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. Here we used the Designed Ankyrin Repeat Protein (DARPin) scaffold to generate binders to fluorescent proteins and used them to modify biological systems directly at the protein level. DARPins binding to GFP or mCherry were selected by ribosome display. For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM. We then verified in cell culture their specific binding in a complex cellular environment and found an affinity cut-off in the mid-nanomolar region, above which binding is no longer detectable in the cell. Next, their binding properties were employed to change the localization of the respective fluorescent proteins within cells. Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations. Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level.

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. Affinities of the selected binders range from 3 to 100 nM. CD studies confirm that the quadruplex fold is maintained upon binding. The DARPins show different specificity profiles: some discriminate human telomere quadruplexes from other quadruplex-forming sequences like ILPR, c-MYC and c-KIT, while others recognize two of the sequences tested or even all quadruplexes. None of them recognizes dsDNA. Quadruplex-binding DARPins constitute valuable tools for specific detection at very small scales and for the in vivo investigation of quadruplex DNA.

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 2.1 A. Analysis of the structures in light of the available binding data and previously reported TetR complexes allows for a dissection of the origins of selectivity and specificity. In all crystal structures solved to date, the ligand-binding position, as well as the positioning of the residues lining the binding site, is extremely well conserved, irrespective of the chemical nature of the ligand. Selective recognition of 4-ddma-atc is achieved through fine-tuned hydrogen-bonding constraints introduced by the His64-->Lys substitution, as well as a combination of hydrophobic effect and the removal of unfavorable electrostatic interactions through the introduction of Leu135 and Ile138.

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. In the second variant, the murine CDRs were grafted to the human acceptor framework huVkappa3-huV(H)3, with 11 additional point mutations introduced to obtain a better match between CDR graft and acceptor framework, to arrive at 4G7-graft. Compared to 4G7-wt, 4G7-mut showed greater thermodynamic stability in guanidinium chloride-induced equilibrium denaturation experiments and somewhat greater stability in human serum. The loop graft maintained the comparatively high stability of the murine loop donor, but did not improve it further. Our analysis indicates that this is due to subtle strain introduced between CDRs and framework, mitigating the otherwise highly favorable properties of the human acceptor framework. This slight strain in the loop graft is also reflected in the binding affinities for CD19 on leukemic cells of 8.4 nM for 4G7-wt, 16.4 nM for 4G7-mut and 30.0 nM for 4G7-graft. This comparison of knowledge-based mutation and loop-grafting-based approaches will be important, when moving molecules forward to therapeutic applications.

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. Mainly, they are focused on prophylaxis and the release of antibacterial agents. However, as buccal delivery, because of its undeniable advantages, has become popular for systemic drug delivery, and prolonged well-controlled release has been identified as beneficial, especially for chronic diseases, a new class of delivery systems is evolving: highly miniaturized computerized delivery systems, integrated into a dental appliance. Dental delivery systems today are used in two ways: the main application is the local treatment of diseases affecting the oral cavity itself like periodontitis or fungal infections. The second is for systemic drug delivery.

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. The two amino acid exchanges in TetR E37A P39K are sufficient for tetO-4C5G specific binding. The E37A mutation increases the affinity of TetR for tetO variants and seems to be essential for binding to modified operator sequences. The Lys39 residue is in a position to directly contact the fourth and fifth bps of tetO thereby creating specificity for tetO-4C5G. Combinations of these mutations with others that lead to a reverse phenotype or altered inducer specificity yielded new TetR mutants with the respective combined activities. Single chain TetR variants were constructed that contain DNA reading heads with two different operator binding specificities. Specific binding of this TetR mutant to the respective mixed tetO-wt/4C5G variants containing one wild type and one double exchange operator half site was only accomplished at a low expression level of TetR variant, while cross-talk with other operator variants were observed at an elevated expression level. This observation emphasizes the importance of the transcription factor expression level for in vivo DNA binding specificity. These new TetR variants can be useful for multigene regulation systems.

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. A telemetry system operating at the 27 MHz ISM band was constructed using a small number of circuit components. The performance of the constructed system was evaluated in a laboratory condition. The transmission range of the realized system was measured up to 20 cm when a biological tissue in a thickness of 1 cm was applied between the transmitter and the receiver.

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.

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.

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.

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.

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.

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.

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.

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.