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In prokaryotes, a large share of the proteins are secreted from the cell through a process that requires their translocation across the cytoplasmic membrane. This process is mediated by the universally conserved Sec system with homologues in the endoplasmic reticulum and thylakoid membranes of eukaryotes. The Sec system also facilitates the membrane insertion of integral membrane proteins, an essential step along their folding pathway. In bacteria, the Sec system consists of the protein-conducting channel (SecYEG) that associates with soluble components, such as the motor protein SecA or translating ribosomes, and with integral membrane proteins, such as the heterotrimeric complex termed SecDFyajC and the YidC insertase. Over the past three decades, biochemical and structural studies have provided a comprehensive view of protein translocation, but the exact mechanistic details of this process remain to be resolved. For a number of other biomolecular systems, single-molecule biophysical analysis has efficiently complemented the conventional biochemical studies conducted in bulk, with high-sensitivity measurements probing the structure and dynamics of individual molecules in vitro and in vivo. Here, we review recent advances in studies of protein translocation employing single-molecule techniques with the aim of resolving molecular mechanisms, thereby providing a new and detailed view of the process.

The lactose permease from Escherichia coli (LacY) is a galactoside/H(+) symporter that catalyzes the coupled stoichiometric transport of a sugar and an H(+) across the cytoplasmic membrane. X-ray crystal structures of WT LacY and the conformationally restricted mutant Cys154→Gly exhibit an inward-facing conformation with a tightly sealed periplasmic side and a deep central cleft or cavity open to the cytoplasm. Although the crystal structures may give the impression that LacY is a rigid molecule, multiple converging lines of evidence demonstrate that galactoside binding to WT LacY induces reciprocal opening and closing of periplasmic and cytoplasmic cavities, respectively. By this means, the sugar- and H(+)-binding sites in the middle of the molecule are exposed alternatively to either side of the membrane. In contrast to the crystal structure, biochemical/biophysical studies with mutant Cys154→Gly show that the periplasmic side is paralyzed in an open-outward conformation. In this study, a rigid, funnel-shaped, maleimide-containing molecule was used to probe the periplasmic cavity of a pseudo-WT and the Cys154→Gly mutant by site-directed alkylation. The findings provide strong support for previous observations and indicate further that the external opening of the periplasmic cleft in the mutant is patent to the extent of at least 8.5 Å in the absence of sugar or about half that of the WT cavity with bound galactoside.

Nisin is a pentacyclic peptide antibiotic active against Gram-positive bacteria. Its thioether rings are formed by two enzymatic steps: nisin dehydratase (NisB)-mediated dehydration of serines and threonines followed by nisin cyclase (NisC)-catalyzed enantioselective coupling of cysteines to the formed dehydroresidues. Here, we report the in vivo activity of NisC to cyclize a wide array of unrelated and designed peptides that were fused to the nisin leader peptide. To assess the role of NisC, leader peptide fusions, secreted by Lactococcus lactis cells containing NisBT with or without NisC were compared. In hexapeptides, a dehydroalanine could spontaneously react with a more C-terminally located cysteine. In contrast, peptides containing dehydrobutyrines require NisC for cyclization. In agreement with in silico predictions NisC could efficiently cyclize the hexapeptides ADhbVECK and lDhbPGCK, but ADhbVWCE was not cyclized. Interestingly, NisC could efficiently catalyze the synthesis of peptides with intertwined rings and of a designed polyhexapeptide containing four thioether rings. Taken together the data demonstrate that NisC can be widely applied for the cyclization and stabilization of nonlantibiotic peptides.

ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains ( NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.

LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[alpha-P-32]ATP, which, at 4 degrees C, occurs predominantly at LmrC, while aluminiumfluoride (AlFx)-induced trapping of the hydrolyzed nucleotide at 30 degrees C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs.

The SecYEG complex is a membrane-embedded channel that permits the passage of precursor proteins (preproteins) across the inner membrane of Escherichia coli. SecA is a molecular motor that associates with the SecYEG pore and drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the conformational state of soluble and SecYEG-bound SecA using single tryptophan mutants of SecA. The fluorescence spectral properties of the single tryptophans of SecA and their accessibility to the quencher acrylamide demonstrate that SecA undergoes a conformational change that results in a more compact structure upon binding of ATP and binding to the SecYEG pore. In addition, SecYEG-bound SecA undergoes ATP-dependent conformational changes that are not observed for soluble SecA. These data support a model in which binding to the SecYEG channel has a major impact on the SecA conformation.

The molecular chaperone SecB targets preproteins to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA recognizes SecB via its carboxyl-terminal 22 aminoacyl residues, a highly conserved domain that contains 3 cysteines and 1 histidine residue that could potentially be involved in the coordination of a metal ion. Treatment of SecA with a zinc chelator resulted in a loss of the stimulatory effect of SecB on the SecA translocation ATPase activity, while the activity could be restored by the addition of ZnCl2. Interaction of SecB with the SecB binding domain of SecA is disrupted by chelators of divalent cations, and could be restored by the addition of Cu2+ or Zn2+. Atomic absorption and electrospray mass spectrometry revealed the presence of one zinc atom per monomeric carboxyl terminus of SecA. It is concluded that the SecB binding domain of SecA is stabilized by a zinc ion that promotes the functional binding of SecB to SecA.

Unfolded outer membrane protein A (OmpA) of Escherichia coli spontaneously inserts and refolds into lipid bilayers upon dilution of denaturing urea. In the accompanying paper, we have developed a new technique, time-resolved distance determination by fluorescence quenching (TDFQ), which is capable of monitoring the translocation across lipid bilayers of fluorescence reporter groups such as tryptophan in real time [Kleinschmidt, J. H., and Tamm, L. K. (1999) Biochemistry 38, 4996-5005]. Specifically, we have shown that wild-type OmpA, which contains five tryptophans, inserts into lipid bilayers via three structurally distinct membrane-bound folding intermediates. To take full advantage of the TDFQ technique and to further dissect the folding pathway, we have made five different mutants of OmpA, each containing a single tryptophan and four phenylalanines in the five tryptophan positions of the wild-type protein. All mutants refolded in vivo and in vitro and, as judged by SDS-PAGE, trypsin fragmentation, and Trp fluorescence, their refolded state was indistinguishable from the native state of OmpA. TDFQ analysis of the translocation across the lipid bilayer of the individual Trps of OmpA yielded the following results: Below 30 degrees C, all Trps started from a far distance from the bilayer center and then gradually approached a distance of approximately 10 A from the bilayer center. In a narrow temperature range between 30 and 35 degrees C, Trp-15, Trp-57, Trp-102, and Trp-143 were detected very close to the center of the lipid bilayer in the first few minutes and then moved to greater distances from the center. When monitored at 40 degrees C, which resolved the last steps of OmpA refolding, these four tryptophans crossed the center of the bilayer and approached distances of approximately 10 A from the center after refolding was complete. In contrast Trp-7 approached the 10 A distance from a far distance at all temperatures and was never detected to cross the center of the lipid bilayer. The translocation rates of Trp-15, Trp-57, Trp-102, and Trp-143 which are each located in different outer loop regions of the four beta-hairpins of the eight-stranded beta-barrel of OmpA were very similar to one another. This result and the common distances of these Trps from the membrane center observed in the third membrane-bound folding intermediate provide strong evidence for a synchronous translocation of all four beta-hairpins of OmpA across the lipid bilayer and suggest that OmpA inserts and folds into lipid bilayers by a concerted mechanism.

Protein translocation in Escherichia coli is mediated by the SecA ATPase bound to the SecYEG membrane protein complex. SecA translocation ATPase activity as well as protein translocation is dependent on the presence of negatively charged lipids. By using a phospholipid with an acyl chain linked photoactivatable group, the lipid accessibility of SecA bound at the translocase was explored. SecA bound to lipid vesicles containing negatively charged lipids was found to be readily accessible for labeling by the photoactivatable phospholipid. The presence of an excess amount of SecYEG complex resulted in a remarkable reduction in the amount of lipid-accessible SecA irrespective of the nucleotide-bound form of SecA. These data demonstrate that the SecYEG-bound SecA is largely shielded from the phospholipid acyl chains and suggest the presence of two distinct pools of membrane-bound SecA that differ in the degree of lipid association.

The homodimeric SecA protein is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade. SecA contains two essential nucleotide binding sites (NBSs), i.e., NBS1 and NBS2 that hind ATP with high and low affinity, respectively. The photoactivatable bifunctional cross-linking agent 3'-arylazido-8-azidoadenosine 5'-triphosphate (diN(3)ATP) was used to investigate the spatial arrangement of the nucleotide binding sites of SecA, DiN(3)ATP is an authentic ATP analogue as it supports SecA-dependent precursor protein translocation and translocation ATPase, UV-induced photo-cross-linking of the diN(3)ATP-bound SecA results in the formation of stable dimeric species of SecA. D209N SecA, a mutant unable to bind nucleotides at NBS1, was also photo-crosslinked by diN(3)ATP, whereas no cross-linking occurred with the NBS2 mutant R509K SecA, We concluded that the low-affinity NBS2, which is located in the carboxyl-terminal half of SecA, Is the site of crosslinking and that NBS2 binds nucleotides at or near the subunit interface of the SecA dimer.

SecA is the peripheral subunit of the preprotein translocase of Escherichia coli. SecA consists of two independently folding domains, i.e., the N-domain bearing the high-affinity nucleotide binding site (NBS-I) and the C-domain that harbors the low-affinity NBS-II. ATP induces SecA insertion into the membrane during preprotein translocation. Domain-specific monoclonal antibodies (mAbs) were developed to analyze the functions of the SecA domains in preprotein translocation. The antigen binding sites of the obtained mAbs were confined to five epitopes. One of the mAbs, i.e., mAb 300-1K5, recognizes an epitope in the C-domain in a region that has been implicated in membrane insertion. This mAb, either as IgG or as Fab, completely inhibits in vitro proOmpA translocation and SecA translocation ATPase activity. It prevents SecA membrane insertion and, more strikingly, reverses membrane insertion and promotes the release of SecA from the membrane. Surface plasmon resonance measurements demonstrate that the mAb recognizes the ADP- and the AMP-PNP-bound state of SecA either free in solution or bound at the membrane at the SecYEG protein. It is concluded that the mAb actively reverses a conformation essential for membrane insertion of SecA. The other mAbs directed to various epitopes in the N-domain were found to be without effect, although all bind the native SecA. These results demonstrate that the C-domain plays an important role in the SecA membrane insertion, providing further evidence that this process is needed for preprotein translocation.

Nisin is a cationic polycyclic bacteriocin secreted by some lactic acid bacteria. Nisin has previously been shown to permeabilize Liposomes. The interaction of nisin was analyzed with liposomes prepared of the zwitterionic phosphatidylcholine (PC) and the anionic phosphatidylglycerol (PG). Nisin induces the release of 6-carboxyfluorescein and other small anionic fluorescent dyes from: PC Liposomes in;a Delta psi-stimulated manner, and not that of neutral and cationic fluorescent dyes. This activity is blocked in PG liposomes. Nisin, however, efficiently dissipates the Delta psi in cytochrome c oxidase proteoliposomes reconstituted with PG, with a threshold Delta psi, requirement of about -100 mV. Nisin associates with the anionic surface of PG liposomes and disturbs the lipid dynamics near the phospholipid polar head group-water interface. Further studies with a novel cationic lantibiotic, epilancin K7, indicate that this molecule penetrates into the hydrophobic carbon region of the Lipid bilayer upon the imposition of a Delta psi. It is concluded that nisin acts as an anion-selective carrier in the absence of anionic phospholipids. In vivo, however, this activity is likely to be prevented by electrostatic interactions with anionic lipids of the target membrane. It is suggested that pore formation by cationic (type A) lantibiotics involves the local perturbation of the bilayer structure and a Delta psi-dependent reorientation of these molecules from a surface-bound into a membrane-inserted configuration.

The molecular chaperone SecB binds to hydrophobic sections of unfolded secretory proteins and thereby prevents their premature folding prior to secretion by the translocase of Escherichia coli. Here, we have Investigated the effect of the single-residue mutation of leucine 42 to arginine (L42R) centrally positioned in the polypeptide binding pocket of SecB on its chaperonin function. The mutant retains its tetrameric structure and SecA targeting function but is defective in its holdase activity. Isothermal titration calorimetry and single-molecule optical tweezer studies suggest that the SecB(L42R) mutant exhibits a reduced polypeptide binding affinity allowing for partial folding of the bound polypeptide chain rendering it translocation-incompetent.

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.

The essential prokaryotic cell division protein FtsZ is a tubulin homologue that forms a ring at the division site. FtsZ forms polymers in a GTP-dependent manner. Recent biochemical evidence has shown that FtsZ forms multimeric structures in vitro and in vivo and functions as a self-activating GTPase. Structural analysis of FtsZ points to an important role for the highly conserved tubulin-like loop 7 (T7-loop) in the self-activation of GTP hydrolysis. The T7-loop was postulated to form the active site together with the nucleotide-binding site on an adjacent FtsZ monomer. To characterize the role of the T7-loop of Escherichia coli FtsZ, we have mutagenized residues M206, N207, D209, D212, and R214. All the mutant proteins, except the R214 mutant, are severely affected in polymerization and GTP hydrolysis. Charged residues D209 and D212 cannot be substituted with a glutamate residue. All mutants interact with wild-type FtsZ in vitro, indicating that the T7-loop mutations do not abolish FtsZ self-association. Strikingly, in mixtures of wild-type and mutant proteins, most mutants are capable of inhibiting wild-type GTP hydrolysis. We conclude that the T7-loop is part of the active site for GTP hydrolysis, formed by the association of two FtsZ monomers.

Ac-MB21-NH(2) (Ac-FASLLGKALKALAKQ-NH(2)) and dermaseptin S3(1-16)-NH(2) (ALWKNMLKGIGKLAGK-NH(2)) are cationic amphipathic peptides with antimicrobial activity against a broad spectrum of microorganisms including various fungi. The interaction of the peptides with liposomes was studied by exploiting the tryptophan fluorescence of F1W-Ac-MB21-NH(2) and dermaseptin S3(1-16)-NH(2). Spectral analysis and the use of quenchers indicate that the tryptophans of both peptides insert more deeply in anionic than in zwitterionic liposomes. Membrane insertion correlates with the formation of an alpha-helical peptide structure. Both peptides permeabilize liposomes composed of anionic, cylindric phospholipids more efficiently than liposomes formed of zwitterionic, conic (phospho)lipids.

Preprotein translocation in Escherichia coli is mediated by translocase, a multimeric membrane protein complex with SecA as the peripheral ATPase and SecYEG as the translocation pore. Unique cysteines were introduced into transmembrane segment (TMS) 2 of SecY and TMS 3 of SecE to probe possible sites of interaction between the integral membrane subunits. The SecY and SecE single-Cys mutants were cloned individually and in pairs into a secYEG expression vector and functionally overexpressed. Oxidation of the single-Cys pairs revealed periodic contacts between SecY and SecE that are confined to a specific alpha-helical face of TMS 2 and 3, respectively. A Cys at the opposite alpha-helical face of TMS 3 of SecE was found to interact with a neighboring SecE molecule. Formation of this SecE dimer did not affect the high-affinity binding of SecA to SecYEG and ATP hydrolysis, but blocked preprotein translocation and thus uncouples the SecA ATPase activity from translocation. Conditions that prevent membrane deinsertion of SecA markedly stimulated the interhelical contact between the SecE molecules. The latter demonstrates a SecA-mediated modulation of the protein translocation channel that is sensed by SecE.

Production of bacteriocins by lactic acid bacteria is in some cases regulated by a quorum sensing mechanism that involves a secreted bacteriocin-like peptide pheromone. In the case of Lactobacillus plantarum C11, this pheromone, the 26-mer plantaricin A (PlnA), has the interesting property of having both bacteriocin and pheromone activities. To gain insight into how PlnA functions as a pheromone and as a bacteriocin, the L- and D-enantiomers of an N-terminally truncated form of PlnA were synthesized (PlnA-22L and PlnA-22D; PlnA-22L has full biological activity). With circular dichroism, it was shown that the two peptides are unstructured in aqueous solution, but they adopt mirror-image amphiphilic helical structures in the presence of trifluoroethanol and membrane-mimicking entities such as micelles of dodecylphosphocholine and negatively charged Ole2GroPGro liposomes, but not in the presence of zwitterionic Ole2GroPCho liposomes. Thus, the negative charge on the membrane is important for structuring of the (positively charged) PlnA peptides. In terms of in vivo antimicrobial activity, PlnA-22L and PlnA-22D behaved almost identically. Likewise, the peptides dissipated the membrane potential and the transmembrane pH gradient in sensitive cells equally effectively. PlnA-22L induced bacteriocin production in L. plantarum C11 (i.e., displayed pheromone activity), the level of induction being clearly dose-dependent. PlnA-22D did not display pheromone activity, but, at high concentrations, was able to inhibit the pheromone activity of PlnA-22L. The results indicate that the antimicrobial activity of PlnA does not require chiral interactions and is mediated through the formation of a strongly amphiphilic alpha-helical structure. In contrast, PlnA's pheromone activity is dependent on a chiral interaction between the amphiphilic helix (PlnA-22L) and a receptor protein. One may speculate that PlnA is an evolutionary intermediate between a true bacteriocin and a pheromone.

The homodimeric SecA protein is the peripheral subunit of the preprotein translocase in bacteria. It binds the preprotein and promotes its translocation across the bacterial cytoplasmic membrane by nucleotide modulated coinsertion and deinsertion into the membrane, SecA has two essential nucleotide binding sites (NBS; Mitchell & Oliver, 1993). The high-affinity NBS-I resides in the amino-terminal domain of the protein, and the low-affinity NBS-II is localized at 2/3 of the protein sequence. The nucleotide-bound stares of soluble SecA were studied by site directed tryptophan fluorescence spectroscopy, tryptic digestion, differential scanning calorimetry, and dynamic light scattering. A nucleotide-induced conformational change of a carboxy-terminal domain of SecA was revealed by Trp fluorescence spectroscopy. The Trp fluorescence of a single Trp SecA mutant containing Trp775 decreased and increased upon the addition of NBS-I saturating concentrations of ADP or AMP-PNP, respectively. DSC measurements revealed that SecA unfolds as a two domain protein finding of ADP to NBS-I increased the interaction between the two domains whereas binding of AMP-PNP did not influence this interaction. When both NBS-I and NBS-II are bound by ADP, SecA seems to have a more compact globular conformation whereas binding of AMP-PNP seems to cause a more extended conformation, it is suggested that the compact: ADP-bound conformation resembles the membrane deinserted state of SecA, while the more extended ATP-bound conformation may correspond to the membrane inserted form of SecA.

In this study, wild-type lac permease and lac permease mutated at each of the eight cysteinyl residues in the molecule were solubilized from the membrane, purified, and reconstituted into proteoliposomes. Lactose equilibrium exchange and efflux activities of mutants with Ser in place of Cys117, Cys176, Cys234, Cys333, Cys353, or Cys355 are essentially the same as wild-type permease. In contrast, mutants in Cys148 and Cys154 exhibit diminished exchange and efflux activities. These mutants in Cys148 and Cys154, except for the C148S mutant, have previously been shown to slow down active transport as well [Van Iwaarden, P.R., Driessen, A. J. M., Menick, D. R., Kaback, H.R.,Konings, W. N. (1991) J. Biol. Chem. 266, 15688-15692]. C148S permease shows monophasic kinetics with a high apparent KM with respect to external lactose in the exchange reaction under nonequilibrium conditions, whereas wild-type permease exhibits biphasic kinetics with both a high and low KM component. Moreover, the absence of the low Km pathway in the C148S permease is correlated with the absence of a high-affinity binding site for p-nitrophenyl alpha-D-galactopyranoside (NPG). Interestingly, the affinity of the permease for NPG appears to increase with the hydrophobicity of the side chain at position 154 (SerCysGlyVal). Finally, the presence of a high-affinity binding site for NPG in C154V is consistent with the biphasic exchange kinetics exhibited by this mutant. The results are discussed in the context of a model in which lac permease has two substrate binding sites, a catalytic site and a regulatory site.