Correlation of pause sites in MDV-1 RNA replication with kinetic refolding of the growing chain. A Monte Carlo simulation of the Markov process

We consider a template-instructed MDV-1 RNA replication catalyzed by Q beta replicase. The relaxation of the effective binding of the growing chain to the enzyme is simulated, considering a Markov process where refolding events in the chain are concomitant with chain elongation events. When realistic transition probabilities are considered, it becomes evident that the pause sites in replication are due to relaxation of the binding by folding of the growing chain.     

Proline can have opposite effects on fast and slow protein folding phases

Proline isomerization is well known to cause additional slow phases during protein refolding. We address a new question: does the presence of prolines significantly affect the very fast kinetics that lead to the formation of folding intermediates? We examined both the very slow (10-100 min) and very fast (4 micro s-2.5 ms) folding kinetics of the two-domain enzyme yeast phosphoglycerate kinase by temperature-jump relaxation. Phosphoglycerate kinase contains a conserved cis-proline in position 204, in addition to several trans-prolines. Native cis-prolines have the largest effect on folding kinetics because the unfolded state favors trans isomerization, so we compared the kinetics of a P204H mutant with the wild-type as a proof of principle. The presence of Pro-204 causes an additional slow phase upon refolding from the cold denatured state, as reported in the literature. Contrary to this, the fast folding events are sped up in the presence of the cis-proline, probably by restriction of the conformational space accessible to the molecule. The wild-type and Pro204His mutant would be excellent models for off-lattice simulations probing the effects of conformational restriction on short timescales.     

Characterization of crystals of the thermostable DNA polymerase I from thermus aquaticus

Thermus aquaticus DNA polymerase I is an enzyme that is of both physiological and technological interest. It carries out template-directed polymerization of DNA at elevated temperatures and is widely used in polymerase chain reaction (PCR). We have obtained crystals of the enzyme that diffracts X-rays to at least 3.0 Å resolution in a cubic space group. Determination of the three-dimensional structure of the native enzyme along with those of relevant complexes will greatly enhance our knowledge of molecular events involved in DNA replication, will permit improvements in PCR, and will add to our knowledge of the structural bases of thermo stability in proteins. © 1995 Wiley-Liss, Inc.     

Affinity precipitation and macroaffinity ligand facilitated three-phase partitioning for refolding and simultaneous purification of urea-denatured pectinase

Protein refolding is an integral step in the recovery of protein activity from inclusion bodies. It is shown that affinity precipitation and macroaffinity ligand facilitated three-phase partitioning (MLFTPP) led to refolding of urea-denatured pectinase present in a commercial preparation, with simultaneous purification. Affinity precipitation consists of precipitation of the desired enzyme by complexing it with a suitable stimulus-sensitive macroaffinity ligand. This ligand in this case was alginate/esterified alginate. The complex of the polymer-pectinase could be precipitated by adding calcium ions. In MLFTPP (carried out by adding tertiary butanol and ammonium sulfate to the aqueous solution of crude enzyme and the polymer), the polymer or its complex with the enzyme form an interfacial precipitate between tert-butyl alcohol phase and aqueous phase. It is believed that in both processes, while molecular recognition of alginate/esterified alginate to pectinase facilitates their selective binding to the enzyme, the correct refolding is facilitated by preventing molecular aggregation of unfolded enzyme molecules. Three-phase partitioning with esterified alginate as the macroaffinity ligand gave 100% recovery with 4-fold purification. Affinity precipitation with 1% alginate gave 52% yield with 18-fold purification. On the other hand, use of 0.5% esterified alginate gave only 7-fold purification but with 75% recovery of activity.     

Molecular characterization of a recombinant replication protein (Rep) from the Antarctic bacterium Psychrobacter sp. TA144

The Antarctic Gram-negative bacterium Psychrobacter sp. TA144 contains two small cryptic plasmids, called pTAUp and pTADw. pTAUp encodes a replication enzyme (PsyRep) whose activity is responsible for plasmid replication via the rolling circle replication pathway. Several attempts to produce the wild-type biologically active PsyRep in Escherichia coli failed, possibly due to auto-regulation of the protein population. However, the serendipitous occurrence of a frameshift mutation during the preparation of an expression vector resulted in the over-production of a recombinant protein, changed in its last 14 amino acid residues (PsyRep*), that precipitates in insoluble form. The purification of PsyRep* inclusion bodies and the successful refolding of the cold adapted enzyme allowed us to carry out its functional characterization. The mutated protein still displays a double stranded DNA nicking activity, while the change at the C-terminus impairs the enzyme specificity for the pTAUp cognate Ori+ sequence.     

Thermal unfolding of nucleoside hydrolases from the hyperthermophilic archaeon Sulfolobus solfataricus: role of disulfide bonds

Nucleoside hydrolases are metalloproteins that hydrolyze the N-glycosidic bond of β-ribonucleosides, forming the free purine/pyrimidine base and ribose. We report the stability of the two hyperthermophilic enzymes Sulfolobus solfataricus pyrimidine-specific nucleoside hydrolase (SsCU-NH) and Sulfolobus solfataricus purine-specific inosineadenosine- guanosine nucleoside hydrolase (SsIAG-NH) against the denaturing action of temperature and guanidine hydrochloride by means of circular dichroism and fluorescence spectroscopy. The guanidine hydrochloride-induced unfolding is reversible for both enzymes as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. The evidence that the denaturation of SsIAG-NH carried out in the presence of reducing agents proved to be reversible indicates that the presence of disulfide bonds interferes with the refolding process of this enzyme. Both enzymes are highly thermostable and no thermal unfolding transition can be obtained up to 108°C. SsIAG-NH is thermally denatured under reducing conditions (T(m)=93°C) demonstrating the contribution of disulfide bridges to enzyme thermostability.     

The role of propeptide in the refolding of human group IB phospholipase A2

Phospholipase A2 (PLA2, EC 3.1.1.4) catalyzes the hy-drolysis of the 2-acyl ester bond of sn-3-phosphoglycerides,producing a free fatty acid and a lysophospholipid [1].Mammalian secretory phospholipase A2 is tentatively sug-gested to be involved in the in…     

Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli.

Cobra venom (Naja naja naja) phospholipase A2 (PLA2) contains 14 cysteines in the form of 7 disulfide bonds amongst its 119 amino acids. A gene encoding the PLA2 was synthesized and inserted into a bacterial expression vector containing the phage lambda pL promoter. In order to obtain protein without the initiating methionine at the N-terminus, a Factor Xa site was engineered upstream from the PLA2 gene. Upon heat-induction of the cells transformed with the expression plasmid, the protein is produced as insoluble inclusion bodies. The enzyme was partially purified by washing the inclusion bodies with Triton X-100 and urea. The expressed protein was first denatured with 8 M guanidine-HCl and 10 mM DTT. After digestion with Factor Xa, formation of disulfide bonds and refolding into the fully active form was carried out in the presence of cysteine and Ca2+. The renatured recombinant protein was purified by Affi-gel blue column chromatography. The purified recombinant enzyme had the same specific activity as the native enzyme when assayed on a variety of substrates and cross-reacted with antisera prepared against the native enzyme. This is the first report of the expression of a recombinant PLA2 from any venom.     

Coexpression, purification and characterization of the E and S subunits of coenzyme B(12) and B(6) dependent Clostridium sticklandii D-ornithine aminomutase in Escherichia coli.

D-Ornithine aminomutase from Clostridium sticklandii comprises two strongly associating subunits, OraS and OraE, with molecular masses of 12,800 and 82,900 Da. Previous studies have shown that in Escherichia coli the recombinant OraS protein is synthesized in the soluble form and OraE as inclusion bodies. Refolding experiments also indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate (PLP) or adenosylcobalamin (AdoCbl) play important roles in the refolding process. In this study, the DNA fragment containing both genes was cloned into the same expression vector and coexpression of the oraE and oraS genes was carried out in E. coli. The solubility of the coexpressed OraS and OraE increases with decreasing isopropyl thio-beta-D-galactoside induction temperature. Among substrate analogues tested, only 2,4-diamino-n-butyric acid displays competitive inhibition of the enzyme with a K(i) of 96 +/- 14 microm. Lys629 is responsible for the binding of PLP. The apparent K(d) for coenzyme B(6) binding to d-ornithine aminomutase is 224 +/- 41 nm as measured by equilibrium dialysis. The mutant protein, OraSE-K629M, is successfully expressed. It is catalytically inactive and unable to bind PLP. Because no coenzyme is involved in protein folding during in vivo translation of OraSE-K629M in E. coli, in vitro refolding of the enzyme employs a different folding mechanism. In both cases, the association of the S and E subunit is important for D-ornithine aminomutase to maintain an active conformation.     

Native-like intermediate on the folding pathway of Escherichia coli succinyl-CoA synthetase

The transition between the native and denatured states of the tetrameric succinyl-CoA synthetase from Escherichia coli has been investigated by circular dichroism, fluorescence spectroscopy, cross-linking by glutaraldehyde and activity measurements. At pH 7.4 and 25 degrees C, both denaturation of succinyl-CoA synthetase by guanidine hydrochloride and refolding of the denatured enzyme have been characterized as reversible reactions. In the presence of its substrate ATP, the denatured enzyme could be successfully reconstituted into the active enzyme with a yield of 71-100%. Kinetically, reacquisition of secondary structure by the denatured enzyme was rapid and occurred within 1 min after refolding was initiated. On the other hand, its reactivation was a slow process which continued up to 25 min before 90% of the native activity could be restored. Both secondary and quaternary structures of the enzyme, reconstituted in the absence of ATP, were indistinguishable from those of the native enzyme but the renatured protein was catalytically inactive. This observation indicates the presence of catalytically inactive tetramer as an intermediate in the reconstitution process. The reconstituted protein could be reactivated by ATP even 10 min after the reacquisition of the native secondary structure by the refolding protein. However, reactivation of the protein by ATP 60 min after the regain of secondary structure was significantly less, suggesting that rapid refolding and reassociation of the monomers into a native-like tetramer and reactivation of the tetramer are sequential events; the latter involving slow and small conformational rearrangements in the refolded enzyme that are likely to be associated with phosphorylation.     

Cell cycle arrest of a Caulobacter crescentus secA mutant.

Cell differentiation is an inherent component of the Caulobacter crescentus cell cycle. The transition of a swarmer cell, with a single polar flagellum, into a sessile stalked cell includes several morphogenetic events. These include the release of the flagellum and pili, the proteolysis of chemotaxis proteins, the biogenesis of the polar stalk, and the initiation of DNA replication. We have isolated a group of temperature-sensitive mutants that are unable to complete this process at the restrictive temperature. We show here that one of these strains has a mutation in a homolog of the Escherichia coli secA gene, whose product is involved in protein translocation at the cell membrane. This C. crescentus secA mutant has allowed the identification of morphogenetic events in the swarmer-to-stalked cell transition that require SecA-dependent protein translocation. Upon shift to the nonpermissive temperature, the mutant secA swarmer cell is able to release the polar flagellum, degrade chemoreceptors, and initiate DNA replication, but it is unable to form a stalk, complete DNA replication, or carry out cell division. At the nonpermissive temperature, the cell cycle blocks prior to the de novo synthesis of flagella and chemotaxis proteins that normally occurs in the predivisional cell. Although interactions between the chromosome and the cytoplasmic membrane are believed to be a functional component of the temporal regulation of DNA replication, the ability of this secA mutant to initiate replication at the nonpermissive temperature suggests that SecA-dependent events are not involved in this process. However, both cell division and stalk formation, which is analogous to a polar division event, require SecA function.     

Stability, refolding and Ca2+ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei.

The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86 +/- 0.29 M for intrinsic fluorescence and 4.90 +/- 0.31 M for far-UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 M guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 M in the first dilution step. Particular attention has been paid to the role of Ca2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca2+ ions with a Kd of 0.080 +/- 0.010 microM and a Hill coefficient H of 1.00 +/- 0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride-induced unfolding and the DeltaGH2OD increased from 6.83 +/- 0.43 to 8.42 +/- 0.55 kcal.mol-1. The refolding of the pullulanase, on the other hand, was not affected by Ca2+.     

An interlocked dimer of the protelomerase TelK distorts DNA structure for the formation of hairpin telomeres

The termini of linear chromosomes are protected by specialized DNA structures known as telomeres that also facilitate the complete replication of DNA ends. The simplest type of telomere is a covalently closed DNA hairpin structure found in linear chromosomes of prokaryotes and viruses. Bidirectional replication of a chromosome with hairpin telomeres produces a catenated circular dimer that is subsequently resolved into unit-length chromosomes by a dedicated DNA cleavage-rejoining enzyme known as a hairpin telomere resolvase (protelomerase). Here we report a crystal structure of the protelomerase TelK from Klebsiella oxytoca phage varphiKO2, in complex with the palindromic target DNA. The structure shows the TelK dimer destabilizes base pairing interactions to promote the refolding of cleaved DNA ends into two hairpin ends. We propose that the hairpinning reaction is made effectively irreversible by a unique protein-induced distortion of the DNA substrate that prevents religation of the cleaved DNA substrate.     

Probing the structural domains and function in vivo of Escherichia coli DNA topoisomerase I by mutagenesis

Insertion and deletion mutagenesis within the gene topA of Escherichia coli encoding DNA topoisomerase I was carried out to test the existence of subdomains in the enzyme and the relationship between the slow-growth topA- phenotype and the known DNA relaxation activity of the enzyme. All mutants that show no detectable DNA relaxation activity in cell extracts fail to complement the temperature-sensitive growth defect of strain AS17 topAam harboring a plasmid-borne temperature-sensitive suppressor tRNA. All mutants that show partial or full levels of DNA relaxation activity in cell extracts (relative to activity in extracts of wild-type cells) can complement this defect. The carboxyl-proximal 25% of the enzyme appears to be in a domain that is dispensable both in terms of the catalytic function of the enzyme and its biological role. Analysis of the mutant enzyme also indicates that the formation of the covalent topoisomerase-DNA complex is correlated with the DNA relaxation activity, which supports the notion that the covalent complex is an obligatory intermediate in the catalysis of DNA topoisomerization.     

Kinetic studies of the refolding of yeast phosphoglycerate kinase: comparison with the isolated engineered domains.

Unfolding and refolding kinetics of yeast phosphoglycerate kinase were studied by following the time-dependent changes of two signals: the ellipticity at 218 nm and 222 nm, and the fluorescence emission at 330 nm (following excitation at 295 nm). The protein is composed of two similar-sized structural domains. Each domain has been produced by recombinant DNA techniques. It has been previously demonstrated that the engineered isolated domains are able to fold into a quasinative structure (Minard, P., et al., 1989b, Protein Eng. 3, 55-60; Missiakas, D., Betton, J.M., Minard, P., & Yon, J.M., 1990, Biochemistry 29, 8683-8689). The behavior of the isolated domains was studied using the same two conformational probes as for the whole enzyme. We found that the refolding kinetics of each domain are multiphasic. In the whole protein, domain folding and pairing appeared to be simultaneous events. However, it was found that some refolding steps occurring during the refolding of the isolated C-domain are masked during the refolding of yeast phosphoglycerate kinase. The N-domain was also found to refold faster when it was isolated than when integrated.     

Structural stability of ribosomes subjected to RNase treatment evidenced by dielectric spectroscopy and differential scanning microcalorimetry

Previous studies from our laboratory demonstrated the existence of at least two levels of structural complexity in E. coli 70S ribosomes. Ribosomal RNA seems to be principally involved in the overall stability of these structures. In this paper we present an investigation of ribosomes subjected to treatment with RNase. The study is based on both differential scanning microcalorimetry and dielectric spectroscopy. In the thermograms obtained on treated ribosomes only the low temperature peak of the two typical denaturation events observed in native ribosomes, is promptly eliminated by the enzyme treatment. Dielectric spectroscopy measurements carried out on the same samples indicate an alteration of the dielectric behavior previously shown to consist of two subsequent relaxation processes. In fact, only the low frequency relaxation is affected by the treatment. The second one, observed at higher frequency, remains unaltered. The same effect on the dielectric parameters is observed if the ribosome particles are heated and then cooled prior to measurement. These results are consistent with the idea that two different structures are present within the ribosome. One is very stable and withstands both temperature and RNase treatment while the second is promptly abolished by both treatments. Data presented here strongly suggest that the RNA domains exposed to the solvent play a fundamental role in the stability of the 3-D structure of the ribosome particle.