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At a functional level, the bifunctional enzyme from Escherichia coli does not have any advantage over the synthesized monofunctional enzyme. The monomeric bifunctional enzyme indoleglycerol phosphate synthase: phosphoribosyl anthranilate isomerase was examined for field interaction through breaking up the domains on the gene level and creating monofunctional proteins. The synthesized monofunctional enzymes were stable and had all catalytic properties. There was no interaction between the enzymes, neither in vitro nor in vivo.

The structure and dynamics of the region 4 of sigma(sup)70 subunit of Escherichia coli RNA polymerase reveal that at pH 2.8 helical structure mostly remain in the protonated protein, which increases upon deprotonation of carboxylic amino acids. Data indicate that three helical regions originate from the helical secondary structure.

The transient folding of domain 4 of an E. coli RNA polymerase $$\sigma^{70}$$ subunit ( $$r{\text{EC}}\sigma_{4}^{70}$$ ) induced by an increasing concentration of 2,2,2-trifluoroethanol (TFE) in an aqueous solution was monitored by means of CD and heteronuclear NMR spectroscopy. NMR data, collected at a 30 % TFE, allowed the estimation of the population of a locally folded $$r{\text{EC}}\sigma_{4}^{70}$$ structure (CSI descriptors) and of local backbone dynamics (N relaxation). The spontaneous organization of the helical regions of the initially unfolded protein into a TFE-induced 3D structure was revealed from structural constraints deduced from N- to C-edited NOESY spectra. In accordance with all the applied criteria, three highly populated α-helical regions, separated by much more flexible fragments, form a transient HLHTH motif resembling those found in PDB structures resolved for homologous proteins. All the data taken together demonstrate that TFE induces a transient native-like structure in the intrinsically disordered protein. [ABSTRACT FROM AUTHOR]

Folding of a recombinant protein rECσ [ABSTRACT FROM AUTHOR]

Abstract: Conflicting experimental data on the influence of Mg2+ ions on the salt dependence of formation/dissociation of open transcription complex (RPo) of Escherichia coli RNA polymerase led us to carry systematic measurements of the dissociation rate constant (k d) and thermodynamic stability of complexes at λPR and Pa promoters in a broad range of [NaCl] and [MgCl2] at 25, 31 and 37 °C, using fluorescence detected abortive transcription assay. Values of k d determined in MgCl2 in the presence of heparin, as a commonly used anionic competitor, were shown to depend on heparin concentration whereas in NaCl this effect was not observed. Kinetics of dissociation was therefore determined in the course of salt-induced down-shift of the binding equilibrium. Salt derivatives of k d''s (n d) appeared to be similar in NaCl (~8.5) and MgCl2 (~10) for both complexes. Isotherms of fractional occupancy of promoters by RNAP as a function of ln [salt] were shown to conform to a sigmoid Boltzman function parameterized to include binding constant of RPo and a net change (n obs) in the number of electrolyte ions associated with complex components upon its formation/dissociation. The fitted values of n obs appeared also similar in NaCl and in MgCl2: ~18 for RPo/λPR and ~20 for RPo/Pa, respectively. Overall unfavorable vant''Hoff enthalpy (ΔH obs) of RPo proved to be much higher in MgCl2 than in NaCl by ca. 20 kcal/mol for both complexes, rendering them profoundly less stable in the former salt. In both salts, ΔH obs was higher by ~30 kcal/mol for RPo/Pa relative to RPo/λPR. Similarity of n obs and n d values for the two salts indicates thermodynamic equivalence of Mg2+ and Na+ in [salt]-controlled binding equilibrium of RPo. This finding remains in disagreement with earlier data and suggests that salt effects on open complex stability should be sought in global compensating changes in distribution of all ionic species around the interacting complex components. [Copyright &y& Elsevier]

Folding of a recombinant protein rECsigma(70) (4) comprising domain 4 of E. coli RNA polymerase sigma(70) subunit, upon addition of 2,2,2-trifluoroethanol (TFE) to its aqueous solution, was monitored by heteronuclear NMR spectroscopy. The TFE-induced migration of resonance signals in a series of (15)N-HSQC spectra displayed sequence-dependent heterogeneity. A common trend of uniform upfield shift in both (1)H and (15)N dimensions, indicative of generation of helical structures, breaks down for some residues almost cooperatively at 10-15% TFE (v/v), pointing to the buildup of non-helical regions separating the initially induced helices. The preferences of residues to assume either helical or non-helical conformation are correlated with the location in the sequence rather than with their type. CSI descriptors and (15)N relaxation data obtained for the protein at 10% TFE allowed characterization of the stability of the pre-folded state of rECsigma(70) (4). By all the criteria applied, three highly populated alpha-helical regions separated by much more flexible residues forming a loop and a turn in the DNA-binding HLHTH motif were identified. The location of the secondary structure elements along the protein sequence coincides with those found in homologous proteins, and with the helix nucleation regions determined in unfolded rECsigma(70) (4) at low pH. The bimodal distribution of the (15)N relaxation parameters enabled identification of residues forming a framework of the folded protein strictly corresponding to the HLHTH motif, bracketed by unfolded terminal regions. Thus, in respect to rECsigma(70) (4) in aqueous solution TFE acts not only as a strong helix inducer, but also as a folding agent.

Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.2 of sigma(70) in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the (13)Calpha, (13)CO, and (1)Halpha nuclei and of vicinal (3)J(HNH)(alpha) coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and (3)J(HNH)(alpha) couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the sigma(A) subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and sigma(70) of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R(1) and R(2), and [(1)H]-(15)N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments. Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of sigma(70)(4) protein in its protonated state.