (Ca_V) channels are multi-subunit membrane complexes that allow depolarization-induced Ca²⁺ influx into cells. The skeletal muscle L-type Ca_V channels consist of an ion-conducting Ca_V1.1 subunit and auxiliary α₂δ−1, β₁ and γ₁ subunits. This complex serves both as a Ca_V channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α₂δ−1 and β₁ subunits regulate Ca_V channel function, there is far less information on the γ₁ subunit. Previously, we characterized the interaction of γ₁ with the other components of the skeletal Ca_V channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca²⁺ current density in myotubes from γ₁ null mice. (3) In the current report, using Western blotting we show that the expression of the Ca_V1.1 protein is significantly lower when it is heterologously co-expressed with γ₁. Consistent with this, patch-clamp recordings showed that transient transfection of γ₁ drastically inhibited macroscopic currents through recombinant N-type (Ca_V2.2/α₂δ−1/β₃) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ₁ subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca²⁺ current density following γ₁ transfection.     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.     

Delayed internalization and lack of recycling in a beta<Subscript>2</Subscript>-adrenergic receptor fused to the G protein alpha-subunit

Background  

Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β₂-adrenergic receptor (β₂AR) and Gα_s indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gα_s-fused β₂AR.     

Characteristics of protection by MgADP and MgATP of α3β3γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a bi-site mechanism of catalysis

MgADP and MgATP binding to catalytic sites of βY341W-α₃β₃Γ subcomplex of F₁-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect βY341W-α₃β₃Γ sub-complex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 μM and 12 mM, and 46 μM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K _d of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with βY341W-α₃β₃Γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F₁-ATPases.     

GFT projection NMR based resonance assignment of membrane proteins: application to subunit c of <Emphasis Type="Italic">E. coli</Emphasis> F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP synthase in LPPG micelles

G-matrix FT projection NMR spectroscopy was employed for resonance assignment of the 79-residue subunit c of the Escherichia coli F₁F₀ ATP synthase embedded in micelles formed by lyso palmitoyl phosphatidyl glycerol (LPPG). Five GFT NMR experiments, that is, (3,2)D HNNCO, L-(4,3)D HNNC ^αβ C ^α, L-(4,3)D HNN(CO)C ^αβ C ^α, (4,2)D HACA(CO)NHN and (4,3)D HCCH, were acquired along with simultaneous 3D ¹⁵N, ¹³C^aliphatic, ¹³C^aromatic-resolved [¹H,¹H]-NOESY with a total measurement time of ∼43 h. Data analysis resulted in sequence specific assignments for all routinely measured backbone and ¹³C^β shifts, and for 97% of the side chain shifts. Moreover, the use of two G²FT NMR experiments, that is, (5,3)D HN{N,CO}{C ^αβ C ^α} and (5,3)D {C ^αβ C ^α}{CON}HN, was explored to break the very high chemical shift degeneracy typically encountered for membrane proteins. It is shown that the 4D and 5D spectral information obtained rapidly from GFT and G²FT NMR experiments enables one to efficiently obtain (nearly) complete resonance assignments of membrane proteins. Qi Zhang, Hanudatta S. Atreya, Douglas E. Kamen, Mark E. Girvin and Thomas Szyperski—New York Consortium on Membrane Protein Structure.     

Effects of amyloid-β peptides on voltage-gated L-type Ca(V)1.2 and Ca(V)1.3 Ca(2+) channels

Overload of intracellular Ca²⁺ has been implicated in the pathogenesis of neuronal disorders, such as Alzheimer’s disease. Various mechanisms produce abnormalities in intracellular Ca²⁺ homeostasis systems. L-type Ca²⁺ channels have been known to be closely involved in the mechanisms underlying the neurodegenerative properties of amyloid-β (Aβ) peptides. However, most studies of L-type Ca²⁺ channels in Aβ-related mechanisms have been limited to Ca_V1.2, and surprisingly little is known about the involvement of Ca_V1.3 in Aβ-induced neuronal toxicity. In the present study, we examined the expression patterns of Ca_V1.3 after Aβ_25–35 exposure for 24 h and compared them with the expression patterns of Ca_V1.2. The expression levels of Ca_V1.3 were not significantly changed by Aβ_25–35 at both the mRNA levels and the total protein level in cultured hippocampal neurons. However, surface protein levels of Ca_V1.3 were significantly increased by Aβ_25–35, but not by Aβ_35–25. We next found that acute treatment with Aβ_25–35 increased Ca_V1.3 channel activities in HEK293 cells using whole-cell patch-clamp recordings. Furthermore, using GTP pulldown and co-immunoprecipitation assays in HEK293 cell lysates, we found that amyloid precursor protein interacts with β₃ subunits of Ca²⁺ channels instead of Ca_V1.2 or Ca_V1.3 α₁ subunits. These results show that Aβ_25–35 chronically or acutely upregulates Ca_V1.3 in the rat hippocampal and human kidney cells (HEK293). This suggests that Ca_V1.3 has a potential role along with Ca_V1.2 in the pathogenesis of Alzheimer’s disease.     

3,5-Diiodo-L-Thyronine increases F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATP synthase activity and cardiolipin level in liver mitochondria of hypothyroid rats

Short-term effects of 3,5-L-diiodothyronine (T₂) administration to hypothyroid rats on F_oF₁-ATP synthase activity were investigated in liver mitochondria. One hour after T₂ injection, state 4 and state 3 respiration rates were noticeably stimulated in mitochondria subsequently isolated. F_oF₁-ATP synthase activity, which was reduced in mitochondria from hypothyroid rats as compared to mitochondria from euthyroid rats, was significantly increased by T₂ administration in both the ATP-synthesis and hydrolysis direction. No change in β-subunit mRNA accumulation and protein amount of the α-β subunit of F_oF₁-ATP synthase was found, ruling out a T₂ genomic effect. In T₂-treated rats, changes in the composition of mitochondrial phospholipids were observed, cardiolipin (CL) showing the greatest alteration. In mitochondria isolated from hypothyroid rats the decrease in the amount of CL was accompanied by an increase in the level of peroxidised CL. T₂ administration to hypothyroid rats enhanced the level of CL and decreased the amount of peroxidised CL in subsequently isolated mitochondria, tending to restore the CL value to the euthyroid level. Minor T₂-induced changes in mitochondrial fatty acid composition were detected. Overall, the enhanced F_oF₁-ATP synthase activity observed following injection of T₂ to hypothyroid rats may be ascribed, at least in part, to an increased level of mitochondrial CL associated with decreased peroxidation of CL.     

Characterization of neuronal zebra finch GABAA receptors: steroid effects

Songbirds are widely studied to investigate the hormonal control of behavior. However, little is known about the effects of steroids on neurotransmission in these birds. We used electrophysiological and pharmacological techniques to characterize γ-aminobutyric acid (GABA) type A receptors (GABA_A) of primary cultured telencephalic and hippocampal neurons from developing zebra finches. Additionally, their modulation by 17β-estradiol(E₂), 5α- and 5β-dihydrotestosterone (DHT), 5α- and 5β-pregnan-3α-ol-20-one, and corticosterone was examined. Whole-cell GABA-evoked currents were inhibited by picrotoxin (10 μmol l⁻¹) and bicuculline methiodide (10 μmol l⁻¹) and potentiated by pentobarbital (100 μmol l⁻¹) and propofol (3 μmol l⁻¹). Loreclezole (10 μmol l⁻¹) potentiated GABA-evoked currents, suggesting the presence of β₂, β₃ and/or β₄ subunits. Diazepam (1 μmol l⁻¹) potentiated currents, while Zn²⁺ (1 μmol l⁻¹) caused no inhibition, indicating the presence of γ subunits. 5α- and 5β-Pregnan-3α-ol-20-one (100 nmol l⁻¹) potentiated currents, whereas E₂ (1 μmol l⁻¹), 5α- and 5β-DHT (1 μmol l⁻¹), and corticosterone (10 μmol l⁻¹) had no detectable effect. We conclude that zebra finch telencephalic and hippocampal GABA_A receptors include α, β, and γ subunits and are similar to their mammalian counterparts in both their biophysical and pharmacological properties. Additionally, GABA-evoked currents are greatly potentiated by 5α- and 5β-pregnan-3α-ol-20-one but show little or no acute modulation by sex steroids or corticosterone. Accepted: 12 November 1997     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

β-Adrenoreceptor agonists and antagonists modulate the activity of α<Subscript>2</Subscript>-adrenoreceptors in rat cortex cerebral membranes

The influence of isoprenaline- and propranolole-induced activation and inhibition of β-adrenoreceptors on the specific nonselective α₂-antagonist [³H]RX821002 binding was studied on rat cerebral cortex subcellular membrane fractions. It was shown that the ligand-receptor interaction for α₂-adrenoreceptors corresponded to the model that assumed the presence of one receptor pool and binding of two ligand molecules to a receptor dimer. The following parameters were determined for [³H]RX821002 binding to α₂-adrenoreceptors: K _d1 = 1.57 ± 0.27 nM, B _max = 7.24 ± 1.63 fmol/mg of protein, n = 2. In the case of isoprenaline-induced activation of β-adrenoreceptors the binding of radiolabeled ligand to α₂-adrenoreceptors was described by the same model. The affinity of α₂-adrenoreceptors for [³H]RX821002 decreased more than twofold (K _d = 3.55 ± 0.02 nM) and the quantity of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg of protein). Propranolole changed the model of ligand binding, and two pools of receptors were detected with the following parameters: K _d1 = 0.61 ± 0.02 nM, K _d2 = 3.41 ± 0.13 nM, B _ml = 1.88 ± 0.028 fmol/mg of protein, B _m2 = 9.27 ± 0.08 fmol/mg of protein, n = 2. The data suggest that α₂-adrenoreceptors in subcellular membrane fractions from rat cerebral cortex exist in dimeric form. Isoprenaline and propranolole exhibit modulating effect on the specific antagonist binding to α₂-adrenoreceptors, which results in the inhibition and alteration of [³H]RX821002 binding parameters.     

Cα-Methyl, Cα-phenylglycine peptides: A structural study

Summary In order to obtain further information on the role played by phenyl ring position in the C^α-methylated α-amino acid side chain on peptide preferred conformation, the crystal-state structural preferences of C^α-methyl, C^α-phenylglycine peptides have been determined by X-ray diffraction. This study shows that either the fully extended conformation or the β-bend/3₁₀-helical structures are adopted by peptides characterized by this C^α-methylated, β-branched, aromatic α-amino acid.     

The Effects of Copper (II) Ions on <Emphasis Type="Italic">Enterococcus hirae</Emphasis> Cell Growth and the Proton-Translocating F<Subscript>o</Subscript>F<Subscript>1</Subscript> ATPase Activity

Enterococcus hirae grow well under anaerobic conditions at alkaline pH (pH 8.0) producing acids by glucose fermentation. Bacterial growth was shown to be accompanied by decrease of redox potential from positive values (~+35 mV) to negative ones (~−220 mV). An oxidizer copper (II) ions (Cu²⁺) affected bacterial growth in a concentration-dependent manner (within the range of 0.05 mM to 1 mM) increasing lag phase duration and decreasing specific growth rate. These effects were observed with the wild-type strain ATCC9790 and the atpD mutant strain MS116 (with absent β subunit of F₁ of the F_oF₁ ATPase) both. Also ATPase activity and proton–potassium ions exchange were assessed with and without N,N′-dicyclohexylcarbodiimide (DCCD), inhibitor of the F_oF₁ ATPase. In both cases (DCCD ±), even low Cu²⁺ concentrations had noticeable effect on ATPase activity, but with less visible concentration-dependent manner. Changes in the number of accessible SH-groups were observed with E. hirae ATCC9790 and MS116 membrane vesicles. In both strains Cu²⁺ markedly decreased the number of SH-groups in the presence of K⁺ ions. The addition of ATP increased the amount of accessible SH-groups in ATCC9790 and decreased this number in MS116; Cu²⁺ blocked ATP-installed increase in SH-groups number in ATCC9790. H⁺–K⁺-exchange of bacteria was markedly inhibited by Cu²⁺, but stronger effects were detected together with DCCD. Moreover, discrimination between Cu²⁺ and other bivalent cation—Ni²⁺ was shown. It is suggested that Cu²⁺ ions inhibit E. hirae cell growth by direct affect on the F_oF₁ ATPase leading to conformational changes in this protein complex and decrease in its activity.     

Selective backbone labeling of proteins using {1,2-<Superscript>13</Superscript>C<Subscript>2</Subscript>}-pyruvate as carbon source

A simple isotope labeling approach for selective ¹³C/¹⁵N backbone labeling of proteins is described. Using {1,2-¹³C₂}-pyruvate as the sole carbon source in bacterial growth media, selective incorporation of ¹³C^α-¹³CO spin-pairs into the backbones of protein molecules with medium-to-high levels of ¹³C-enrichment is possible for a subset of 12 amino acids. The isotope labeling scheme has been tested on a pair of proteins—a 7-kDa immunoglobulin binding domain B1 of streptococcal protein G and an 82-kDa enzyme malate synthase G. A number of protein NMR applications are expected to benefit from the {1,2-¹³C₂}-pyruvate based protein production.