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1.
D Mercadante LD Melton GE Norris TS Loo MA Williams RC Dobson GB Jameson 《Biophysical journal》2012,103(2):303-312
The oligomerization of β-lactoglobulin (βLg) has been studied extensively, but with somewhat contradictory results. Using analytical ultracentrifugation in both sedimentation equilibrium and sedimentation velocity modes, we studied the oligomerization of βLg variants A and B over a pH range of 2.5–7.5 in 100 mM NaCl at 25°C. For the first time, to our knowledge, we were able to estimate rate constants (koff) for βLg dimer dissociation. At pH 2.5 koff is low (0.008 and 0.009 s−1), but at higher pH (6.5 and 7.5) koff is considerably greater (>0.1 s−1). We analyzed the sedimentation velocity data using the van Holde-Weischet method, and the results were consistent with a monomer-dimer reversible self-association at pH 2.5, 3.5, 6.5, and 7.5. Dimer dissociation constants KD2-1 fell close to or within the protein concentration range of ∼5 to ∼45 μM, and at ∼45 μM the dimer predominated. No species larger than the dimer could be detected. The KD2-1 increased as |pH-pI| increased, indicating that the hydrophobic effect is the major factor stabilizing the dimer, and suggesting that, especially at low pH, electrostatic repulsion destabilizes the dimer. Therefore, through Poisson-Boltzmann calculations, we determined the electrostatic dimerization energy and the ionic charge distribution as a function of ionic strength at pH above (pH 7.5) and below (pH 2.5) the isoelectric point (pI∼5.3). We propose a mechanism for dimer stabilization whereby the added ionic species screen and neutralize charges in the vicinity of the dimer interface. The electrostatic forces of the ion cloud surrounding βLg play a key role in the thermodynamics and kinetics of dimer association/dissociation. 相似文献
2.
Liu L Mushero N Hedstrom L Gershenson A 《Protein science : a publication of the Protein Society》2007,16(11):2403-2411
Serpins inhibit serine proteases by mechanically disrupting the protease active site. The protease first reacts with the serpin''s reactive center loop (RCL) to form an acylenzyme. Then the RCL inserts into a β-sheet in the body of the serpin, translocating the attached protease ∼70 Å and deforming the protease active site, thereby trapping the acylenzyme. Loop insertion (∼1 s−1) is an order of magnitude slower than hydrolysis of a typical substrate acylenzyme (∼50 s−1), indicating that the protease is inhibited during translocation. We have previously trapped a partially translocated covalent complex of rat trypsin and α1-proteinase inhibitor (EpartI*) resulting from attractive interactions between cationic dyes and anionic rat trypsin. Here, using single pair Förster resonance energy transfer, we demonstrate that EpartI* is a metastable complex that can dissociate to free protease and cleaved serpin (I*) as well as convert to the canonical fully translocated complex EfullI*. The partitioning between these two pathways is pH dependent, with conversion favored at low pH and dissociation favored at high pH. The short lifetime of EpartI* (∼3 h at pH 7.4) and the pH dependence of EpartI* dissociation suggest that, unlike in EfullI*, the catalytic triad is intact in EpartI*. These results also demonstrate that interactions between target proteases and the body of the serpin can hinder protease translocation leading to short-lived covalent complexes. 相似文献
3.
Gokarn YR Fesinmeyer RM Saluja A Razinkov V Chase SF Laue TM Brems DN 《Protein science : a publication of the Protein Society》2011,20(3):580-587
Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li+, Na+, K+, Rb+, Cs+, GdnH+, and Ca2+), identity. The Q* decreased in the order F− > Cl− > Br− > NO3− ∼ I− > SCN− > ClO4− ≫ SO42−, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO42− anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface. 相似文献
4.
Tongsook C Sucharitakul J Thotsaporn K Chaiyen P 《The Journal of biological chemistry》2011,286(52):44491-44502
p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxyphenylacetate to form 3,4-dihydroxyphenylacetate. Based on structures of the oxygenase component (C2), both His-120 and Ser-146 are located ∼2.8 Å from the hydroxyl group of HPA. The variants H120N, H120Q, H120Y, H120D, and H120E can form C4a-hydroperoxy-FMN (a reactive intermediate necessary for hydroxylation) but cannot hydroxylate HPA. The impairment of H120N is not due to substrate binding because the variant can still bind HPA. In contrast, the H120K variant catalyzes hydroxylation with efficiency comparable with that of the wild-type enzyme; the hydroxylation rate constant for H120K is 5.7 ± 0.6 s−1, and the product conversion ratio is 75%, compared with values of 16 s−1 and 90% for the wild-type enzyme. H120R can also catalyze hydroxylation, suggesting that a positive charge on residue 120 can substitute for the hydroxylation function of His-120. Because the hydroxylation reaction of wild-type C2 is pH-independent between pH 6 and 10, the protonation status of key components required for hydroxylation likely remains unchanged in this pH range. His-120 may be positively charged for selective binding to the phenolate form of HPA, i.e. to form the Hisδ+·HPAδ− complex, which in turn promotes oxygen atom transfer via an electrophilic aromatic substitution mechanism. Analysis of Ser-146 variants revealed that this residue is necessary for but not directly engaged in hydroxylation. Product formation in S146A is pH-independent and constant at ∼70% over a pH range of 6–10, whereas product formation for S146C decreased from ∼65% at pH 6.0 to 27% at pH 10.0. These data indicate that the ionization of Cys-146 in the S146C variant has an adverse effect on hydroxylation, possibly by perturbing formation of the Hisδ+·HPAδ− complex needed for hydroxylation. 相似文献
5.
Hern��n Alonso Michelle J. Blayney Jennifer L. Beck Spencer M. Whitney 《The Journal of biological chemistry》2009,284(49):33876-33882
Like many enzymes, the biogenesis of the multi-subunit CO2-fixing enzyme ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) in different organisms requires molecular chaperones. When expressed in Escherichia coli, the large (L) subunits of the Rubisco from the archaeabacterium Methanococcoides burtonii assemble into functional dimers (L2). However, further assembly into pentamers of L2 (L10) occurs when expressed in tobacco chloroplasts or E. coli producing RuBP. In vitro analyses indicate that the sequential assembly of L2 into L10 (via detectable L4 and L6 intermediates) occurs without chaperone involvement and is stimulated by protein rearrangements associated with either the binding of substrate RuBP, the tight binding transition state analog carboxyarabinitol-1,5-bisphosphate, or inhibitory divalent metal ions within the active site. The catalytic properties of L2 and L10 M. burtonii Rubisco (MbR) were indistinguishable. At 25 °C they both shared a low specificity for CO2 over O2 (1.1 mol·mol−1) and RuBP carboxylation rates that were distinctively enhanced at low pH (∼4 s−1 at pH 6, relative to 0.8 s−1 at pH 8) with a temperature optimum of 55 °C. Like other archaeal Rubiscos, MbR also has a high O2 affinity (Km(O2) = ∼2.5 μm). The catalytic and structural similarities of MbR to other archaeal Rubiscos contrast with its closer sequence homology to bacterial L2 Rubisco, complicating its classification within the Rubisco superfamily. 相似文献
6.
Avila RL D'Antonio M Bachi A Inouye H Feltri ML Wrabetz L Kirschner DA 《The Journal of biological chemistry》2010,285(53):42001-42012
P0 constitutes 50–60% of protein in peripheral nerve myelin and is essential for its structure and stability. Mutations within the P0 gene (MPZ) underlie a variety of hereditary neuropathies. MpzS63C transgenic mice encode a P0 with a serine to cysteine substitution at position 34 in the extracellular domain of mature P0 (P0S34C), associated with the hypomyelinating Déjérine-Sottas syndrome in human. S63C mice develop a dysmyelinating neuropathy, with packing defects in peripheral myelin. Here, we used x-ray diffraction to examine time-dependent packing defects in unfixed myelin. At ∼7 h post-dissection, WT and S63C(+/+) myelin showed native periods (175 Å) with the latter developing at most a few percent swollen myelin, whereas up to ∼50% of S63C(+/−) (mutant P0 on heterozygous P0 null background) or P0(+/−) myelin swelled to periods of ∼205 Å. In the same time frame, S63C(−/−) myelin was stable, remaining swollen at ∼210 Å. Surprisingly, treatment of whole S63C(−/−) nerves with a reducing agent completely reverted swollen arrays to native spacing and also normalized the swollen arrays that had formed in S63C(+/−) myelin, the genotype most closely related to the human disorder. Western blot revealed P0-positive bands at ∼27 and ∼50 kDa, and MALDI-TOF mass spectrometry showed these bands consisted of Ser34-containing peptides or P0 dimers having oxidized Cys34 residues. We propose that P0S34C forms ectopic disulfide bonds in trans between apposed Cys34 side chains that retard wrapping during myelin formation causing hypomyelination. Moreover, the new bonds create a packing defect by stabilizing swollen membrane arrays that leads to demyelination. 相似文献
7.
Paternoster L Lorentzon M Vandenput L Karlsson MK Ljunggren O Kindmark A Mellstrom D Kemp JP Jarett CE Holly JM Sayers A St Pourcain B Timpson NJ Deloukas P Davey Smith G Ring SM Evans DM Tobias JH Ohlsson C 《PLoS genetics》2010,6(11):e1001217
Previous genome-wide association (GWA) studies have identified SNPs associated with areal bone mineral density (aBMD). However, this measure is influenced by several different skeletal parameters, such as periosteal expansion, cortical bone mineral density (BMDC) cortical thickness, trabecular number, and trabecular thickness, which may be under distinct biological and genetic control. We have carried out a GWA and replication study of BMDC, as measured by peripheral quantitative computed tomography (pQCT), a more homogenous and valid measure of actual volumetric bone density. After initial GWA meta-analysis of two cohorts (ALSPAC n = 999, aged ∼15 years and GOOD n = 935, aged ∼19 years), we attempted to replicate the BMDC associations that had p<1×10−5 in an independent sample of ALSPAC children (n = 2803) and in a cohort of elderly men (MrOS Sweden, n = 1052). The rs1021188 SNP (near RANKL) was associated with BMDC in all cohorts (overall p = 2×10−14, n = 5739). Each minor allele was associated with a decrease in BMDC of ∼0.14SD. There was also evidence for an interaction between this variant and sex (p = 0.01), with a stronger effect in males than females (at age 15, males −6.77mg/cm3 per C allele, p = 2×10−6; females −2.79 mg/cm3 per C allele, p = 0.004). Furthermore, in a preliminary analysis, the rs1021188 minor C allele was associated with higher circulating levels of sRANKL (p<0.005). We show this variant to be independent from the previously aBMD associated SNP (rs9594738) and possibly from a third variant in the same RANKL region, which demonstrates important allelic heterogeneity at this locus. Associations with skeletal parameters reflecting bone dimensions were either not found or were much less pronounced. This finding implicates RANKL as a locus containing variation associated with volumetric bone density and provides further insight into the mechanism by which the RANK/RANKL/OPG pathway may be involved in skeletal development. 相似文献
8.
Ellinghaus D Ellinghaus E Nair RP Stuart PE Esko T Metspalu A Debrus S Raelson JV Tejasvi T Belouchi M West SL Barker JN Kõks S Kingo K Balschun T Palmieri O Annese V Gieger C Wichmann HE Kabesch M Trembath RC Mathew CG Abecasis GR Weidinger S Nikolaus S Schreiber S Elder JT Weichenthal M Nothnagel M Franke A 《American journal of human genetics》2012,90(4):636-647
Psoriasis (PS) and Crohn disease (CD) have been shown to be epidemiologically, pathologically, and therapeutically connected, but little is known about their shared genetic causes. We performed meta-analyses of five published genome-wide association studies on PS (2,529 cases and 4,955 controls) and CD (2,142 cases and 5,505 controls), followed up 20 loci that showed strongest evidence for shared disease association and, furthermore, tested cross-disease associations for previously reported PS and CD risk alleles in additional 6,115 PS cases, 4,073 CD cases, and 10,100 controls. We identified seven susceptibility loci outside the human leukocyte antigen region (9p24 near JAK2, 10q22 at ZMIZ1, 11q13 near PRDX5, 16p13 near SOCS1, 17q21 at STAT3, 19p13 near FUT2, and 22q11 at YDJC) shared between PS and CD with genome-wide significance (p < 5 × 10−8) and confirmed four already established PS and CD risk loci (IL23R, IL12B, REL, and TYK2). Three of the shared loci are also genome-wide significantly associated with PS alone (10q22 at ZMIZ1, prs1250544 = 3.53 × 10−8, 11q13 near PRDX5, prs694739 = 3.71 × 10−09, 22q11 at YDJC, prs181359 = 8.02 × 10−10). In addition, we identified one susceptibility locus for CD (16p13 near SOCS1, prs4780355 = 4.99 × 10−8). Refinement of association signals identified shared genome-wide significant associations for exonic SNPs at 10q22 (ZMIZ1) and in silico expression quantitative trait locus analyses revealed that the associations at ZMIZ1 and near SOCS1 have a potential functional effect on gene expression. Our results show the usefulness of joint analyses of clinically distinct immune-mediated diseases and enlarge the map of shared genetic risk loci. 相似文献
9.
Li J Yang D He Y Wang M Wen Z Liu L Yao J Matsuda K Nakamura Y Yu J Jiang X Sun S Liu Q Jiang X Song Q Chen M Yang H Tang F Hu X Wang J Chang Y He X Chen Y Lin J 《PloS one》2011,6(8):e24221
Background
Human leukocyte antigen DP (HLA-DP) locus has been reported to be associated with hepatitis B virus (HBV) infection in populations of Japan and Thailand. We aimed to examine whether the association can be replicated in Han Chinese populations.Methodology/Principal Findings
Two HLA-DP variants rs2395309 and rs9277535 (the most strongly associated SNPs from each HLA-DP locus) were genotyped in three independent Han cohorts consisting of 2 805 cases and 1 796 controls. By using logistic regression analysis, these two SNPs in the HLA-DPA1 and HLA-DPB1 genes were significantly associated with HBV infection in Han Chinese populations (P = 0.021∼3.36×10−8 at rs2395309; P = 8.37×10−3∼2.68×10−10 at rs9277535). In addition, the genotype distributions of both sites (rs2395309 and rs9277535) were clearly different between southern and northern Chinese population (P = 8.95×10−5 at rs2395309; P = 1.64×10−9 at rs9277535). By using asymptomatic HBV carrier as control group, our study showed that there were no associations of two HLA-DP variants with HBV progression (P = 0.305∼0.822 and 0.163∼0.881 in southern Chinese population, respectively; P = 0.097∼0.697 and 0.198∼0.615 in northern Chinese population, respectively).Conclusions
Our results confirmed that two SNPs (rs2395309 and rs9277535) in the HLA-DP loci were strongly associated with HBV infection in southern and northern Han Chinese populations, but not with HBV progression. 相似文献10.
To estimate the proficiency of inorganic pyrophosphatase as a catalyst, 31P NMR was used to determine rate constants and thermodynamics of activation for the spontaneous hydrolysis of inorganic pyrophosphate (PPi) in the presence and absence of Mg2+ at elevated temperatures. These values were compared with rate constants and activation parameters determined for the reaction catalyzed by Escherichia coli inorganic pyrophosphatase using isothermal titration calorimetry. At 25 °C and pH 8.5, the hydrolysis of MgPPi2− proceeds with a rate constant of 2.8 × 10−10 s−1, whereas E. coli pyrophosphatase was found to have a turnover number of 570 s−1 under the same conditions. The resulting rate enhancement (2 × 1012-fold) is achieved entirely by reducing the enthalpy of activation (ΔΔH‡ = −16.6 kcal/mol). The presence of Mg2+ ions or the transfer of the substrate from bulk water to dimethyl sulfoxide was found to increase the rate of pyrophosphate hydrolysis by as much as ∼106-fold. Transfer to dimethyl sulfoxide accelerated PPi hydrolysis by reducing the enthalpy of activation. Mg2+ increased the rate of PPi hydrolysis by both increasing the entropy of activation and reducing the enthalpy of activation. 相似文献
11.
Eugenia Mileykovskaya Andrea C. Ryan Xi Mo Chun-Chieh Lin Khaled I. Khalaf William Dowhan Teresa A. Garrett 《The Journal of biological chemistry》2009,284(5):2990-3000
The pgsA null Escherichia coli strain, UE54, lacks the
major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite
these alterations the strain exhibits relatively normal cell division.
Analysis of the UE54 phospholipids using negativeion electrospray ionization
mass spectrometry resulted in identification of a new anionic phospholipid,
N-acylphosphatidylethanolamine. Staining with the fluorescent dye
10-N-nonyl acridine orange revealed anionic phospholipid membrane
domains at the septal and polar regions. Making UE54 null in minCDE
resulted in budding off of minicells from polar domains. Analysis of lipid
composition by mass spectrometry revealed that minicells relative to parent
cells were significantly enriched in phosphatidic acid and
N-acylphosphatidylethanolamine. Thus despite the absence of
cardiolipin, which forms membrane domains at the cell pole and division sites
in wild-type cells, the mutant cells still maintain polar/septal localization
of anionic phospholipids. These three anionic phospholipids share common
physical properties that favor polar/septal domain formation. The findings
support the proposed role for anionic phospholipids in organizing amphitropic
cell division proteins at specific sites on the membrane surface.A unique lipid composition and lipid-protein interactions appear to exist
at the transient membrane domain that defines the division site in bacterial
cells (1). Using the
cardiolipin
(CL)4-specific
fluorescent dye 10-N-nonyl acridine orange (NAO), we previously found
CL-enriched membrane domains located at cell poles and near potential division
sites in Escherichia coli
(2). Subsequently others
reported similar CL domains in Bacillus subtilis
(3) and Pseudomonas
putida (4). In addition,
cell pole and division site enrichment in CL in E. coli was confirmed
by lipid analysis of minicells spontaneously budded off from the cell poles of
a ΔminCDE mutant
(5). We suggested that
formation of CL domains at cell pole/division sites plays an important role in
selection and recognition of the division site by amphitropic cell cycle and
cell division proteins, such as DnaA (initiation of DNA replication at
oriC), MinD (a part of MinCDE system preventing positioning of the
divisome at cell poles in E. coli), and FtsA (bacterial actin, which
is a linker protein for cytoskeletal protein FtsZ (bacterial tubulin),
responsible for targeting the Z-ring to the mid-cell membrane domain). They
interact directly with membrane phospholipids through specific amphipathic
motifs enriched in basic amino acids, which confers the preference for anionic
lipids (for references see Ref.
1). In E. coli the
ATP-bound form of MinD recruits an inhibitor of Z-ring formation, MinC, to the
membrane, whereas the topological regulator, MinE, induces hydrolysis of ATP
bound to MinD resulting in release of MinD, and consequently MinC, from the
membrane into the cytoplasm. As a result, all three proteins oscillate between
the cell poles maintaining the maximum concentration of the inhibitor MinC at
the cell poles and its minimum concentration at the cell center. Pole-to-pole
oscillation of Min proteins occurs by dynamic redistribution of the proteins
within a helical oligomeric structure that winds around the cell (for recent
review and references see Ref.
6).Our previous study of a mutant lacking phosphatidylethanolamine (PE) and
containing highly elevated levels of phosphatidylglycerol (PG) and CL
demonstrated a strong inhibition of cell division and aggregation of MinD and
FtsZ/FtsA proteins at domains enriched in CL
(7,
8). To further investigate the
role of lipids in the process of cell division, we chose an E. coli
mutant with an opposite extreme in phospholipid composition to PE-lacking
mutants, namely a ΔpgsA mutant (pgsA encodes
phosphatidylglycerol phosphate synthase, which catalyzes the committed step to
PG and CL synthesis (9)). This
mutant is devoid of PG and CL (contribute ∼20 mole % of phospholipids in
wild type) and contains higher levels of PE (∼90 mole % versus 80
mole % in wild type) (10,
11). Interestingly, the
ΔpgsA null mutant accumulates elevated amounts of the
phospholipid precursors, phosphatidic acid (PA) (∼4 mole %) and
CDP-diacylglycerol (∼3 mole %), which are also anionic lipids that were
proposed to fulfill the structural and functional roles of PG and CL
(10,
11). These results suggest
that a minimum of 5–10% anionic lipid is required to support viability.
Another minor anionic phospholipid, N-acylphosphatidylethanolamine
was suggested to be present in wild-type E. coli
(12), which, if proven true,
might be also elevated in this mutant. Finally, if anionic lipids are
essential for cell division, then we would expect these normally minor lipids
to segregate into similar anionic lipid domains, as does CL.In this report we identified N-acyl-PE in E. coli and,
along with PA, its enrichment in polar/septal membrane domains of the
ΔpgsA mutant UE54
(11) lacking PG and CL. Thus
E. coli has a mechanism for preferential segregation of anionic
phospholipids to the polar/septal regions where several amphitropic proteins,
which show preference for interaction with anionic phospholipids in
vitro, are functionally located. 相似文献
12.
Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent''s disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl−/1 H+. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO3−/H+ antiporter atClCa, led to coupled NO3−/H+ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO3−/H+ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism. 相似文献
13.
Lanthanide DOTA-tetraglycinate (LnDOTA-(gly)4
−) complexes contain four magnetically equivalent amide protons that exchange with protons of bulk water. The rate of this base catalyzed exchange process has been measured using chemical exchange saturation transfer (CEST) NMR techniques as a function of solution pH for various paramagnetic LnDOTA-(gly)4
− complexes to evaluate the effects of lanthanide ion size on this process. Complexes with Tb(III), Dy(III), Tm(III) and Yb(III) were chosen because these ions induce large hyperfine shifts in all ligand protons, including the exchanging amide protons. The magnitude of the amide proton CEST exchange signal differed for the four paramagnetic complexes in order, Yb>Tm>Tb>Dy. Although the Dy(III) complex showed the largest hyperfine shift as expected, the combination of favorable chemical shift and amide proton CEST linewidth in the Tm(III) complex was deemed most favorable for future in vivo applications where tissue magnetization effects can interfere. TmDOTA-(gly)4
− at various concentrations was encapsulated in the core interior of liposomes to yield lipoCEST particles for molecular imaging. The resulting nanoparticles showed less than 1% leakage of the agent from the interior over a range of temperatures and pH. The pH versus amide proton CEST curves differed for the free versus encapsulated agents over the acidic pH regions, consistent with a lower proton permeability across the liposomal bilayer for the encapsulated agent. Nevertheless, the resulting lipoCEST nanoparticles amplify the CEST sensitivity by a factor of ∼104 compared to the free, un-encapsulated agent. Such pH sensitive nano-probes could prove useful for pH mapping of liposomes targeted to tumors. 相似文献
14.
The dynamics of amyloid fibrils, including their formation and dissociation, could be of vital importance in life. We studied the kinetics of dissociation of the amyloid fibrils from wild-type hen lysozyme at 25°C in vitro as a function of pressure using Trp fluorescence as a probe. Upon 100-fold dilution of 8 mg ml−1 fibril solution in 80 mM NaCl, pH 2.2, no immediate change occurred in Trp fluorescence, but at pressures of 50–450 MPa the fluorescence intensity decreased rapidly with time (kobs = 0.00193 min−1 at 0.1 MPa, 0.0348 min−1 at 400 MPa). This phenomenon is attributable to the pressure-accelerated dissociation of amyloid fibrils into monomeric hen lysozyme. From the pressure dependence of the rates, which reaches a plateau at ∼450 MPa, we determined the activation volume ΔV0‡ = −32.9 ± 1.7 ml mol(monomer)−1 and the activation compressibility Δκ‡ = −0.0075 ± 0.0006 ml mol(monomer)−1 bar−1 for the dissociation reaction. The negative ΔV0‡ and Δκ‡ values are consistent with the notion that the amyloid fibril from wild-type hen lysozyme is in a high-volume and high-compressibility state, and the transition state for dissociation is coupled with a partial hydration of the fibril. 相似文献
15.
Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (∼5). Hydrogen production by biocathodes poised at −600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ∼5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ∼6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm2 at −765 mV (0.065 mA/cm2 sterile control at −800 mV) by the Acetobacterium-dominated community. Supplying −800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m3/day hydrogen (≈2.6 gallons gasoline equivalent), 0.7 kg/m3/day formate, and 3.1 kg/m3/day acetate ( = 4.7 kg CO2 captured). 相似文献
16.
Michael G. Duncan Michael D. Williams Bruce E. Bowler 《Protein science : a publication of the Protein Society》2009,18(6):1155-1164
Evolutionary conservation of substructure architecture between yeast iso-1-cytochrome c and the well-characterized horse cytochrome c is studied with limited proteolysis, the alkaline conformational transition and global unfolding with guanidine-HCl. Mass spectral analysis of limited proteolysis cleavage products for iso-1-cytochrome c show that its least stable substructure is the same as horse cytochrome c. The limited proteolysis data yield a free energy of 3.8 ± 0.4 kcal mol−1 to unfold the least stable substructure compared with 5.05 ± 0.30 kcal mol−1 for global unfolding of iso-1-cytochrome c. Thus, substructure stabilities of iso-1-cytochrome c span only ∼1.2 kcal mol−1 compared with ∼8 kcal mol−1 for horse cytochrome c. Consistent with the less cooperative folding thus expected for the horse protein, the guanidine-HCl m-values are ∼3 kcal mol−1M−1 versus ∼4.5 kcal mol−1M−1 for horse versus yeast cytochrome c. The tight free energy spacing of the yeast cytochrome c substructures suggests that its folding has more branch points than for horse cytochrome c. Studies on a variant of iso-1-cytochrome c with an H26N mutation indicate that the least and most stable substructures unfold sequentially and the two least stable substructures unfold independently as for horse cytochrome c. Thus, important aspects of the substructure architecture of horse cytochrome c, albeit compressed energetically, are preserved evolutionally in yeast iso-1-cytochrome c. 相似文献
17.
Herbeck JT Gottlieb GS Li X Hu Z Detels R Phair J Rinaldo C Jacobson LP Margolick JB Mullins JI 《PloS one》2008,3(2):e1525
Background
Several long-term cohort studies and in-vitro fitness assays have resulted in inconsistent reports on changes in HIV-1 virulence, including reports of decreasing, stable, and increasing virulence over the course of the AIDS pandemic. We tested the hypothesis of changing HIV-1 virulence by examining trends in prognostic clinical markers of disease progression from 1984 to 2005 among nearly 400 antiretroviral-naïve participants in the United States Multicenter AIDS Cohort Study (MACS), a longitudinal study of HIV infection in men who have sex with men (MSM).Methodology/Principal Findings
Because clinical AIDS endpoints could not be used (due to antiretroviral therapies and prophylaxis), three prognostic markers of disease progression were used as proxies for HIV-1 virulence: plasma viral RNA load and CD4+ T cell count at “set point” (between ∼9 and ∼15 months after seroconversion), and rate of CD4 cell decline within three years after seroconversion. We performed multivariate analyses of the association between these markers and seroconversion year, with covariates including MACS site, race/ethnic group, seroconversion age, and CCR5Δ32 status. No statistically significant association was found between year of seroconversion and “set point” plasma viral load (at ∼9 months after seroconversion: slope = −0.004 log10 copies/mL/year, p = 0.76; at ∼15 months: slope = −0.005 log10 copies/mL/year, p = 0.71), CD4 cell count after seroconversion (at ∼9 months: slope = −0.112 cells/µL/year, p = 0.22; at ∼15 months: slope = −0.047 cells/µL/year, p = 0.64), or rate of CD4 cell decline over the first three years after seroconversion (slope = −0.010 cells/ul/yr2, p = 0.88).Conclusions/Significance
The lack of significant trends from 1984 to 2005 in these prognostic markers of HIV disease progression suggests no major change in HIV-1 virulence over the AIDS pandemic in MSM in the US. 相似文献18.
The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2+/+, ACAT2+/−, and ACAT2−/− mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [3H]sitosterol and [14C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2−/− mice was 16% compared with 46–47% in ACAT2+/+ and ACAT2+/− mice. Chylomicrons from ACAT2+/+ and ACAT2+/− mice carried ∼80% of total sterol mass as CE, whereas ACAT2−/− chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to ∼1% in ACAT2−/− mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing ∼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption. 相似文献
19.
Thermodynamic examination of the pyrophosphate sensor helix in the thiamine pyrophosphate riboswitch
Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 × 2 bulged RNA and one 2 × 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 × 2 bulge (C65 and G66). The RNA WT construct was among the most stable (ΔG°37 = −7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A•G mismatch of the bulge decreases the stability of the construct ∼0.5–1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA–Mg2+ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/mol relative to 1 M salt. Formation of a single A+•C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+•C mismatch base pairs together contribute about 2 kcal/mol. 相似文献
20.
R Pardoux S Sauge-Merle D Lemaire P Delangle L Guilloreau JM Adriano C Berthomieu 《PloS one》2012,7(8):e41922
To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. 相似文献