Major anticodon-binding region missing from an archaebacterial tRNA synthetase.

The small size of the archaebacterial Methanococcus jannaschii tyrosyl-tRNA synthetase may give insights into the historical development of tRNAs and tRNA synthetases. The L-shaped tRNA has two major arms-the acceptor.TpsiC minihelix with the amino acid attachment site and the anticodon-containing arm. The structural organization of the tRNA synthetases parallels that of tRNAs. The more ancient synthetase domain contains the active site and insertions that interact with the minihelix portion of the tRNA. A second, presumably more recent, domain interacts with the anticodon-containing section of tRNA. The small size of the M. jannaschii enzyme is due to the absence of most of the second domain, including a segment thought to bind to the anticodon. Consistent with the absence of an anticodon-binding motif, a mutation of the central base of the anticodon had a relatively small effect on the aminoacylation efficiency of the M. jannaschii enzyme. In contrast, others showed earlier that the same mutation severely reduced charging by a normal-sized bacterial enzyme that has the aforementioned anticodon-binding motif. However, the M. jannaschii enzyme has a peptide insertion into its catalytic domain. This insertion is shared with all other tyrosyl-tRNA synthetases and is needed for a critical minihelix interaction. We show that the M. jannaschii enzyme is active on minihelix substrates over a wide temperature range and has preserved the same peptide-dependent minihelix specificity seen in other tyrosine enzymes. These findings are consistent with the concept that anticodon interactions of tRNA synthetases were later adaptations to the emerging synthetase-tRNA complex that was originally framed around the minihelix.

Domain-domain communication in a miniature archaebacterial tRNA synthetase.

The three-dimensional structure of tRNA is organized into two domains-the acceptor-TPsiC minihelix with the amino acid attachment site and a second, anticodon-containing, stem-loop domain. Aminoacyl-tRNA synthetases have a structural organization that roughly recapitulates the two-domain organization of tRNAs-an older primary domain that contains the catalytic center and interacts with the minihelix and a secondary, more recent, domain that makes contacts with the anticodon-containing arm. The latter contacts typically are essential for enhancement of the catalytic constant k(cat) through domain-domain communication. Methanococcus jannaschii tyrosyl-tRNA synthetase is a miniature synthetase with a tiny secondary domain suggestive of an early synthetase evolving from a one-domain to a two-domain structure. Here we demonstrate functional interactions with the anticodon-containing arm of tRNA that involve the miniaturized secondary domain. These interactions appear not to include direct contacts with the anticodon triplet but nonetheless lead to domain-domain communication. Thus, interdomain communication may have been established early in the evolution from one-domain to two-domain structures.

Colicin E1 forms a dimer after urea-induced unfolding.

Unfolding of the soluble colicin E1 channel peptide was examined with the use of urea as a denaturant; it was shown that it unfolds to an intermediate state in 8.5 M urea, equivalent to a dimeric species previously observed in 4 M guanidinium chloride. Single tryptophan residues, substituted into the peptide at various positions by site-directed mutagenesis, were employed as fluorescent probes of local unfolding. Unfolding profiles for specific sites within the peptide were obtained by quantifying the shifts in the fluorescence emission maxima of single tryptophan residues on unfolding and plotting them against urea concentration. Unfolding reported by tryptophan residues in the C-terminal region was not characteristic of complete peptide denaturation, as evidenced by the relatively blue-shifted values of the fluorescence emission maxima. Unfolding was also monitored by using CD spectroscopy and the fluorescent probe 2-(p-toluidinyl)-naphthalene 6-sulphonic acid; the results indicated that unfolding of helices is concomitant with the exposure of protein non-polar surface. Unfolding profiles were evaluated by non-linear least-squares curve fitting and calculation of the unfolding transition midpoint. The unfolding profiles of residues located in the N-terminal region of the peptide had lower transition midpoints than residues in the C-terminal portion. The results of unfolding analysis demonstrated that urea unfolds the peptide only partly to an intermediate state, because the C-terminal portion of the channel peptide retained significant structure in 8.5 M urea. Characterization of the peptide's global unfolding by size-exclusion HPLC revealed that the partly denatured structure that persists in 8.5 M urea is a dimer of two channel peptides, tightly associated by hydrophobic interactions. The presence of the dimerized species was confirmed by SDS/PAGE and intermolecular fluorescence resonance energy transfer.

Different adaptations of the same peptide motif for tRNA functional contacts by closely homologous tRNA synthetases.

The N73 nucleotide at the end of the tRNA acceptor stem is commonly used by tRNA synthetases for discrimination. Because only a few synthetase-tRNA cocrystal structures have been determined, understanding of the molecular basis for N73 discrimination is limited. Here we investigated the possibility that, for at least some synthetases, the capacity to recognize different N73 nucleotides resides in the variable sequence of the loop of motif 2, a motif found in all class II enzymes. In the cocrystal of the class II yeast aspartyl-tRNA synthetase, atomic groups of the G73 discriminator of tRNAAsp interact with three side chains of the enzyme. We examined lysyl-tRNA synthetase, a close structural homologue of the aspartyl enzyme. Different substitutions were introduced into the Escherichia coli enzyme (A73 discriminator) to make its loop more like that of the human enzyme (G73 discriminator). Our data show that the loop of motif 2 of the lysine enzyme makes tRNA functional contacts, as predicted from the structural comparison. And yet, the E. coli enzyme with the "humanized" loop sequence had the same quantitative kinetic preference for A73 versus G as the wild-type enzyme. We conclude that discriminator base selectivity in the lysine enzyme requires residues in addition to or other than those in the loop of motif 2. Thus, even tRNA synthetases that are close structural homologues may use the same RNA binding element to make functional contacts with places (in the acceptor stem) that are idiosyncratic to each synthetase-tRNA pair.

Genetic code origins: tRNAs older than their synthetases?

We present a phylogenetic analysis to determine whether a given tRNA molecule was established in evolution before its cognate aminoacyl-tRNA synthetase. The earlier appearance of tRNA versus their metabolically related enzymes is a prediction of the RNA world theory, but the available synthetase and tRNA sequences previously had not allowed a formal comparison of their relative time of appearance. Using data recently obtained from the emerging genome projects, our analysis points to the extant forms of lysyl-tRNA synthetase being preceded in evolution by the establishment of the identity of lysine tRNA.

Identification of a chameleon-like pH-sensitive segment within the colicin E1 channel domain that may serve as the pH-activated trigger for membrane bilayer association.

In vitro, the channel-forming domain of colicin E1 requires activation by acidic pH (<4.5) or detergents. The activation of this domain to its insertion-competent state results in an increased ability of the protein to dock onto and to form channels in artificial membranes. Fluorescence methods were used to characterize the conformational changes occurring in a channel-forming peptide of colicin E1 in solution with pH. The 178-residue thermolytic fragment of colicin E1 contains three Trp residues, W-424, W-460, and W-495. In order to study the structural and dynamic requirements for activation of the C-terminal domain of colicin E1, single-Trp-containing peptides were prepared by site-directed mutagenesis. All of the mutant peptides displayed in vitro channel activity and cellular cytotoxicity similar to the those of wild-type peptide. Two Trp residues, W-413 and W-424, exhibited pH-sensitive fluorescence parameters. Upon acidification (pH 6.0 --> 3.5), the fluorescence quantum yield of W-413 and W-424 increased 50% and 80%, respectively, indicating a significant change in the local environment of the peptide segment containing these two Trp residues. The fluorescence decay of W-413 and W-424 was best fit by three fluorescence decay components, two of which were sensitive to pH. However, only small changes in spectral shape and position were observed for W-424 fluorescence, whereas there were larger changes in these fluorescence parameters for W-413. The quantum yields for the Trp residues in the seven other single-Trp mutant peptides and the wild-type peptide were distinct but only slightly affected by changes in pH. Time-resolved fluorescence measurements showed that W-460, -484, and -495 each had two fluorescence decay components with similar decay times, with one component dominating the fluorescence decay behavior. Furthermore, the individual fluorescence decay times for all the single-Trp peptides, except for W-413 and W-424, were insensitive to pH changes. At pH 3.5, the fluorescence of the wild-type peptide was fit by three decay time components, with the two longer decay times being quite different from the fluorescence decay times of the single-Trp mutant proteins (W-424, -460, and -495, the naturally occurring Trp residues). In contrast, at pH 6.0, the wild-type peptide showed double-exponential decay kinetics. Time-resolved fluorescence anisotropy decay measurements of the three single-Trp mutant proteins, containing a naturally occurring Trp residue, suggest that local segmental motion of the peptide as reported by each of the three tryptophans is highly restricted and largely insensitive to changes in pH. On the other hand, the anisotropy decay profiles of the wild-type protein were consistent with energy transfer occurring between Trp residues, likely between W-460 and W-495. These steady-state and time-resolved fluorescence results show that W-413 and W-424 report conformational changes which may be associated with the insertion-competent state and reside on the protein segment(s) which form the pH-activated trigger of the channel peptide.

Characterization of an unfolding intermediate and kinetic analysis of guanidine hydrochloride-induced denaturation of the colicin E1 channel peptide.

The equilibrium unfolding pathway of the colicin E1 channel peptide was shown in a previous study to involve an unfolding intermediate, stable in approximately 4 M guanidine hydrochloride, which comprised primarily the C-terminal hydrophobic alpha-helical hairpin segment of the peptide [Steer, B. A., & Merrill, A. R. (1995) Biochemistry 34, 7225-7233]. In this study, the structural nature of this unfolding intermediate was investigated further, and it was found that the intermediate primarily consists of a dimer species and is comprised of two partially denatured monomeric peptides, which appear to be associated by hydrophobic interactions. The dimerized structure was detected by size-exclusion high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical cross-linking, and intermolecular fluorescence energy transfer. Using stopped-flow fluorescence spectroscopy, the kinetics of the denaturation and dimerization of the colicin E1 channel peptide in 4 M guanidine hydrochloride were examined. Denaturation kinetics were also investigated by wild-type peptide Trp fluorescence and 1-anilinonaphthalene-8-sulfonic acid binding. The kinetics of dimer formation were examined by monitoring the time dependence of intermolecular Trp to 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid fluorescence resonance energy transfer upon denaturation in 4 M guanidine hydrochloride. In addition, single Trp mutant peptides were employed as site-specific fluorescent probes of unfolding kinetics and reported diverse and characteristic unfolding kinetics. However, it was shown that following a rapid and major unfolding transition the peptide's core residues cluster slowly, by hydrophobic association, forming an intermediate species which is a prerequisite to dimerization. These equilibrium and kinetic unfolding data describe a unique unfolding mechanism where the channel peptide forms a partially unfolded dimerized structure in 4 M guanidine hydrochloride.

Guanidine hydrochloride-induced denaturation of the colicin E1 channel peptide: unfolding of local segments using genetically substituted tryptophan residues.

The soluble colicin E1 channel peptide has a roughly spherical, highly alpha-helical, compact structure. The structural unfolding properties of the colicin E1 channel peptide were analyzed using fluorescence techniques. The guanidine hydrochloride-induced unfolding pattern of the wild-type channel peptide was examined by monitoring intrinsic tryptophan fluorescence. Additionally, peptide unfolding was examined with the fluorophore, 1-anilinonaphthalene-8-sulfonic acid. In order to probe the unfolding of local segments, single-tryptophan channel peptides were constructed by site-directed mutagenesis. Shifts in fluorescence emission maxima of the single tryptophan residues were used to monitor site-specific unfolding events, in the presence of guanidine hydrochloride. The unfolding patterns reported by tryptophans in different regions of the peptide were diverse. The concentration of guanidine hydrochloride at the unfolding transition midpoint for each mutant peptide and the free energy of unfolding were calculated in order to estimate local segment stabilities. Also, secondary structure unfolding was monitored using circular dichroism spectroscopy. The results of unfolding analysis showed that the channel peptide's unfolding mechanism involves an intermediate structure stabilized by the C-terminal hydrophobic core of the peptide. Knowledge of the unfolding pattern of the soluble channel peptide will aid in the understanding of the secondary and tertiary structural interactions within the channel peptide and the mechanism of colicin E1 activation.

Genes encoding osmoregulatory proline/glycine betaine transporters and the proline catabolic system are present and expressed in diverse clinical Escherichia coli isolates.

Sixty-three clinical isolates identified as Escherichia coli, 30 from the human urinary tract and 33 derived from other human origins, were screened for proline/glycine betaine transporters similar to those that support proline catabolism and proline- or glycine betaine-based osmoregulation in E. coli K-12. Both molecular (DNA- and protein-based) analyses and physiological tests were performed. All tests were calibrated with E. coli K-12 derivatives from which genetic loci putP (encoding a proline transporter required for proline catabolism), proP, and (or) proU (loci encoding osmoregulatory proline/glycine betaine transporters) had been deleted. All clinical isolates showed both enhanced sensitivity to the toxic proline analogue azetidine-2-carboxylate on media of high osmolality and growth stimulation by glycine betaine in an artificial urine preparation of high osmolality. DNA sequences similar to the putP, proP, and proU loci of E. coli K-12 were detected by DNA amplification and (or) hybridization and protein specifically reactive with antibodies raised against the ProX protein of E. coli K-12 (a ProU constituent) was detected by western blotting in over 95% of the isolates. Two anomalous isolates were reclassified as non-E. coli on the basis of the API 20E series of tests. A protein immunochemically cross-reactive with the ProP protein of E. coli K-12 was also expressed by the clinical isolates. Since all three transporters were ubiquitous, no particular correlation between clinical origin and PutP, ProP, or ProU activity was observed. These data suggest that the transporters encoded in loci putP, proP, and proU perform housekeeping functions essential for the survival of E. coli cells in diverse habitats.

The colicin E1 insertion-competent state: detection of structural changes using fluorescence resonance energy transfer.

The single cysteine residue (Cys-505) located in the hydrophobic membrane anchor domain in the colicin E1 COOH-terminal channel peptide was labeled with the thiol-specific fluorescent reagent IAEDANS [5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid]. The labeling stoichiometry was nearly 1:1 [AEDANS: peptide (mol:mol)]. Eleven single Trp mutants of the channel peptide were prepared, and the FRET efficiency for each Trp residue (donor) and the AEDANS chromophore (acceptor), covalently attached to Cys-505, was measured. The FRET efficiencies for the various donor-acceptor pairs ranged from 15% to approximately 100% for the native peptide in solution (pH6.0). The FRET efficiency for the W-507 channel peptide-AEDANS adduct approached 100% since this adduct showed no detectable Trp fluorescence. Activation of the channel peptide to the insertion-competent state upon addition of the nonionic detergent octyl beta-D-glucoside [10,000:1 detergent: peptide (mol:mol)] resulted in decreased FRET efficiencies. The detergent-activated colicin E1 channel peptide-AEDANS adducts possessed significant in vitro channel activity at pH 6.0. The relative changes in the FRET efficiencies upon peptide activation ranged from -1% (W-495 channel peptide-AEDANS adduct) to 48% (W-355 channel peptide-AEDANS adduct). A direct correlation existed between the relative change in FRET efficiency upon channel peptide activation and the position of the Trp (donor) residue within the channel peptide primary sequence (higher relative delta E the closer the Trp donor was to the NH2 terminus), except for the W-484 channel peptide-AEDANS adduct, which showed a higher relative delta E than either W-443 or W-460 channel peptide-AEDANS adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and sequencing of Escherichia coli gene proP reveals unusual structural features of the osmoregulatory proline/betaine transporter, ProP.

Transporters encoded in genetic loci putP, proP and proU mediate proline and/or betaine accumulation by Escherichia coli K-12. The ProP and ProU systems are osmoregulatory. Activation of ProP in response to hyperosmotic stress has been demonstrated both in vivo and in vitro. It therefore serves as a model experimental system for the analysis of osmosensory and osmoregulatory mechanisms. We developed methodologies which will facilitate the identification of proline transporter genes by functional complementation of putP proP proU bacteria. E. coli gene proP was isolated and located within a chromosomal DNA fragment. Deletion, complementation and sequence analysis revealed putative promoter and transcription termination signals flanking a 1500 base-pair open reading frame. The predicted 55 kDa ProP protein was hydrophobic. In vitro expression of proP yielded a protein whose apparent molecular mass was determined to be 42 kDa by polyacrylamide gel electrophoresis under denaturing conditions. Database searches and cluster analysis defined relationships among the ProP sequence and those of integral membrane proteins that comprise a transporter superfamily. Members of the superfamily catalyze facilitated diffusion or ion linked transport of organic solutes in prokaryotes and eukaryotes. Multiple alignment revealed particularly close correspondence among the ProP protein, citrate transporters from E. coli and Klebsiella pneumoniae and an alpha-ketoglutarate transporter from E. coli. The predicted ProP sequence differed from those closely similar sequences in possessing an extended central hydrophilic loop and a carboxyl terminal extension. Unlike other protein sequences within the transporter superfamily, the carboxyl terminal extension of ProP was strongly predicted to participate in formation of an alpha-helical coiled coil. These data suggest that the ProP protein catalyzes solute-ion cotransport. Its unusual structural features may be related to osmoregulation of its activity.