Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry.

Protein-protein interactions are crucial for cellular homeostasis and play important roles in the dynamic execution of biological processes. While antibodies represent a well-established tool to study protein interactions of extracellular domains and secreted proteins, as well as in fixed and permeabilized cells, they usually cannot be functionally expressed in the cytoplasm of living cells. Non-immunoglobulin protein-binding scaffolds have been identified that also function intracellularly and are now being engineered for synthetic biology applications.

G-quadruplexes are specifically recognized and distinguished by selected designed ankyrin repeat proteins.

We introduce designed ankyrin repeat binding proteins (DARPins) as a novel class of highly specific and structure-selective DNA-binding proteins, which can be functionally expressed within all cells. Human telomere quadruplex was used as target to select specific binders with ribosome display. The selected DARPins discriminate the human telomere quadruplex against the telomeric duplex and other quadruplexes.

Structural origins for selectivity and specificity in an engineered bacterial repressor-inducer pair.

The bacterial tetracycline transcription regulation system mediated by the tetracycline repressor (TetR) is widely used to study gene expression in prokaryotes and eukaryotes. To study multiple genes in parallel, a triple mutant TetR(K(64)L(135)I(138)) has been engineered that is selectively induced by the synthetic tetracycline derivative 4-de-dimethylamino-anhydrotetracycline (4-ddma-atc) and no longer by tetracycline, the inducer of wild-type TetR. In the present study, we report the crystal structure of TetR(K(64)L(135)I(138)) in the absence and in complex with 4-ddma-atc at resolutions of 2.

Stabilization and humanization of a single-chain Fv antibody fragment specific for human lymphocyte antigen CD19 by designed point mutations and CDR-grafting onto a human framework.

A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutions predicted to correct destabilizing residues in the 4G7-wt sequence to create 4G7-mut.

Drug delivery from the oral cavity: focus on a novel mechatronic delivery device.

Dental drug delivery systems have been used for a long time, in particular for the local therapy of diseases affecting the oral cavity. Research today concentrates on the design of formulations to increase their retention time. Even today, however, prosthetic devices incorporating drug delivery are rarely used.

Engineered Tet repressors with recognition specificity for the tetO-4C5G operator variant.

We created a new DNA recognition specificity for tetracycline repressor (TetR) binding to the tet operator variant tetO-4C5G containing four bp exchanges compared to tetO. TetR variants created by doped oligonucleotide mutagenesis of residues in the DNA recognition helix yielded several mutants binding to tetO-4C5G. These variants contained exchanges of the amino acids at positions 36, 37, 39 and 42.

Implantable Active Telemetry System using Microcoils.

The use of microcoils in an active telemetry system that is intended to be implanted in the body for biomonitoring applications is presented in this paper. To achieve a moderate transmission range, active telemetry was realized instead of passive telemetry by load modulation, which is often used in systems incorporating microcoils. Sample microcoils with a diameter of 1 cm and 16 windings were fabricated and used for tests.

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.

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.

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.

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.

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.

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.

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.

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.