Alicja Wawrzynów

The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone.

All major classes of protein chaperones, including DnaK (the Hsp70 eukaryotic equivalent) and GroEL (the Hsp60 eukaryotic equivalent) have been found in Escherichia coli. Molecular chaperones enhance the yields of correctly folded polypeptides by preventing aggregation and even by disaggregating certain protein aggregates. Previously, we identified the ClpX heat‐shock protein of E. coli because it enables the ClpP catalytic protease to degrade the bacteriophage lambda O replication protein. Here we report that ClpX alone possesses all the properties expected of a molecular chaperone protein. Specifically, it can protect the lambda O protein from heat‐induced aggregation, disaggregate preformed lambda O aggregates, and even promote efficient binding of lambda O to its DNA recognition sequence. A lambda O‐ClpX specific protein‐protein interaction can be detected either by a modified ELISA assay or through the stimulation of ClpXs weak ATPase activity by lambda O. Unlike the behaviour of the major DnaK and GroEL chaperones, ClpX requires the presence of ATP or its non‐hydrolysable analogue ATP‐gamma‐S for efficient interaction with other proteins including the protection of lambda O from aggregation. However, ClpXs ability to disaggregate lambda O aggregates requires hydrolysable ATP. We propose that the ClpX protein is a bona fide chaperone, whose biological role includes the maintenance of certain polypeptides in a form competent for proteolysis by the ClpP protease. Furthermore, our results suggest that the ClpX protein also performs typical chaperone protein functions independent of ClpP.

THE CLP ATPASES DEFINE A NOVEL CLASS OF MOLECULAR CHAPERONES

The Clp ATPases were originally identified as a regulatory component of the bacterial ATP‐dependent Clp serine proteases. Proteins homologous to the Escherichia coli Clp ATPases (ClpA, B, X or Y) have been identified in every organism examined so far. Recent data suggest that the Clp ATPases are not only specificity factors which help to ‘present’ various protein substrates to the ClpP or other catalytic proteases, but are also molecular chaperones which can function independently of ClpP. This review discusses the recent evidence that the Clp ATPases are indeed molecular chaperones capable of either repairing proteins damaged during stress conditions or activating the initiation proteins for Mu, λ or P1 DNA replication. A mechanism is suggested to explain how the Clp ATPases ‘decide’ whether to repair or destroy their protein substrates.

Site-directed mutagenesis of the HtrA(DegP) serine protease, whose proteolytic activity is indispensable for Escherichia coli survival at elevated temperatures

The HtrA(DegP) 48-kDa serine protease of Escherichia coli is indispensable for bacterial survival at elevated temperatures. It contains the amino-acid sequence Gly208AnsSerGlyGlyAlaLeu, which is similar to the consensus sequence GlyAspSerGlyGlyProLys surrounding the active Ser residue of trypsin-like proteases. Mutational alteration of Ser210 eliminated proteolytic activity of HtrA. An identical effect was observed when His105 was mutated. The mutated HtrA were unable to suppress thermosensitivity of the htrA bacteria. These results suggest that Ser210 and His105 may be important elements of the catalytic domain and indicate that the proteolytic activity of HtrA is essential for the survival of cells at elevated temperatures.

Recognition, Targeting, and Hydrolysis of the λ O Replication Protein by the ClpP/ClpX Protease

It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage λ O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of λ O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly. Consistent with these results, deletion of the first 18 amino acids of λ O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease. Based on our results with λ O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX.

Structure-function analysis of the Escherichia coli GrpE heat shock protein

We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda. To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified. All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence. The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild‐type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography. Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N‐terminal region, most likely through a computer predicted coiled‐coil region. Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function. Our results also demonstrate that both the N‐terminal and C‐terminal regions are important for GrpE function in lambda DNA replication and its co‐chaperone activity with DnaK.

Ionic equilibria of pyridine N-oxide perchlorates in acetonitrile

Abstract Ionic association constants of pyridine N-oxide perchlorate and semiperchlorate have been determined conductometrically as well as limiting molar conductances of PyOH+ and PyOHOPy+ cations in acetonitrile. The acid dissociation constant of the protonated pyridine N-oxide and the cationic homoconjugation constant of pyridine N-oxide with conjugated cationic acid have been found from potentiometric measurements. On the bass of determined values of the equilibrium constants, a scheme of fundamental ionic equilibria in the acetonitrile solutions of the perchlorates has been suggested.

The Rz1 gene product of bacteriophage lambda is a lipoprotein localized in the outer membrane of Escherichia coli

The Rz1 gene of bacteriophage lambda is located within the Rz1 lysis gene. It codes for the 6.5-kDa prolipoprotein (Rz1) which undergoes N-terminal signal sequence cleavage and post-translational lipid modification of the N-terminal Cys of the mature protein. Globomycin, the antibiotic which inhibits bacterial signal peptidase II, specific for prolipoproteins containing diacylglyceryl cysteine [Hayashi and Wu, J. Bioenerg. Biomembr. 22 (1990) 451-471] inhibits the N-terminal sequence cleavage of the Rz1 precursor. The mature protein is rich in Pro, which constitutes 25% of its amino acids (aa). Using a computer-predicted, synthetic, 15-aa antigenic determinant of Rz1 polyclonal anti-Rz[46-60] antibodies, were obtained, and employed to localize Rz1 in bacterial fractions. In induced Escherichia coli lambda lysogens Rz1 was found almost exclusively in the outer membrane (OM). In a strain overproducing Rz1 from the pSB54 plasmid, it was distributed in all the fractions, OM, fraction A and inner membrane (IM). Expression of Rz1 from the pSB54 caused enlargement of fraction A, corresponding to the adhesion sites of OM and IM. Such an enlargement was previously observed in induced lambda lysogens, shortly before the onset of lysis.

Proton transfer in acetonitrile: homo- and heteroassociation +N-bases and trimethyl-N-oxide

Abstract From electrometric measurements on protonated N -bases and trimethyl- N -oxide in acetonitrile (AN), the following constants have been determined: K a for the acidic dissociation BH + ⇌ B + H + and K f for the homo- (or heteroassociation, BH + + B (or R) ⇌ BHB + (or BHR + ). The homoassociation constants of N -bases (log K f ∼ 2.5) do not provide any information about the effects of acidity. Trimethyl- N -oxide, (CH 3 ) 3 NO, is different from other bases. It has a very high association constant (log K f = 5.51) and the protonated form is five units of pKa higher in acidity than protonated N -bases with the same acidity in water. If both partners (BH + + R) in heterocomplexes are aliphatic or aliphatic—aromatic the paH ( C B  C RH + ) in the complexes is about three units higher than for complexes formed by two aromatic N -base molecules.

Proton-transfer equilibria for N-base–trimethyl-N-oxide cation systems in acetonitrile

The electrometric behaviour of homo- and hetero-complexes, (Me3NOHONMe3)+ and (Me3NOHB)+(where B is an N-base), in acetonitrile have been investigated by e.m.f. measurements.The N-bases, B. studied were: 2-chloropyridine, 3-chloropyridine, pyridine, quinoline, N-methylimidazole, morpholine, N-methylpiperidine, triethylamine and trimethyl-N-oxide.The formation constant, Kf, and the proton-transfer equilibrium constant, KPT, for the reactions (Me3NO + BH+) and (B + Me3NOH+) have been evaluated. The sigmoidal titration curve for (Me3NOH++Me3NO) and some (Me3NOH++B) suggest that (—OHO—)+ and (—OHN)+ hydrogen bridges are very strong. The experimental formation constants of the complexes (Me3NO)2H+, log Kf= 5.51, and (Me3NOHB)+, log Kf=≈ 4.0, with (NHN)+ bridges showing weaker interaction, log Kf≈ 2.0.

Theoretical and experimental studies on the UV spectra of pyridine N-oxide perchlorates

Abstract The UV-spectra of pyridine N-oxide /PyO/, pyridinium N-oxide perchlorate /PyOHClO 4 /, and pyridinium N-oxide semiperchlorate //PyO/2HClO 4 / in acetonitrile have been discussed in details. In the series PyO - PyOHClO 4 - /PyO/ 2 HClO 4 the blue shift of the longest intensive band is observed. CNDO/S-CI calculations have been carried out to explain this. In the case of PyO the first intensive band /276 nm/ involves the charge-transfer from oxygen to the pyridine ring, while the second one /216 nm/ corresponds to 1L b transition of pyridine. For PyOHClO 4 and /PyO/ 2 HClO 4 we have found the opposite sequence. The variation of the positions of the above bands can be explained in terms of the charge-transfer during the transition.