Biomedical subjects

Oliver Scholz

Publications and source records attributed to Oliver Scholz.

8 recordsLinked to original sources

PubMed2004

Structure-based design of Tet repressor to optimize a new inducer specificity.

We constructed a mutant of the tetracycline-inducible repressor protein TetR with specificity for the tc analogue 4-de(dimethylamino)anhydrotetracycline (4-ddma-atc), which is neither an antibiotic nor an inducer for the wild-type protein. The previously published relaxed specificity mutant TetR H64K S135L displays reduced induction by tc but full induction by doxycycline (dox), anhydrotetracycline (atc), and 4-de(dimethylamino)-6-demethyl-6-deoxytetracycline (cmt3). To create induction specificity for tc derivatives lacking the 4-dimethylamino grouping such as cmt3 and 4-ddma-atc, the residues at positions 82 and 138, which are located close to that moiety in the crystal structure of the TetR-[tc-Mg](+)(2) complex, were randomized. We anticipated that a residue with increased size may lead to sterical hindrance, and screening for 4-ddma-atc-specific induction indeed revealed the mutant TetR H64K S135L S138I. Out of 24 exchanges only the addition of S138I to TetR H64K S135L yielded a mutant with a pronounced reduction of affinity for atc and dox, while the one for 4-ddma-atc is not affected. The ratio of binding constants revealed a 200-fold specificity increase for 4-ddma-atc over atc. The contributions of each single mutant to specificity indicate that the tc variants bind slightly different positions in the TetR tc binding pocket.

Models, Molecular↗

PubMed2004

Two mutations in the tetracycline repressor change the inducer anhydrotetracycline to a corepressor.

We report for the first time the in vitro characterization of a reverse tetracycline repressor (revTetR). The dimeric wild-type repressor (TetR) binds to tet operator tetO in the absence of the inducer anhydrotetracycline (atc) to confer tight repression. We have isolated the revTetR G96E L205S mutant, which, contrary to TetR, binds tetO only in the presence of atc. This reverse acting mutant was overproduced and purified. Effector and DNA binding properties were analyzed by EMSA and quantified by fluorescence titration and surface plasmon resonance. The association constant K(A) of revTetR for binding of [atcMg](+) is approximately 10(8) M(-1), four orders of magnitude lower than that of TetR. The affinity of TetR for tetO is 5.6 +/- 2 x 10(9) M(-1) and that for revTetR in the presence of atc is 1 +/- 0.2 x 10(8) M(-1). Both induced forms, the atc-bound TetR and the free revTetR, have the same low affinity of 4 +/- 1 x 10(5) M(-1) for DNA. Therefore, atc does not act as a dimerization agent for revTetR. We discuss the structural differences between TetR and revTetR potentially underlying this reversal of activity.

Circular Dichroism↗

PubMed2004

How does Mg(2+) affect the binding of anhydrotetracycline in the TetR protein?

The binding of anhydrotetracycline (atc) in wild-type TetR(D), TetR(B), and four single tryptophan mutants of TetR(B) was investigated by UV/vis absorption, steady state, and time-resolved fluorescence spectroscopy. From absorption titration experiments with Mg2+, we conclude that binding of one [atc-Mg]+ complex in the homodimer causes changes in the protein conformation around the second binding pocket. In the presence of absence of Mg2+, several different groups of atc-protein arrangements must exist, each with a characteristic atc fluorescence decay time. Taking into account the results of molecular dynamics (MD) simulations, we propose as one possible origin for such a differentiation teh extent of hydrogen bonding between atc and the surrounding amino acids. Binding of Mg2+ should change the arrangement of the surrounding amino acids such that some of the excited atc molecules do not undergo the relaxation process typical for free atc. The MD simulations also show that the pattern of intra- and intermolecular hydrogen bonding in the two monomeric units is no correlated, thereby leading to different fluorescence kinetics for atc in the two monomeric units. Furthermore, it is suggested that hydrogen bonding between Arg104 and O10 of anhydrotetracycline could regulate the relaxation processes of excited anhydrotetracycline.

Bacterial Proteins↗

PubMed2004

Activity reversal of Tet repressor caused by single amino acid exchanges.

We explore by extensive mutagenesis regions in the sequence allowing reversal of the allosteric response of Tet repressor. The wild type requires anhydrotetracycline for induction. About 100 mutants are presented, which, in contrast, require the drug for repression. Their mutations are clustered at the interface of the DNA- and inducer-binding domains. This interface consists of a central hydrophobic region surrounded by several hydrogen bonds. While most of the mutants described here contain two to five mutations, we found five positions in this region of TetR, at which single amino acid exchanges lead to activity reversal. They may disrupt the hydrogen-bonding network bordering the domain interface. We assume that the mutations cause a repositioning of the DNA reading head with respect to the effector binding core so that the same conformational change can result in opposite activities.

Amino Acid Sequence↗

PubMed2004

Independent regulation of two genes in Escherichia coli by tetracyclines and Tet repressor variants.

We report a regulation system in Escherichia coli for independent regulation of two distinct reporter genes by application of Tet repressors with different specificities. One Tet repressor variant comprises wild-type tet operator (tetO) recognition and exclusive induction with the novel inducer 4-dedimethylamino-anhydrotetracycline. The other Tet repressor variant shows tetO-4C recognition and induction with tetracycline. We demonstrate that both variants are independently active in vivo and allow selective regulation of two genes in the same cell without any cross talk.

Endo-1,4-beta Xylanases↗

PubMed2004

Phenotypes of combined tet repressor mutants for effector and operator recognition and allostery.

Tet repressor mutants with a shifted effector specificity, preference for a mutant operator sequence or reversion of activity were combined to construct variants bearing two or three phenotypic alterations. TetR alleles with combinations of altered operator and effector specificities can be created by merging the respective residues in a single polypeptide. The mutations giving rise to revTetR, on the other hand, show drastic influences on the ligand binding phenotypes when combined with respective alterations. One TetR variant displays all three phenotypic alterations and thus demonstrates the general possibility of implementing them in one protein.

Allosteric Regulation↗

PubMed2003

Teaching TetR to recognize a new inducer.

Tet Repressor (TetR) recognizes the inducer tetracycline (tc) with high affinity. The tc analog 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) is not an inducer for TetR. Induction specificity for cmt3 was generated by employing a directed evolution approach to screen appropriate TetR mutants in four successive steps. The specificity of the best TetR mutant is more than 20,000-fold increased for cmt3 over tc as judged by the ratio of their respective binding constants. Two rounds of directed evolution via DNA shuffling revealed His64 as a key residue for inducer specificity. The best TetR mutant with cmt3 specificity contains the H64K exchange, leading to a 300-fold decreased tc and a 20-fold increased cmt3 affinity. Another round of directed evolution made use of randomized oligonucleotides to mutate selected residues close to the tc-binding pocket of TetR and yielded TetR S135L with a 250-fold increased cmt3 affinity. The double mutant TetR H64K S135L was constructed and again subjected to directed evolution using randomized oligonucleotides to alter residues in the "secondary shell" of the tc-binding pocket. The resulting best mutants TetR H64K E114Q S135L, TetR A61V H64K Q109E Q116E S135L and TetR H64K T112K S135L are fully inducible by cmt3 and not by tc. Thus, their inducer specificity has been redesigned. The molecular mechanism of changed inducer recognition is discussed, based on binding constants with several tc analogs and in light of the TetR crystal structure.

Amino Acid Sequence↗

PubMed2002

Pressure effect on the structure of the Tet repressor protein TetR(B).

The fluorescence of two single tryptophan (trp) mutants of the TetR(B)dimer protein was monitored for hydrostatic pressures 0.1 MPa < p < 300 MPa. In mutant W170, in which trp interacts with segments of both subunits, the fitted fluorescence lifetimes vary both with pressure and observation wavelength. In contrast, the lifetimes are fairly independent of both parameters in the case of W171, in which trp is completely solvent exposed. This difference in fluorescence behaviour is in agreement with the fact that only trp 170 experiences a strong alteration of its direct environment upon a movement of alpha-helix 9 induced by increasing static pressure. The conformational changes induced by pressure in this protein region are only partly reversible. After pressure release, the originally more solvent exposed trp 171 ends up, at least in part, in a more solvent shielded environment and the originally protein embedded trp 170 ends up in a more solvent exposed conformation. These observations provide evidence for heterogeneity in chromophore-protein arrangement under normal, biologically relevant conditions. Furthermore, they indicate that no complete dissociation into monomers occurs at pressures below 300 MPa.

Bacterial Proteins↗