Mutations in HlyD, part of the type 1 translocator for hemolysin secretion, affect the folding of the secreted toxin.

HlyD, a member of the membrane fusion protein family, is essential for the secretion of the RTX hemolytic toxin HlyA from Escherichia coli. Random point mutations affecting HlyA secretion were obtained, distributed in most periplasmic regions of the HlyD molecule. Analysis of the secretion phenotypes of different mutants allowed the identification of regions in HlyD involved in different steps of HlyA translocation. Four mutants, V349-I, T85-I, V334-I and L165-Q, were conditionally defective, a phenotype shown to be linked to the presence of inhibitory concentrations of Ca2+ in extracellular medium. Hly mutant T85-I was defective at an early stage in secretion, while mutants V334-I and L165-Q appeared to accumulate HlyA in the cell envelope, indicating a block at an intermediate step. Mutants V349-I, V334-I, and L165-Q were only partially defective in secretion, allowing significant levels of HlyA to be transported, but in the case of V349-I and L165-Q the HlyA molecules secreted showed greatly reduced hemolytic activity. Hemolysin molecules secreted from V349-I and V334-I are defective in normal folding and can be reactivated in vitro to the same levels as HlyA secreted from the wild-type translocator. Both V349-I and V334-I mutations mapped to the C-terminal lipoyl repeat motif, involved in the switching from the helical hairpin to the extended form of HlyD during assembly of the functional transport channel. These results suggest that HlyD is an integral component of the transport pathway, whose integrity is essential for the final folding of secreted HlyA into its active form.

Requirements for osmosensing and osmotic activation of transporter ProP from Escherichia coli.

Transporter ProP of Escherichia coli, a solute-H+ symporter, can sense and respond to osmotic upshifts imposed on cells, on membrane vesicles, or on proteoliposomes that incorporate purified ProP-(His)6. In this study, proline uptake catalyzed by ProP was used as a measure of its osmotic activation, and the requirements for osmosensing were defined using the proteoliposome system. The initial rate of proline uptake increased with decreasing external pH and increasing DeltaPsi, lumen negative. Osmotic upshifts increased DeltaPsi by concentrating lumenal K+, but osmotic activation of ProP could be distinguished from this effect. Osmotic activation of ProP resulted from changes in Vmax, though osmotic shifts also increased the KM for proline. Osmotic activation could be described as a reversible, osmotic upshift-dependent transition linking (at least) two transporter protein conformations. No correlation was observed between ProP activation and the position of the anions of activating sodium salts within the Hofmeister series of solutes. Both the magnitude of the osmotic upshift required to activate ProP and the ProP activity attained were similar for membrane-impermeant osmolytes, including NaCl, glucose, and PEG 600. The membrane-permeant osmolytes glycerol, urea, PEG 62, and PEG 106 failed to activate ProP. Two poly(ethylene glycol)s, PEG 150 and PEG 200, were membrane-permeant and did not cause liposome shrinkage, but they did partially activate ProP-(His)6.

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids.

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.

The role of the carboxyl terminal alpha-helical coiled-coil domain in osmosensing by transporter ProP of Escherichia coli.

Concentrative uptake of osmoprotectants via transporter ProP contributes to the rehydration of Escherichia coli cells that encounter high osmolality media. A member of the major facilitator superfamily, ProP is activated by osmotic upshifts in whole bacteria, in cytoplasmic membrane vesicles and in proteoliposomes prepared with the purified protein. Soluble protein ProQ is also required for full osmotic activation of ProP in vivo. ProP is differentiated from structural and functional homologues by its osmotic activation and its C-terminal extension, which is predicted to form an alpha-helical coiled-coil. A synthetic polypeptide corresponding to the C-terminus of ProP (ProP-p) formed a dimeric alpha-helical coiled-coil. A derivative of transporter ProP lacking 26 C-terminal amino acids was expressed but inactive. A derivative harbouring amino acid changes K460I, Y467I and H495I (each at the core, coiled-coil 'a' position) required a larger osmotic upshift for activation than did the wild type transporter. The same changes extended, stabilized and altered the oligomeric state of the coiled-coil formed by ProP-p. Amino acid change R488I (also at the 'a' position) further increased the magnitude of the osmotic upshift required to activate ProP, reduced the activity attained and rendered ProP activation transient. Unexpectedly, replacement R488I destabilized the coiled-coil formed by ProP-p. The activity and osmotic activation of ProP were even more strongly attenuated by helix-destabilizing change I474P. These data demonstrate that the carboxyl terminal domain of ProP can form a homodimeric alpha-helical coiled-coil with unusual properties. They implicate the C-terminal domain in the osmotic activation of ProP.

Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts.

The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by DeltapH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.

Properties of isolated recombinant N and C domains of chicken troponin C.

The two globular N and C domains of chicken troponin C (TnC) are connected by an exposed alpha-helix (designated D/E; residues 86-94). Recombinant N (residues 1-90) and C (residues 88-162) domains containing either F29 or W29 and F105 or W105 have been engineered and expressed in Escherichia coli. These termination and initiation sites were chosen to minimize disruption of side-chain interactions between the D/E helix and other residues. W29 and W105 served as useful spectral probes for monitoring Ca(2+)-induced structural transitions of the N and C domains, respectively [Pearlstone et al. (1992) Biochemistry 31, 6545-6553; Trigo-Gonzalez et al. (1992) Biochemistry 31, 7009-7015]. By all criteria tested, the properties of the isolated F29W/N domain (1-90) were identical to those of the N domain in intact F29W. These included fluorescence emission spectra in the absence and presence of Ca2+/Mg2+, far-UV CD spectra, and Ca2+ affinity as monitored by fluorescence and ellipticity at 221 nm. Similar but not identical properties were observed for isolated F105W/C domain (88-162) and intact F105W. A summation of the far-UV CD spectra (+/- Ca2+) of the two domains was virtually superimposable on that of the intact protein. Of the total Ca(2+)-induced ellipticity change at 221 nm, 27% could be assigned to the N domain and 73% to the C domain. The data suggest a significant Ca(2+)-induced transition involving secondary structural elements of the N domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Helix variants of troponin C with tailored calcium affinities.

Muscle fiber contraction is regulated through calcium-induced changes in the conformation of troponin C. In this study, we explored the relationship between the stability of a specific helix in the protein and the metal ion affinity of associated binding sites. Serial replacement of the amino acid at position 130 caused the calcium affinity of the paired Ca2+/Mg2+ sites to be attenuated. In the crystal structures of chicken and turkey troponin C, position 130 is the N-cap residue of the G-helix. The ion affinities of variant proteins were shifted in the order Ile < Gly < Asp < Asn < Thr < Ser. Although differing in ion affinities, the variant proteins all exhibited high cooperativity. The results of this study point to a specific relationship between alpha-helix stability and ion affinity in troponin C and suggest that troponin C may be a paradigm for protein folding problems.

A comparative spectroscopic study of tryptophan probes engineered into high- and low-affinity domains of recombinant chicken troponin C.

The spectral properties of three tryptophan-substituted mutants of recombinant chicken troponin C are compared. Site-specific mutagenesis was used to introduce a tryptophan residue into the high-affinity (Ca2+/Mg2+) domain of troponin C at residue position 105, thereby creating the mutant phenylalanine-105 to tryptophan (F105W). The spectral properties of F105W and a double mutant, F29W/F105W, were compared with the mutant phenylalanine-29 to tryptophan (F29W). Since wild-type chicken troponin C does not naturally contain either tyrosine or tryptophan residues, the tryptophan substitutions behaved as site-specific reporters of metal ion binding and conformational change. The residues that occupy positions 29 and 105 are at homologous locations in low-affinity and high-affinity domains, preceding the first liganding residues of binding loops I and III, respectively. Mutant proteins were examined by a combination of absorbance and fluorescence methods. Calcium induced significant changes in the near-UV absorbance spectra, fluorescence emission spectra, and far-UV circular dichroism of all three mutant proteins. Magnesium induced significant changes in the spectral properties of only F105W and F29W/F105W proteins. Tryptophan substitutions allowed Ca(2+)-specific and Ca(2+)/Mg(2+) sites to be titrated independently of one another. Results indicate that there is no interaction between the two binding domains under conditions where troponin C is isolated from the troponin complex. Magnesium-induced changes in the environment of the tryptophan reporter at position 105 were significantly different from those induced by calcium. This suggests that calcium and magnesium differ in their influence on the conformation of the high-affinity, Ca(2+)/Mg(2+) sites.

Expression and characterization of a recombinant yeast isoleucyl-tRNA synthetase.

We describe the heterologous expression of a recombinant Saccharomyces cerevisiae isoleucyl-tRNA synthetase (IRS) gene in Escherichia coli, as well as the purification and characterization of the recombinant gene product. High level expression of the yeast isoleucyl-tRNA synthetase gene was facilitated by site-specific mutagenesis. The putative ribosome-binding site of the yeast IRS gene was made to be the consensus of many highly expressed genes of E. coli. Mutagenesis simultaneously created a unique BclI restriction site such that the gene coding region could be conveniently subcloned as a "cassette." The variant gene was cloned into the expression vector pKK223-3 (Brosius, J., and Holy, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6929-6933) thereby creating the plasmid pKR4 in which yeast IRS expression is under the control of the isopropyl-thio-beta-galactopyranoside (IPTG)-inducible tac promoter. Recombinant yeast IRS, on the order of 10 mg/liter of cell culture, was purified from pKR4-infected and IPTG-induced E. coli strain TG2. Yeast IRS was purified to homogeneity by a combination of anion-exchange and hydroxyapatite gel chromatography. Inhibition of yeast IRS activity by the antibiotic pseudomonic acid A was tested. The yeast IRS enzyme was found to be 10(4) times less sensitive to inhibition by pseudomonic acid A (Ki = 1.5 x 10(-5) M) than the E. coli enzyme. E. coli strain TG2 infected with pKR4, and induced with IPTG, had a plating efficiency of 100% at inhibitor concentrations in excess of 25 micrograms/ml. At the same concentration of pseudomonic acid A, E. coli strain TG2 infected with pKK223-3 had a plating efficiency less than 1%. The ability of yeast IRS to rescue E. coli from pseudomonic acid A suggests that the eukaryotic synthetase has full activity in its prokaryotic host and has specificity for E. coli tRNA(ile).