Study on conformational states of Escherichia coli tRNAPhe in solution by a modulation-free ESR-spectrometer

A modulation free Electron Spin Resonance spectrometer was used for the registration of spectral absorption lines of a spin-labeled Escherichia coli phenylalanine tRNA in solution with low (less than 0.1%) line shape distortion. The analysis of line shape of two different spin-labels introduced into position 8 revealed that phenylalanine tRNA in solution exists as a mixture of two conformers, the equilibria between conformers being dependent on pH, concentration of magnesium and functional state of tRNA (deacylated, aminoacylated or peptidylated). There are no overall structural rearrangements upon aminoacylation or peptidylation of tRNA. The observed small changes of spectral line shape can be assigned to shifts in conformational equilibria.     

A fluorescent probe-labeled Escherichia coli aspartate transcarbamoylase that monitors the allosteric conformational state

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.     

Inhibition of RecA protein by the Escherichia coli RecX protein: modulation by the RecA C terminus and filament functional state

The RecX protein is a potent inhibitor of RecA activities. We identified several factors that affect RecX-RecA interaction. The interaction is enhanced by the RecA C terminus and by significant concentrations of free Mg(2+) ion. The interaction is also enhanced by an N-terminal His(6) tag on the RecX protein. We conclude that RecX protein interacts most effectively with a RecA functional state designated A(o) and that the RecA C terminus has a role in modulating the interaction. We further identified a C-terminal point mutation in RecA protein (E343K) that significantly alters the interaction between RecA and RecX proteins.     

A study of the complex-formation of phenylalanyl-tRNA-synthetase from Escherichia coli using the tRNA-Phe method of small-angle x-ray scattering

The method of small-angle X-ray scattering was employed to analyse the equilibrium enzyme-substrate complexes in solution. A new approach of analysis of the experimental data was developed. This type of analysis provides the determination of dissociation constants and structural parameters of enzyme-substrate complexes. The radius of gyration (Rg) and dimensions of half-axis of the equivalent prolonged ellipsoid (a, b) of E. coliphenylalanyl-tRNA synthetase and its complexes with one or two tRNA(Phe) molecules have been determined. The values of these parameters speak in favour of structural rearrangements due to the interaction of the enzyme with tRNA(Phe). The thermodynamic characteristics of phenylalanyl-tRNA synthetase complexes with tRNA(Phe) testify to the negative cooperativity in binding of two tRNA molecules with the enzyme.     

Methionyl-tRNA synthetase-induced conformational change of Escherichia coli tRNAfMet

Ribonuclease T2, nuclease S1, and snake venom phosphodiesterase were used as a structural probe for investigation of the interaction between Escherichia coli tRNAfMet and methionyl-tRNA synthetase, and the cleavage sites were analyzed by a rapid sequencing gel electrophoresis of 5'-32P-labeled tRNA. Both endonucleases cleaved the D-loop of synthetase-bound tRNA much more extensively than that of the free tRNA. Positions of A14, G15, A22, and G23 in the D-loop and C35 in the anticodon of the synthetase-bound tRNA were more susceptible to RNase T2. The synthetase-bound tRNA was predominantly cleaved by nuclease S1 at position of G15, G19, G20, and G23 in the D-loop and G2 in the acceptor stem. In contrast, the synthetase-bound tRNA was more resistant to the 3'-exonuclease, snake venom phosphodiesterase, than was the free tRNA molecule. These results suggest conformational change of the tRNA by the synthetase binding which weakened tertiary interaction between the D-loop and T psi C-loop/extra-loop. Production of acid-soluble radioactivity was also examined in the limited digestion of 5'-32P-labeled tRNA or 3'-14C-labeled methionyl-tRNA. The synthetase enhanced the release of acid-soluble oligonucleotides from the 5'-end of the tRNA but suppressed that from the 3'-end of the molecule. These results are consistent with that obtained by gel electrophoresis.     

Manipulating conformational equilibria in the lactose permease of Escherichia coli.

A mechanism proposed for lactose/H(+) symport by the lactose permease of Escherichia coli indicates that lactose permease is protonated prior to ligand binding. Moreover, in the ground state, the symported H(+) is shared between His322 (helix X) and Glu269 (helix VIII), while Glu325 (helix X) is charge-paired with Arg302 (helix IX). Substrate binding at the outer surface between helices IV (Glu126) and V (Arg144, Cys148) induces a conformational change that leads to transfer of the H(+) to Glu325 and reorientation of the binding site to the inner surface. After release of substrate, Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302. The conservative mutation Glu269-->Asp causes a 50-100-fold decrease in substrate binding affinity and markedly reduced active lactose transport, as well as decreased rates of equilibrium exchange and efflux. Gly-scanning mutagenesis of helix VIII was employed systematically with mutant Glu269-->Asp in an attempt to rescue function, and two mutants with increased activity are identified and characterized. Mutant Thr266-->Gly/Met267-->Gly/Glu269-->Asp binds ligand with increased affinity and catalyzes active lactose transport with a marked increase in rate; however, little improvement in efflux or equilibrium exchange is observed. In contrast, mutant Gly262-->Ala/Glu269-->Asp exhibits no improvement in ligand binding but a small increase in the rate of active transport; however, an increase in the steady-state level of accumulation, as well as efflux and equilibrium exchange is observed. Remarkably, when the two sets of mutations are combined, all translocation reactions are rescued to levels approximating those of wild-type permease. The findings support the contention that Glu269 plays a pivotal role in the mechanism of lactose/H(+) symport. Moreover, the results suggest that the two classes of mutants rescue activity by altering the equilibrium between outwardly and inwardly facing conformations of the permease such that impaired protonation and/or H(+) transfer is enhanced from one side of the membrane or the other. When the two sets of mutants are combined, the equilibrium between outwardly and inwardly facing conformations and thus protonation and H(+) transfer are restored.     

Substrate-dependent conformational dynamics of the Escherichia coli membrane insertase YidC

The binding of Pf3 coat protein to the membrane insertase YidC from Escherichia coli induces a conformational change in the tertiary structure of the insertase, resulting in a quenching of the intrinsic tryptophan (Trp) fluorescence. Tryptophan mutants of YidC were generated to examine such conformational movements in detail with time-resolved and steady-state fluorescence spectroscopy. Ten of the 11 Trp residues within YidC were substituted to phenylalanines generating single Trp mutants either at position 354, 454, or 508. In addition, a double mutant with Trp residues at 332 and 334 was studied. Purified YidC mutants were reconstituted into DOPC/DOPG vesicles and titrated with a Trp-free mutant of Pf3 coat, enabling a detailed conformational study of the periplasmic P1, P2, and P3 domains of YidC before and after binding of substrate. Time-resolved fluorescence anisotropy revealed that the mobility of the residues W332/W334 and W508 was considerably increased after binding of Pf3 coat to the insertase. Furthermore, analysis of the fluorescence emission spectra and the decay times showed that all Trp residues are embedded in an equivalent environment that is a membrane/water interface.     

Method of the anomalous time dependence of viscosity for registration of changes in conformational state of Escherichia coli cell genome

The sensitivity of the anomalous time dependence of viscosity to the concentration of the DNA-protein complexes (DNA + histone-like proteins of bacteria or, in other words, the genome) such as chromatin and the conformations of these complexes in lysates of E. coli AB1157 cells were studied. A linear region of the anomalous viscosity time dependence on the concentration of E. coli cells was found in which the interactions between single DNA-protein complexes can be neglected. The response of the genome of E. coli to ethidium bromide at concentrations of 0.0003-3 mg/ml was studied. Significant differences in the effect of ethidium bromide on E. coli cells in the stationary and logarithmic growth phases were found. The effect of heating cell lysates, the molar concentration of NaCl in lysates, and the addition of proteins into lysates on the parameters of the anomalous viscosity time dependence was studied. It was shown that proteins do not contribute significantly to the effect of anomalous viscosity time dependence. The results obtained confirm that the method is sensitive to changes in the conformational state of the genome of E. coli cells.     

Eclipse period without sequestration in Escherichia coli

The classical Meselson-Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC. Populations of bacteria growing exponentially in heavy ((15)NH(4)+ and (13)C(6)-glucose) medium were shifted to light ((14)NH(4)+ and (12)C(6)-glucose) medium. The HH-, HL- and LL-DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene-specific probes. The eclipse period, estimated from the kinetics of conversion of HH-DNA to HL- and LL-DNA, turned out to be 0.60 generation times for the wild-type strain. This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured. For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively. Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised. The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.     

Energetics of ligand-induced conformational flexibility in the lactose permease of Escherichia coli

Isothermal titration calorimetry has been applied to characterize the thermodynamics of ligand binding to wild-type lactose permease (LacY) and a mutant (C154G) that strongly favors an inward facing conformation. The affinity of wild-type or mutant LacY for ligand and the change in free energy (DeltaG) upon binding are similar. However, with the wild type, the change in free energy upon binding is due primarily to an increase in the entropic free energy component (TDeltaS), whereas in marked contrast, an increase in enthalpy (DeltaH) is responsible for DeltaG in the mutant. Thus, wild-type LacY behaves as if there are multiple ligand-bound conformational states, whereas the mutant is severely restricted. The findings also indicate that the structure of the mutant represents a conformational intermediate in the overall transport cycle.     

Interhelical packing modulates conformational flexibility in the lactose permease of Escherichia coli

A key to obtaining an X-ray structure of the lactose permease of Escherichia coli (LacY) (Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., and Iwata, S. (2003) Science 301, 549-716) was the use of a mutant in which Cys154 (helix V) is replaced with Gly. LacY containing this mutation strongly favors an inward-facing conformation, which binds ligand with high affinity, but catalyzes little transport and exhibits few if any of the ligand-dependent conformational changes observed with wild-type LacY. The X-ray structure demonstrates that helix V crosses helix I in the approximate middle of the membrane in such a manner that Cys154 lies close to Gly24 (helix I). Therefore, it seems likely that replacing Cys154 with Gly may lead to tighter packing between helices I and V, thereby resulting in the phenotype observed. Consistently, replacement of Gly24 with Cys in the C154G mutant rescues significant transport activity, and the mutant exhibits properties similar to wild-type LacY with respect to substrate binding and thermostability. However, the only other replacements that rescue transport to any extent whatsoever are Val and Asp, both of which are much less effective than Cys. The results suggest that, although helix packing probably plays an important role with respect to the properties of the C154G mutant, the ability of Cys at position 24 to rescue transport activity of C154G is more complicated than simple replacement of bulk between positions 24 and 154. Rather, activity is dependent on more subtle interactions between the helices, and mutations that disrupt interactions between helix IV and loop 6-7 or between helices II and IV also rescue transport in the C154G mutant.     

Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion

Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme. A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP. In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit. These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin. The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed. ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2. In a similar manner to native E. coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide. Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.     

Substrate-induced conformational changes in Escherichia coli arginyl-tRNA synthetase observed by 19F NMR spectroscopy

The 19F nuclear magnetic resonance (NMR) spectra of 4-fluorotryptophan (4-F-Trp)-labeled Escherichia coli arginyl-tRNA synthetase (ArgRS) show that there are distinct conformational changes in the catalytic core and tRNA anticodon stem and loop-binding domain of the enzyme, when arginine and tRNA(Arg) are added to the unliganded enzyme. We have assigned five fluorine resonances of 4-F-Trp residues (162, 172, 228, 349 and 446) in the spectrum of the fluorinated enzyme by site-directed mutagenesis. The local conformational changes of E. coli ArgRS induced by its substrates observed herein by 19F NMR are similar to those of crystalline yeast homologous enzyme.     

Negative modulation of Escherichia coli NAD kinase by NADPH and NADH.

NAD kinase was purified 93-fold from Escherichia coli. The enzyme was found to have a pH optimum of 7.2 and an apparent Km for NAD+, ATP, and Mg2+ of 1.9, 2.1, and 4.1 mM, respectively. Several compounds including quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, AMP, ADP, and NADP+ did not affect NAD kinase activity. The enzyme was not affected by changes in the adenylate energy charge. In contrast, both NADH and NADPH were potent negative modulators of the enzyme, since their presence at micromolar concentrations resulted in a pronounced sigmoidal NAD+ saturation curve. In addition, the presence of a range of concentrations of the reduced nucleotides resulted in an increase of the Hill slope (nH) to 1.7 to 2.0 with NADH and to 1.8 to 2.1 with NADPH, suggesting that NAD kinase is an allosteric enzyme. These results indicate that NAD kinase activity is regulated by the availability of ATP, NAD+, and Mg2+ and, more significantly, by changes in the NADP+/NADPH and NAD+/NADH ratios. Thus, NAD kinase probably plays a role in the regulation of NADP turnover and pool size in E. coli.     

NMR solution structure of the acylphosphatase from Escherichia coli

The solution structure of Escherichia coli acylphosphatase (E. coli AcP), a small enzyme catalyzing the hydrolysis of acylphosphates, was determined by (1)H and (15)N NMR and restrained modelling calculation. In analogy with the other members of AcP family, E. coli AcP shows an alpha/beta sandwich domain composed of four antiparallel and one parallel beta-strand, assembled in a five-stranded beta-sheet facing two antiparallel alpha-helices. The pairwise RMSD values calculated for the backbone atoms of E. coli and Sulfolobus solfataricus AcP, Bovine common type AcP and Horse muscle AcP are 2.18, 5.31 and 5.12 A, respectively. No significant differences are present in the active site region and the catalytic residue side chains are consistently positioned in the structures.     

Oligomeric state of the Escherichia coli metal transporter YiiP

YiiP is a 32.9-kDa metal transporter found in the plasma membrane of Escherichia coli (Chao, Y., and Fu, D. (2004) J. Biol. Chem. 279, 17173-17180). Here we report the determination of the YiiP oligomeric state in detergent-lipid micelles and in membranes. Molecular masses of YiiP solubilized with dodecyl-, undecyl-, decyl-, or nonyl-beta-d-maltoside were measured directly using size-exclusion chromatography coupled with laser light-scattering photometry, yielding a mass distribution of YiiP homo-oligomers within a narrow range (68.0-68.8 kDa) that equals the predicted mass of a YiiP dimer within experimental error. The detergent-lipid masses associated with YiiP in the mixed micelles were found to increase from 135.5 to 232.6 kDa, with an apparent correlation with the alkyl chain length of the maltoside detergents. Cross-linking the detergent-solubilized YiiP with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) resulted in a dimeric cross-linked product in an EDC concentration-dependent manner. The oligomeric state of the purified YiiP in reconstituted membranes was determined by electron microscopic analysis of two-dimensional YiiP crystals in negative stain. A projection structure calculated from measurable optical diffractions to 25 A revealed a pseudo-2-fold symmetry within a molecular boundary of approximately 75 x 40 A, indicative of the presence of YiiP dimers in membranes. These data provide direct structural evidence for a dimeric association of YiiP both in detergent-lipid micelles and in the reconstituted lipid bilayer. The functional relevance of the dimeric association in YiiP is discussed.     

Simulating the temporal modulation of inducible DNA damage response in Escherichia coli

Living organisms make great efforts to maintain their genetic information integrity. However, DNA is vulnerable to many chemical or physical agents. To rescue the cell timely and effectively, the DNA damage response system must be well controlled. Recently, single cell experiments showing that after DNA damage, expression of the key DNA damage response regulatory protein oscillates with time. This phenomenon is observed both in eukaryotic and bacterial cells. We establish a model to simulate the DNA damage response (SOS response) in bacterial cell Escherichia coli. The simulation results are compared to the experimental data. Our simulation results suggest that the modulation observed in the experiment is due to the fluctuation of inducing signal, which is coupled with DNA replication. The inducing signal increases when replication is blocked by DNA damage and decreases when replication resumes.