Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC) and under high pressure conditions at low temperature (3.75 kbar, -13ºC). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Thermodynamic evaluation and modeling of proton and water exchange associated with benzamidine and berenil binding to ß-trypsin

Serine-proteases are involved in vital processes in virtually all species. They are important targets for researchers studying the relationships between protein structure and activity, for the rational design of new pharmaceuticals. Trypsin was used as a model to assess a possible differential contribution of hydration water to the binding of two synthetic inhibitors. Thermodynamic parameters for the association of bovine ß-trypsin (homogeneous material, observed 23,294.4 ± 0.2 Da, theoretical 23,292.5 Da) with the inhibitors benzamidine and berenil at pH 8.0, 25ºC and with 25 mM CaCl2, were determined using isothermal titration calorimetry and the osmotic stress method. The association constant for berenil was about 12 times higher compared to the one for benzamidine (binding constants are K = 596,599 ± 25,057 and 49,513 ± 2,732 M-1, respectively; the number of binding sites is the same for both ligands, N = 0.99 ± 0.05). Apparently the driving force responsible for this large difference of affinity is not due to hydrophobic interactions because the variation in heat capacity (DCp), a characteristic signature of these interactions, was similar in both systems tested (-464.7 ± 23.9 and -477.1 ± 86.8 J K-1 mol-1 for berenil and benzamidine, respectively). The results also indicated that the enzyme has a net gain of about 21 water molecules regardless of the inhibitor tested. It was shown that the difference in affinity could be due to a larger number of interactions between berenil and the enzyme based on computational modeling. The data support the view that pharmaceuticals derived from benzamidine that enable hydrogen bond formation outside the catalytic binding pocket of ß-trypsin may result in more effective inhibitors.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

Effect of pentoxifylline on lung inflammation and gas exchange in a sepsis-induced acute lung injury model

Experimental models of sepsis-induced pulmonary alterations are important for the study of pathogenesis and for potential intervention therapies. The objective of the present study was to characterize lung dysfunction (low PaO2 and high PaCO2, and increased cellular infiltration, protein extravasation, and malondialdehyde (MDA) production assessed in bronchoalveolar lavage) in a sepsis model consisting of intraperitoneal (ip) injection of Escherichia coli and the protective effects of pentoxifylline (PTX). Male Wistar rats (weighing between 270 and 350 g) were injected ip with 10(7) or 10(9) CFU/100 g body weight or saline and samples were collected 2, 6, 12, and 24 h later (N = 5 each group). PaO2, PaCO2 and pH were measured in blood, and cellular influx, protein extravasation and MDA concentration were measured in bronchoalveolar lavage. In a second set of experiments either PTX or saline was administered 1 h prior to E. coli ip injection (N = 5 each group) and the animals were observed for 6 h. Injection of 10(7) or 10(9) CFU/100 g body weight of E. coli induced acidosis, hypoxemia, and hypercapnia. An increased (P < 0.05) cell influx was observed in bronchoalveolar lavage, with a predominance of neutrophils. Total protein and MDA concentrations were also higher (P < 0.05) in the septic groups compared to control. A higher tumor necrosis factor-alpha (P < 0.05) concentration was also found in these animals. Changes in all parameters were more pronounced with the higher bacterial inoculum. PTX administered prior to sepsis reduced (P < 0.05) most functional alterations. These data show that an E. coli ip inoculum is a good model for the induction of lung dysfunction in sepsis, and suitable for studies of therapeutic interventions.

Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

Streptococcus mutans GlnK protein: an unusual PII family member

Streptococcus mutans is a Gram-positive bacterium present in the oral cavity, and is considered to be one of the leading causes of dental caries. S. mutans has a glnK gene, which codes for a PII-like protein that is possibly involved in the integration of carbon, nitrogen and energy metabolism in several organisms. To characterize the GlnK protein of S. mutans, the glnK gene was amplified by PCR, and cloned into the expression vectors pET29a(+) and pET28b(+). The native GlnK-Sm was purified by anion exchange (Q-Sepharose) and affinity (Hi-Trap Heparin) chromatography. The GlnK-His-Sm protein was purified using a Hi-Trap Chelating-Ni2+ column. The molecular mass of the GlnK-His-Sm proteins was 85 kDa as determined by gel filtration, indicating that this protein is a hexamer in solution. The GlnK-His-Sm protein is not uridylylated by the Escherichia coli GlnD protein. The activities of the GlnK-Sm and GlnK-His-Sm proteins were assayed in E. coli constitutively expressing the Klebsiella pneumoniae nifLA operon. In K. pneumoniae, NifL inhibits NifA activity in the presence of high ammonium levels and the GlnK protein is required to reduce the inhibition of NifL in the presence of low ammonium levels. The GlnK-Sm protein was unable to reduce NifL inhibition of NifA protein. Surprisingly, the GlnK-His-Sm protein was able to partially reduce NifL inhibition of the NifA protein under nitrogen-limiting conditions, in a manner similar to the GlnK protein of E. coli. These results suggested that S. mutans GlnK is functionally different from E. coli PII proteins.

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 ± 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.

Destabilizing Mutations Alter the Hydrogen Exchange Mechanism in Ribonuclease A

The effect of strongly destabilizing mutations, I106A and V108G of Ribonuclease A (RNase A), on its structure and stability has been determined by NMR. The solution structures of these variants are essentially equivalent to RNase A. The exchange rates of the most protected amide protons in RNase A (35ºC), the I106A variant (35ºC), and the V108G variant (10ºC) yield stability values of 9.9, 6.0, and 6.8 kcal/mol, respectively, when analyzed assuming an EX2 exchange mechanism. Thus, the destabilization induced by these mutations is propagated throughout the protein. Simulation of RNase A hydrogen exchange indicates that the most protected protons in RNase A and the V108G variant exchange via the EX2 regime, whereas those of I106A exchange through a mixed EX1 1 EX2 process. It is striking that a single point mutation can alter the overall exchange mechanism. Thus, destabilizing mutations joins high temperatures, high pH and the presence of denaturating agents as a factor that induces EX1 exchange in proteins. The calculations also indicate a shift from the EX2 to the EX1 mechanism for less protected groups within the same protein. This should be borne in mind when interpreting exchange data as a measure of local stability in less protected regions

AtTRB1, a telomeric DNA-binding protein from Arabidopsis, is concentrated in the nucleolus and shows highly dynamic association with chromatin

AtTRB1, 2 and 3 are members of the SMH (single Myb histone) protein family, which comprises double-stranded DNA-binding proteins that are specific to higher plants. They are structurally conserved, containing a Myb domain at the N-terminus, a central H1/H5-like domain and a C-terminally located coiled-coil domain. AtTRB1, 2 and 3 interact through their Myb domain specifically with telomeric double-stranded DNA in vitro, while the central H1/H5-like domain interacts non-specifically with DNA sequences and mediates protein–protein interactions. Here we show that AtTRB1, 2 and 3 preferentially localize to the nucleus and nucleolus during interphase. Both the central H1/H5-like domain and the Myb domain from AtTRB1 can direct a GFP fusion protein to the nucleus and nucleolus. AtTRB1–GFP localization is cell cycle-regulated, as the level of nuclear-associated GFP diminishes during mitotic entry and GFP progressively re-associates with chromatin during anaphase/telophase. Using fluorescence recovery after photobleaching and fluorescence loss in photobleaching, we determined the dynamics of AtTRB1 interactions in vivo. The results reveal that AtTRB1 interaction with chromatin is regulated at two levels at least, one of which is coupled with cell-cycle progression, with the other involving rapid exchange.

Isolation of lactoperoxidase using different cation exchange resins by batch and column procedures

Lactoperoxidase (LP) was isolated from whey protein by cation-exchange using Carboxymethyl resin (CM-25C) and Sulphopropyl Toyopearl resin (SP-650C). Both batch and column procedures were employed and the adsorption capacities and extraction efficiencies were compared. The resin bed volume to whey volume ratios were 0.96:1.0 for CM-25C and ≤ 0.64:1.0 for SP-650 indicating higher adsorption capacity of SP-650 compared to CM-25C. The effluent LP activity depended on both the enzyme activity in the whey and the amount of whey loaded on the column within the saturation limits of the resin. The percentage recovery was high below the saturation point and fell off rapidly with over-saturation. While effective recovery was achieved with column extraction procedures, the recovery was poor in batch procedures. The whey-resin contact time had little impact on the enzyme adsorption. SDS PAGE and HPLC analyses were also carried out, the purity was examined and the proteins characterised in terms of molecular weights. Reversed phase HPLC provided clear distinction of the LP and lactoferrin (LF) peaks. The enzyme purity was higher in column effluents compared to batch effluents, judged on the basis of the clarity of the gel bands and the resolved peaks in HPLC chromatograms.

Extracellular disulfide exchange and the regulation of cellular function

An emerging concept is that disulfide bonds can act as a dynamic scaffold to present mature proteins in different conformational and functional states on the cell surface. Two examples are the conversion of the receptor, integrin a alpha(IIb)beta(3), from a low affinity to a high affinity state, and the interaction of CD4 receptor with the HIV-1 envelope glycoprotein gp120 to promote virus-cell fusion. In both of these cases there is a remodeling of the protein disulfide bonding pattern. The formation and rearrangement of disulfide bonds is modulated by a family of enzymes known as the thiol isomerases, which include protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72. While these enzymes were reported originally to be restricted in location to the endoplasmic reticulum, in some cells thiol isomerases are found on the cell surface. This may indicate a wider role for these enzymes in cell function. In platelets it has been shown that reagents that react with cell surface sulfhydryl groups are capable of blocking a number of functional responses, including integrin-mediated aggregation, adhesion, and granule secretion. Furthermore, the use of function blocking antibodies to either PDI or ERp5 causes inhibition of these functional responses. This review summarizes current knowledge of the extracellular regulation of disulfide exchange and the implications of this in the regulation of cell function.

Selective separation of the major whey proteins using ion exchange membranes

Synthetic microporous membranes with functional groups covalently attached were used to selectively separate beta-lactoglobulin, BSA, and alpha-lactalbumin from rennet whey. The selectivity and membrane performance of strong (quaternary ammonium) and weak (diethylamine) ion-exchange membranes were studied using breakthrough curves, measurement of binding capacity, and protein composition of the elution fraction to determine the binding behavior of each membrane. When the weak and strong anion exchange membranes were saturated with whey, they were both selective primarily for beta-lactoglobulin with less than 1% of the eluate consisting of alpha-lactalbumin or BSA. The binding capacity of a pure alpha-lactoglobulin solution was in excess of 1.5 mg/cm(2) of membrane. This binding capacity was reduced to approximately 1.2 mg/cm(2) when using a rennet whey solution (pH 6.4). This reduction in protein binding capacity can be explained by both the competitive effects of other whey proteins and the effect of ions present in whey. Using binary solution breakthrough curves and rennet whey breakthrough curves, it was shown that alpha-lactalbumin and BSA were displaced from the strong and weak anion exchange membranes by beta-lactoglobulin. Finally, the effect of ionic strength on the binding capacity of individual proteins for each membrane was determined by comparing model protein solutions in milk permeate (pH 6.4) and a 10 mM sodium phosphate buffer (pH 6.4). Binding capacities of beta-lactoglobulin, alpha-lactalbumin, and BSA in milk permeate were reduced by as much as 50%. This reduction in capacity coupled with the low binding capacity of current ion exchange membranes are 2 serious considerations for selectively separating complex and concentrated protein solutions.

Expression of the Rap1 guanine nucleotide exchange factor, MR-GEF, is altered in individuals with bipolar disorder

In the rodent forebrain GABAergic neurons are generated from progenitor cells that express the transcription factors Dlx1 and Dlx2. The Rap-1 guanine nucleotide exchange factor, MR-GEF, is turned on by many of these developing GABAergic neurons. Expression of both Dlx1/2 and MR-GEF is retained in both adult mouse and human forebrain where, in human, decreased Dlx1 expression has been associated with psychosis. Using in situ hybridization studies we show that MR-GEF expression is significantly down-regulated in the forebrain of Dlx1/2 double mutant mice suggesting that MR-GEF and Dlx1/2 form part of a common signalling pathway during GABAergic neuronal development. We therefore compared MR-GEF expression by in situ hybridization in individuals with major psychiatric disorders (schizophrenia, bipolar disorder, major depression) and control individuals. We observed a significant positive correlation between layers II and IV of the dorso-lateral prefrontal cortex (DLPFC) in the percentage of MR-GEF expressing neurons in individuals with bipolar disorder, but not in individuals with schizophrenia, major depressive disorder or in controls. Since MR-GEF encodes a Rap1 GEF able to activate G-protein signalling, we suggest that changes in MR-GEF expression could potentially influence neurotransmission.