1H, 15N and 13C NMR assignments of the 434 repressor fragments 1-63 and 44-63 unfolded in 7 M urea.

An E. coli overexpression system for the N-terminal domain of the 434 repressor with residues 1-63 (434 repressor(1-63)) was constructed and used to produce this polypeptide with uniform 15N-labeling, and with 13C-labeling of the methyl groups of valine and leucine. Using these protein preparations almost complete sequence-specific resonance assignments were obtained for the urea-unfolded form of the 434 repressor(1-63). In addition, the isotope-labeled tryptic peptide, 44-63, was produced by enzymatic cleavage of the recombinant 434 repressor(1-63), and its NMR spectrum was assigned. Corresponding residues in 434 repressor(1-63) and 434 repressor(44-63) in 7 M urea were found to have nearly identical chemical shifts, and in both species similar deviations from 1H random coil shifts were found as previously in 434 repressor(1-69). These indicate the presence of residual non-random structure in the polypeptide segment 50-60. The present NMR assignments, which include stereospecific assignments for the diastereotopic methyl groups of Val and Leu, are the basis for detailed studies of this residual structure in the urea-unfolded form of the 434 repressor.     

The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. I. Evidence for two binding sites with different affinities

F Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe binding to rabbit peritoneal neutrophils and purified membranes were measured at 4 degrees C silicone oil centrifugation assays, and the results were analyzed by the LIGAND computer program, which permits analysis of ligand binding to multiple classes of binding sites. LIGAND analysis of peptide binding to intact neutrophil indicated that both f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe detected two population of binding sites. The apparent Kd values for f Met-Leu-[3H]Phe binding were 1.6 +/- 1.0 X 10(-9) M and 2.2 +/- 0.9 X 10(-8) M, respectively, and 3.1 +/- 0.2 X 10(-9) M and 1.2 +/- 0.6 X 10(-7) M for f Nle-Leu-[3H]Phe. Furthermore, the higher affinity sites detected on whole cells comprised approximately 15 to 30% of the total sites. Two populations of binding sites were also detected on purified neutrophil plasma membranes by both radiolabeled chemotactic peptides. LIGAND analysis of peptide binding to purified membranes yielded apparent Kd values of 5.0 +/- 2.5 X 10(-10) M and 4.8 +/- 0.6 X 10(-8) M for f Met-Leu-[3H]Phe binding, and 4.7 +/- 4.2 X 10(-10) M and 3.0 +/- 1.3 X 10(-8) M for f Nle-Leu-[3H]Phe. The percentage of higher affinity sites detected by f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe on purified membranes were 1 to 5% of the total sites detected. These data are consistent either with the existence of two independent binding sites for formylpeptides on rabbit neutrophils or receptor negative cooperativity.     

Two-dimensional 1H-NMR studies of phospholipase-A2-inhibitor complexes bound to a micellar lipid-water interface

One- and two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2 with substrate analogs bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine. The interactions between the inhibitor and the enzyme were localized by comparison of the two-dimensional NOE spectra recorded for the enzyme-inhibitor complex using both protonated and selectively deuterated inhibitors. These experiments led us to the following conclusions for the phospholipase-A2-micelle complex: (i) the 38-kDa phospholipase A2 complex gives NMR spectra with relatively narrow lines, which is indicative of high mobility of the enzyme; (ii) the residues Ala1, Trp3, Phe63 and Tyr69 located in the interface recognition site, as well as Phe22, Tyr75, Phe106 and Tyr111 are involved in the micelle-binding process; (iii) when present on the micelle, phospholipase A2 is stereospecific for the inhibitor binding; (iv) the inhibitor, (R)-dodecyl-2-aminohexanol-1-phosphoglycol, binds stoichiometrically to phospholipase A2 with high affinity (Kd less than or equal to 10 microM); (v) the inhibitor binds in the active site of the enzyme, which is evidenced by large chemical-shift differences for Phe5, Ile9, Phe22, His48, Tyr52 and Phe106; (vi) the acyl chain of the inhibitor makes hydrophobic contacts (less than 0.4 nm) near Phe5, Ile9, Phe22 and Phe106. Comparison of our results on the enzyme-inhibitor-micelle ternary complex with the crystal structure of the enzyme-inhibitor complex [Thunnissen, M. M. G. M., AB, E., Kalk, K. H., Drenth, J., Dijkstra, B. W., Kuipers, O. P., Dijkman, R., de Haas, G. H. & Verheij, H. M. (1990) Nature 347, 689-691] shows that the mode of inhibitor binding is similar.     

Extracellular hydrolysis of formyl peptides and subsequent uptake of liberated amino acids by alveolar macrophages

The mechanism of accumulation of radioactive label from fNle-Leu-[3H]Phe by guinea pig alveolar macrophages was investigated. The binding of fNle-Leu-[3H]Phe to macrophages reached equilibrium within 5 min at 4 degrees C, but equilibrium could not be achieved at temperatures where fNle-Leu-Phe stimulated superoxide anion production is observed (e.g., 21-23 degrees C). At this temperature a rapid phase of initial binding of fNle-Leu-[3H]Phe to its receptor was followed by continued accumulation of cell-associated radioactivity which was linear and was dependent on the extracellular pH, i.e., the rate increased as the pH was lowered from pH 8 to pH 6. Examination for possible intracellular hydrolysis of fNle-Leu-[3H]Phe revealed the presence of extensive amounts of [3H]phenylalanine, both cell-associated and in the medium. The increases in cell-associated [3H]phenylalanine correlated in time and pH with cell-associated radioactivity that was accumulated after stimulation with fNle-Leu-[3H]Phe. The addition of 1 mM unlabelled phenylalanine blocked the long term accumulation of label from fNle-Leu-[3H]Phe by macrophages. 1 mM phenylalanine had no measureable effect on fNle-Leu-Phe stimulated O2- production, fNle-Leu-[3H]Phe hydrolysis or on fNle-Leu-[3H]Phe binding to its receptor. These results indicated that the long term accumulation of radioactivity by alveolar macrophages was due to extracellular hydrolysis of fNle-Leu-[3H]Phe followed by transport of liberated [3H]phenylalanine into the cells. A high affinity (Km = 3.56 X 10(-8) M) transport system for phenylalanine was measured in alveolar macrophages, which was not stimulated by the addition of fNle-Leu-Phe. The extracellular hydrolysis of fNle-Leu-[3H]Phe could not be attributed to release of macrophage enzymes into the medium. The responsible proteinase appears to be membrane bound and has a Km for the hydrolysis of fNle-Leu-[3H]Phe of 2.6 X 10(-7) M which is similar to the Kd (1.5 X 10(-7) M) for fNle-Leu-Phe binding. Taken together, these data suggest that for the alveolar macrophage: (1) formyl peptides are not internalized by a receptor-mediated process; (2) a surface proteinase can catalyze the hydrolysis of formyl peptides; and (3) [3H]phenylalanine formed by fNle-Leu-[3H]Phe hydrolysis is transported into the interior of the macrophage.     

Folding propensities of synthetic peptide fragments covering the entire sequence of phage 434 Cro protein.

The phage 434 Cro protein, the N-terminal domain of its repressor (R1-69) and that of phage lambda (lambda6-85) constitute a group of small, monomeric, single-domain folding units consisting of five helices with striking structural similarity. The intrinsic helix stabilities in lambda6-85 have been correlated to its rapid folding behavior, and a residual hydrophobic cluster found in R1-69 in 7 M urea has been proposed as a folding initiation site. To understand the early events in the folding of 434 Cro, and for comparison with R1-69 and lambda6-85, we examined the conformational behavior of five peptides covering the entire 434 Cro sequence in water, 40% (by volume) TFE/water, and 7 M urea solutions using CD and NMR. Each peptide corresponds to a helix and adjacent residues as identified in the native 434 Cro NMR and crystal structures. All are soluble and monomeric in the solution conditions examined except for the peptide corresponding to the 434 Cro helix 4, which has low water solubility. Helix formation is observed for the 434 Cro helix 1 and helix 2 peptides in water, for all the peptides in 40% TFE and for none in 7 M urea. NMR data indicate that the helix limits in the peptides are similar to those in the native protein helices. The number of side-chain NOEs in water and TFE correlates with the helix content, and essentially none are observed in 7 M urea for any peptide, except that for helix 5, where a hydrophobic cluster may be present. The low intrinsic folding propensities of the five helices could account for the observed stability and folding behavior of 434 Cro and is, at least qualitatively, in accord with the results of the recently described diffusion-collision model incorporating intrinsic helix propensities.     

Folding kinetics of phage 434 Cro protein

Folding kinetics for phage 434 Cro protein are examined and compared with those reported for lambda(6-85), the N-terminal domain of the repressor of phage lambda. The two proteins have similar all-helical structures consisting of five helices but different stabilities. In contrast to lambda(6-85), sharp and distinct aromatic (1)H NMR signals without exchange broadening characterize the native and urea-denatured 434 Cro forms at equilibrium at 20 degrees C, indicating slow interconversion on the NMR time scale. Stopped-flow fluorescence data using the single 434 Cro tryptophan indicate strongly urea-dependent refolding rates and smaller urea dependencies of the unfolding rates, suggesting a native-like transition state ensemble. Refolding rates are slower and unfolding rates considerably faster at pH 4 than at pH 6. This accounts for the lower stability of 434 Cro at pH 4 and suggests the existence of pH-dependent, possibly salt bridge interactions that are more stabilizing at pH 6. At <2 M urea, decreased folding amplitudes and nonlinear urea dependencies that are apparent at pH 6 indicate deviation from two-state behavior and suggest the formation of an early folding intermediate. The folding behavior of 434 Cro and why it folds 2 orders of magnitude slower than lambda(6-85) are rationalized in terms of the lower intrinsic helix stabilities and putative charge interactions in 434 Cro.     

Refolding and purification of human papillomavirus type 16 E7-lacZ fusion protein expressed in Escherichia coli.

Human papillomavirus (HPV) type 16 E7-lacZ fusion protein was produced in Escherichia coli, extracted as inclusion bodies, refolded with reducing reagents, and subjected to gel filtration. The refolded protein was purified by ion-exchange column chromatography, resulting in a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1H nuclear magnetic resonance spectral changes were observed in the high field methyl region in the presence of Zn2+ ion, suggesting that the refolded form of the fusion protein is possibly renaturated into the putative zinc finger motif (C. Edmond and K. H. Vousden, 1989, J. Virol. 63, 2650-2656) and supporting the data of J. A. Rawls, R. Pusztai, and M. Green (1990, J. Virol. 64, 6121-6129) on zinc binding to E7 protein using radioisotopically labeled zinc ion.     

Association of the radiosensitizers metronidazole and misonidazole (RO-07-0582) with bovine serum albumin. Effect of urea and ionic strength

The stability of association of nitroimidazole radiosensitizers (metronidazole and misonidazole) with bovine serum albumin (BSA) was examined in aqueous solutions by 1H n.m.r. spectroscopy in the presence of urea (0-8M) as denaturant, or high salt concentration (NaCl0-5M). A broadening of n.m.r. lines of the two radiosensitizers observed in the presence of BSA disappeared with increasing urea concentration. An especially large narrowing effect was observed for the lines attributed to the methylene group near to the hydroxyl in the side chain of misonidazole. The results suggest a release of both radiosensitizers from their binding sites on unfolding by urea of the polypeptide chain of BSA. The increase of ionic strength I caused a monotonic enhancement of broadening by BSA of the metronidazole lines. For misonidazole, the enhancement of broadening was observed at values of I greater than 1, but at low (less than 1 M) concentrations of NaCl the broadening disappeared. Thus, the results obtained in the systems with salt reflect quantitative changes in hydrophobic and hydrogen-bonded contributions to binding of aliphatic moieties of radiosensitizers to BSA.     

NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning,sequence determination and overexpression in Escherichia coli

A gene encoding NADP-dependent Ds-threo-isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a lambda EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45837 Da molecular mass. This sequence is highly similar to previously sequenced isocitrate dehydrogenases. In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP-dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved. We have developed methods for the expression in Escherichia coli and purification of the enzyme from H. volcanii. This expression was carried out in E. coli as inclusion bodies using the cytoplasmic expression vector pET3a. The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl(2) and 3 M NaCl. Maximal activity was obtained after several hours incubation at room temperature.     

Effects of salts on the function and conformational stability of shikimate kinase

The unfolding of shikimate kinase (SK) from Erwinia chrysanthemi by urea and its subsequent refolding on dilution of the denaturing agent has been studied in detail [Eur. J. Biochem. 269 (2002) 2124]. Comparison of the effects of urea on the enzyme with those of guanidinium chloride (GdmCl) and NaCl indicated that chloride ions significantly weakened the binding of shikimate. This finding prompted a more detailed examination of the effects of salts on the structure, function and stability of the enzyme; the effects of NaCl and Na(2)SO(4) were investigated in detail. These salts have very small effects on the secondary structure as judged by far UV CD circular dichroism (CD), although the near UV CD spectra suggest that some limited changes in the environment of aromatic amino acids may occur. Both salts inhibit SK activity and analysis of the kinetic and substrate binding parameters point to a complex mechanism for the inhibition. Inclusion of salts leads to a marked stabilisation against unfolding of the enzyme by urea. When the enzyme is unfolded by incubation in 4 M urea, addition of NaCl or Na(2)SO(4) leads to a relatively slow refolding of the enzyme as judged by the regain of native-like CD and fluorescence. In addition, the refolded enzyme can bind shikimate, though more weakly than the native enzyme. However, the refolded enzyme does not appear to be capable of binding nucleotides, nor does it possess detectable catalytic activity. The refolding process brought about by addition of salt in the presence of 4 M urea is not associated with any change in the fluorescence of the probe 8-anilino-1-naphthalenesulfonic acid (ANS), indicating that an intermediate formed by hydrophobic collapse is unlikely to be significantly populated. The results point to both specific and general effects of salts on SK. These are discussed in the light of the structural information available on the enzyme.     

Structure of phenylalanine-accepting transfer ribonucleic acid and of its environment in aqueous solvents with different salts

Yeast tRNA(Phe) was studied in different salt-containing solvents by UV absorbance and small-angle neutron scattering (SANS). This extends results obtained previously in NaCl and KCl solutions [Li, Z.-Q., Giegé, R., Jacrot, B., Oberthür, R., Thierry, J. C., & Zaccai, G. (1983) Biochemistry 22, 4380-4388]. As expected, at low concentrations of all salts studied, the tRNA molecule is unfolded. The importance of specific counterion interactions and the flexibility of the macromolecule are emphasized by the observation that it cannot take up its folded structure in N(CH3)4Cl solvents, even when that salt concentration is increased to 1 M, in the absence of Mg ions. In CsCl solvents, on the other hand, the folded conformation is obtained in salt concentrations above about 0.2 M, similar to NaCl or KCl. By a comparison of SANS results in CsCl H2O and CsCl 2H2O solvents with the data from NaCl and KCl solvents, thermodynamic and structural parameters were derived for the solvated macromolecule. All the data are accounted for, quantitatively, by a model for the particle in NaCl, KCl, or CsCl solution made up of tRNA76-, closely associated with 76 positive hydrated counterions, surrounded by an aqueous solvent layer that excludes salt (and, therefore, of density different from that of bulk solvent). The mass of water in that layer depends on salt concentration, and the values found are consistent with those predicted by the Donnan effect.     

Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies

The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21(DE3) cells. The insoluble aggregate was dissolved in 8M urea and, following extraction with Triton X-100, the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2M NaCl. After two buffer changes, chromatography on Blue Sepharose was used as a final step in the purification procedure. Approximately 54mg active protein was recovered from a 1L culture and the refolded enzyme had a specific activity of 75U/mg. The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK). The K(m) values of ATP and ADP were also similar to those of HMCK, while the K(m) values for both phosphocreatine and creatine were approximately 5-10-fold higher. The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1mg/L culture, enzyme with a specific activity of ca. 5U/mg was obtained.     

Involvement of cysteine residues and domain interactions in the reversible unfolding of lipoxygenase-1

Urea-induced unfolding of lipoxygenase-1 (LOX1) at pH 7.0 was followed by enzyme activity, spectroscopic measurements, and limited proteolysis experiments. Complete unfolding of LOX1 in 9 M urea in the presence of thiol reducing or thiol modifying reagents was observed. The aggregation and oxidative reactions prevented the reversible unfolding of the molecule. The loss of enzyme activity was much earlier than the structural loss of the molecule during the course of unfolding, with the midpoint concentrations being 4.5 and 7.0 M for activity and spectroscopic measurements, respectively. The equilibrium unfolding transition could be adequately fitted to a three-state, two-step model (N left arrow over right arrow I left arrow over right arrow U) and the intermediate fraction was maximally populated at 6.3 M urea. The free energy change (DeltaG(H(2)O)) for the unfolding of native (N) to intermediate (I) was 14.2 +/- 0.28 kcal/mol and for the intermediate to the unfolded state (U) was 11.9 +/- 0.12 kcal/mol. The ANS binding measurements as a function of urea concentration indicated that the maximum binding of ANS was in 6.3 M urea due to the exposure of hydrophobic groups; this intermediate showed significant amount of tertiary structure and retained nearly 60% of secondary structure. The limited proteolysis measurements showed that the initiation of unfolding was from the C-terminal domain. Thus, the stable intermediate observed could be the C-terminal domain unfolded with exposed hydrophobic domain-domain interface. Limited proteolysis experiments during refolding process suggested that the intermediate refolded prior to completely unfolded LOX1. These results confirmed the role of cysteine residues and domain-domain interactions in the reversible unfolding of LOX1. This is the first report of the reversible unfolding of a very large monomeric, multi-domain protein, which also has a prosthetic group.     

The Enterococcus faecalis MSCRAMM ACE binds its ligand by the Collagen Hug model

We have determined the crystal structure of the ligand binding segment of the Enterococcus faecalis collagen binding MSCRAMM ACE (microbial surface components recognizing adhesive matrix molecules adhesin of collagen from enterococci). This segment is composed of two subdomains, N(1) and N(2), each adopting an IgG-like fold and forming a putative collagen binding surface at the interface between the two subdomains. This structure is very similar to that recently reported for CNA, the collagen binding MSCRAMM of Staphylococcus aureus, for which a unique ligand binding mechanism called the Collagen Hug was proposed. We suggest that ACE binds collagen by a similar mechanism and present the first biochemical evidence for this binding model. Replacing residues in the putative collagen binding trench of ACE N(2) with Ala residues affected collagen binding. A closed conformation of ACE stabilized by an engineered disulfide bond is unable to bind collagen. Finally, the importance of the residues in the N(2) extension in stabilizing the MSCRAMM-ligand complex is demonstrated by selected point and truncation mutations.     

Labeling and quality control of 188Re-lanreotide.

Lanreotide was labelled with 188Re obtained from 188W/188Re generator, using stannous ion as reducing agent, ascorbic acid as stabilizers and hydroxy ethylidene bisphosphonate (HEDP) as intermediary ligand at different molar ratios, pH and incubation times. Best yields (>95%) were obtained using molar ratios SnF2/lanreotide, ascorbic/lanreotide and HEDP/lanreotide of 40, 12 and 260, respectively, pH 1-2 with an incubation at 100 degrees C for 30 min. Quality control evaluation and stability of the radiolabel compound was done by the following selected methods: chromatography in Whatman 3 MM with MEK and NaCl 0.15 M as solvents, ITLC-SG with ethanol-HCl 0.01N (90:10); reverse phase extraction cartridge (Sep-pak C18, Waters Associated) and RP-HPLC with radiometric and UV detection (220 nm) using MCH-5 n-capp column with linear gradient from 90% H2O (TFA 0.1%): 10% ACN (TFA 0.1%) up to 10% H2O (TFA 0.1%):90% ACN (TFA 0.1%) in 30 min, at flow 1 ml/min. Biodistribution in normal mice showed that 188Re-lanreotide is excreted mainly through the hepatobiliary system: more than 70% I.D. is present in gallbladder and intestines at 2 hr post injection. The stability of the 188Re-peptide bond by cysteine challenge test at 37 degrees C, during 2 and 24 hr of incubation time, reveals that approximately 300 and 100 molar ratio cys/peptide is required to displace 50% of the 188Re from the complex. In vitro stability of 188Re-lanreotide at room temperature (Rt) was demonstrated during 24 hr Future works must be done in order to investigate its binding capacity to somatostatin receptors.     

A recombinant peptide model of the first nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator: comparison of wild-type and delta F508 mutant forms.

A series of recombinant peptides, each including the sequence proposed to be the first nucleotide-binding fold of cystic fibrosis transmembrane conductance regulator (CFTR), has been produced in an attempt to find a model peptide that would autologously fold into a soluble structure with native-like properties. The peptide NBDIF, which contains the 267-amino acid sequence of CFTR from 384 to 650, meets these requirements. The peptide was produced with a high expression bacterial plasmid pRSET, purified from inclusion bodies following solubilization with 6 M guanidine-HCl and refolded from 8 M urea. Competitive displacement of trinitrophenol-ATP by nucleotides reveals binding of ATP and related nucleotides with KDs in the low micromolar range; the KD for ATP gamma S is 1.0 +/- 0.4 microM and for ADP 8.8 +/- 3.1 microM. The native-like character of the model peptide's structure is further supported by the findings that the KD for the ATP analog, 5'-adenylimidodiphosphate, is fourfold lower than the KD for the methylene analog, 5'-adenylmethylenediphosphonate, and that ATP binding slows the trypsin proteolysis of NBDIF. The CD spectra of NBDIF and the parallel peptide containing the most common cystic fibrosis mutation, deletion of Phe 508, are essentially indistinguishable, both spectra indicating 28% alpha-helix and 23% beta-sheet, with insignificant differences in the amounts of beta-turns and random structure. Extensive investigation using multiple conditions with highly purified preparations of the model peptides demonstrates that they do not support ATP hydrolysis. These large recombinant peptides offer practical models for the investigation of the first nucleotide-binding domain of CFTR.     

Partial characterization of a nuclear proteolytic activity from fertilized sea urchin eggs

The nuclei from fertilized sea urchin eggs, obtained 80 min after fertilization, contains a neutral proteolytic activity. Optimal action on casein was observed at pH 7-8 and a Km value of 1.2 mg/ml was determined for this substrate. The proteolytic activity was stimulated 1.5 fold by the addition of 3 M urea and decreased at higher urea concentrations. NaCl and CaCl2 were inhibitory whereas MgCl2 increased the enzyme activity. Isolated histones from sea urchin sperms, and especially histones H1, H2A, H2B and H3, were degraded by the nuclear activity. A partial inhibition of histones degradation was caused by sodium bisulfite and NaCl. The proteolytic activity was found associated to the chromatin of fertilized sea urchin eggs.     

The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases

The extremely halophilic Archae require near-saturating concentrations of salt in the external environment and in their cytoplasm, potassium being the predominant intracellular cation. The proteins of these organisms have evolved to function in concentrations of salt that inactivate or precipitate homologous proteins from non-halophilic species. It has been proposed that haloadaptation is primarily due to clustering of acidic residues on the surface of the protein, and that these clusters bind networks of hydrated ions. The dihydrofolate reductases from Escherichia coli (ecDHFR) and two DHFR isozymes from Haloferax volcanii (hvDHFR1 and hvDHFR2) have been used as a model system to compare the effect of salts on a mesophilic and halophilic enzyme. The KCl-dependence of the activity and substrate affinity was investigated. ecDHFR is largely inactivated above 1M KCl, with no major effect on substrate affinity. hvDHFR1 and hvDHFR2 unfold at KCl concentrations below approximately 0.5M. Above approximately 1M, the KCl dependence of the hvDHFR activities can be attributed to the effect of salt on substrate affinity. The abilities of NaCl, KCl, and CsCl to enhance the stability to urea denaturation were determined, and similar efficacies of stabilization were observed for all three DHFR variants. The DeltaG degrees (H(2)O) values increased linearly with increasing KCl and CsCl concentrations. The increase of DeltaG degrees (H(2)O) as a function of the smallest cation, NaCl, is slightly curved, suggesting a minor stabilization from cation binding or screening of electrostatic repulsion. At their respective physiological ionic strengths, the DHFR variants exhibit similar stabilities. Salts stabilize ecDHFR and the hvDHFRs by a common mechanism, not a halophile-specific mechanism, such as the binding of hydrated salt networks. The primary mode of salt stabilization of the mesophilic and halophilic DHFRs appears to be through preferential hydration and the Hofmeister effect of salt on the activity and entropy of the aqueous solvent. In support of this conclusion, all three DHFRs are similarly stabilized by the non-ionic cosolute, sucrose.     

Prevention of aggregation after refolding by balanced stabilization-destabilization: production of the Arabidopsis thaliana protein APG8a (At4g21980) for NMR structure determination

The gene coding for APG8a (At4g21980), a protein from Arabidopsis thaliana, is involved in the autophagy process. The protein is an interesting candidate for structure determination by NMR spectroscopy. Toward this end, APG8a fused to an N-terminal His-tag has been expressed in Escherichia coli under a T7 expression system, refolded in vitro, and kept soluble by slight destabilization. The expressed protein appeared in both the soluble and the insoluble fractions. The whole-cell lysate was denatured by the addition of guanidinium chloride. The protein was immobilized on nickel-agarose resin and refolded by stepwise decrement of the denaturant. The elution buffer was 20 mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5 M urea, 300 mM NaCl, and 1 M imidazole. After the removal of imidazole by ultrafiltration, the His-tag was cleaved with biotinylated thrombin. The protein product was kept in 20 mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5 M urea, and 300 mM NaCl. The protein was found to aggregate extensively over time if any one of the three ingredients (sodium chloride, urea, or glycerol) was omitted. The yield of the protein was around 20 mg/L Luria-Bertani culture medium. The (1)H-(15)N NMR correlation spectrum of (15)N-labeled APG8a showed the characteristic signature of a folded protein; thus, the solutes appear to have no deleterious effect on the sample. These solution conditions kept the protein soluble and unaggregated for at least 2 days (enough time for NMR data collection). This approach of balanced stabilization-destabilization may offer a general approach for structural investigations of proteins that tend to aggregate.