Molecular Cross‐Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions

Nonribosomal peptide synthetases (NRPSs) employ multiple domains separated by linker regions to incorporate substrates into natural products. During synthesis, substrates are covalently tethered to carrier proteins that translocate between catalytic partner domains. The molecular parameters that govern translocation and associated linker remodeling remain unknown. Here, we used NMR to characterize the structure, dynamics, and invisible states of a peptidyl carrier protein flanked by its linkers. We showed that the N‐terminal linker stabilizes and interacts with the protein core while modulating dynamics at specific sites involved in post‐translational modifications and/or domain interactions. The results detail the molecular communication between peptidyl carrier proteins and their linkers and could guide efforts in engineering NRPSs to obtain new pharmaceuticals.     

Multimerization behaviour of single chain Fv variants for the tumour-binding antibody B72.3

A systematic study has been performed on the relationship betweenlinker length, relative orientation of variable domains, multimerizationbehaviour and antigen binding activity for single chain Fvs(scFvs) of the tumour-binding antibody B72.3. Thirteen scFvvariants with linkers comprising up to six repeats of the motifGly-Gly-Gry-Gly-Ser were studied. All these scFvs showed a tendencyto form dimers or higher molecular weight species, and thistendency decreased with increasing linker length. The dimersand higher molecular weight forms may arise from head to tailintermolecular association of V_H and V_L domains. For each linkerlength, scFvs with the organization VL-linker-VH showed greaterbinding activity than those with the organization VH-linker-VL.In fact, for the latter organization only the variant with a30 amino acid linker showed good binding activity, suggestingthat (0 for B72.3 the C-tenninus of VH or the N-tenninus ofVL makes a structural contribution to antigen binding, and (ii)shorter linkers interfere with this contribution. Antigen bindingstudies on scFvs should be interpreted with caution becauseof their tendency to multlmerize. Such multimerization can beminimized by using linkers longer than those in common use     

The role of hydrophilic linkers

In microemulsion formulations, linker molecules are additives that can enhance the surfactant-oil interaction (lipophilic linkers) or the surfactant-water interaction (hydrophilic linkers). In this paper, the role of the hydrophilic linker is elucidated through solubilization studies, interfacial tension studies, and by studying the partitioning of the hydrophilic linker into an optimum middle phase. This research used alkyl naphthalene sulfonates as the hydrophilic linkers, sodium dihexyl sulfosuccinate as the surfactant, and trichloroethylene as the oil phase. The hydrophilic linkers were found to have interfacial properties between a hydrotrope and a cosurfactant. More specifically, the data show that a hydrophilic linker is an amphiphile that coadsorbs with the surfactant at the oil/water interface but that has negligible interaction with the oil phase. The role of the hydrophilic linker can thus be interpreted as opening “holes” in the interface. Based on the characteristics of alkyl naphthalene linkers, carboxylic molecules were evaluated as hydrophilic linkers. For trichloroethylene microemulsions, sodium octanoate was found to be an alternative hydrophilic linker to sodium mono- and dimethyl naphthalene sulfonates.     

A new photoactive building block for investigation of DNA backbone interactions: photoaffinity labeling of human DNA polymerase beta

The cross-linking of target proteins or nucleic acids to light-activatable ligands is an important tool for elucidating molecular interactions. Through the use of photoaffinity-labeling reagents, several new insights into nucleic acid interactions have been obtained, for example in DNA replication and repair. In most known photoprobes, the applied light-sensitive functionalities are placed directly at the nucleobase or are attached via linkers to either the nucleobase or the phosphate backbone. Here we describe the first photoprobe that bears a light-sensitive aryl(trifluoromethyl)diazirine at the sugar moiety of a DNA oligonucleotide. We devised a route for the synthesis of the modified nucleoside and its incorporation into an oligonucleotide. The photoactive species was proven to be stable under the conditions employed in routine automated DNA synthesis. The modified oligonucleotide was shown by subsequent photolabeling studies of human DNA polymerase beta to form a covalent complex to the enzyme upon irradiation with near-UV light.     

Thermal transport across carbon nanotubes connected by molecular linkers

Nonequilibrium molecular dynamics is applied to investigate thermal transport across two CNTs connected longitudinally by molecular linkers, which is a basic building-block for CNT network structures. We show the effect of different numbers, monomer types, and lengths of molecular linkers on the interfacial thermal conductance between CNTs and molecular linkers. We also analyze the density of vibrational normal modes to further understand the interfacial thermal conductance between different molecular linkers and CNTs. For most of the molecular linker type we simulated, the interfacial thermal conductance decreases with the increasing chain length. We find that aromatic-backbone structures are better than aliphatic-backbone structures to obtain higher interfacial thermal conductance with CNTs. Incorporating double bonds, oxygen atoms and amide groups into polymer chains shifts or redistributes of the density of vibrational normal modes, which in turn tunes the interfacial thermal conductance of molecular linker with CNTs. These results provide guidance for choosing molecular linkers to build up large-scale CNT-based network structures with tunable thermal properties.     

Latent cytokines: development of novel cleavage sites and kinetic analysis of their differential sensitivity to MMP-1 and MMP-3

We have engineered a latent mouse interferon beta (mIFNbeta) using the latency associated peptide (LAP) of transforming growth factor beta1 (TGF-beta1) to protect the cytokine and avoid its interaction with its receptors. This approach improves the pharmacokinetic properties and reduces the pleiotropic effects limiting the current therapeutic use of cytokines. IFNbeta was fused to the LAP using two flexible linkers flanking a matrix metalloproteinase (MMP) cleavage site for the specific release of IFNbeta at disease sites. In order to improve the hydrolysis rate of the cleavage site, 15 different cleavable linkers were introduced in the LAP-mIFNbeta construct. The kinetic parameters relative to the linker cleavage by MMP-1 and MMP-3 were measured by an ELISA method. Among the modifications done, one of the constructs bearing the activation site of pro-MMPs was the best substrate for both enzymes. The introduction of a hydrophilic sequence derived from the furin cleavage site of the anthrax toxin protective antigen increased the sensitivity to MMP-3 to up to 29-fold. These data suggest that this strategy could be useful for improving the effectiveness of the delivery and targeting of protein therapeutics.     

Optical Control of Antibody Activity by Using Photocleavable Bivalent Peptide–DNA Locks

Antibody-based molecular recognition plays a central role in today's life sciences, ranging from immunoassays to molecular imaging and antibody-based therapeutics. Control over antibody activity by using external triggers such as light could further increase the specificity of antibody-based targeting. Here we present bivalent peptide–DNA ligands containing photocleavable linkers as a noncovalent approach by which to allow photoactivation of antibody activity. Light-triggered cleavage of the 3-amino-3-(2-nitrophenyl)propionic acid peptide linker converted the high-affinity bivalent peptide–DNA lock into weakly binding monovalent ligands, effectively restoring antibody targeting of cell-surface receptors. In this work, a proof of principle was provided with an anti-hemagglutinin antibody, but the molecular design of the lock is generic and applicable to any monoclonal antibody for which an epitope or mimotope of sufficient affinity is available.     

Rapid synthesis of new DNMT inhibitors derivatives of procainamide

DNA methyltransferases (DNMTs) are responsible for DNA methylation, an epigenetic modification involved in gene regulation. Families of conjugates of procainamide, an inhibitor of DNMT1, were conceived and produced by rapid synthetic pathways. Six compounds resulted in potent inhibitors of the murine catalytic Dnmt3A/3L complex and of human DNMT1, at least 50 times greater than that of the parent compounds. The inhibitors showed selectivity for C5 DNA methyltransferases. The cytotoxicity of the inhibitors was validated on two tumour cell lines (DU145 and HCT116) and correlated with the DNMT inhibitory potency. The inhibition potency of procainamide conjugated to phthalimide through alkyl linkers depended on the length of the linker; the dodecane linker was the best.     

Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies

Bispecific single-chain diabodies (scDb) consist of the variableheavy and light chain domains of two antibodies connected bythree linkers. The structure of an scDb in the V_H–V_L orientationis V_HA–linkerA–V_LB–linkerM–V_HB–linkerB–V_LA,with linkers A and B routinely chosen to be 5–6 residuesand linker M 15–20 residues. Here, we applied displayof scDb on filamentous phage to analyse the composition of optimallinker sequences. The three linkers were randomized in lengthand sequence using degenerated triplets coding for only sixhydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly,Ala). Antigen-binding clones were then isolated by one to tworounds of selection on the two different antigens recognizedby the bispecific scDb. Using an scDb directed against carcinoembryonicantigen (CEA) and ß-galactosidase (Gal), we foundthat monomeric scDb had a preferred length of 15 or more aminoacid residues for the middle linker M and of 3–6 residuesfor the linkers A and B. No obvious bias towards a preferredlinker sequence was observed. Reduction of the middle linkerbelow 13 residues led to the formation of dimeric scDb, whichmost likely results from interchain pairing between all theV_H and V_L domains. Dimeric scDb were also formed by fragmentspossessing a long linker M and linkers A and B of 0 or 1 residue.We assume that these dimeric scDb are formed by intrachain pairingof the central variable domains and interchain pairing of theflanking variable domains. Thus, the latter molecules representa novel format of bispecific and tetravalent molecules. Thedescribed strategy allows for the isolation of both optimizedand minimal linker sequences for the assembly of monomeric ordimeric single-chain diabodies.     

Native Chemical Ligation Directed by Photocleavable Peptide Nucleic Acid (PNA) Templates

A novel peptide–peptide ligation strategy is introduced that has the potential to provide peptide libraries of linearly or branched coupled fragments and will be suited to introduce simultaneous protein modifications at different ligation sites. Ligation is assisted by templating peptide nucleic acid (PNA) strands, and therefore, ligation specificity is solely encoded by the PNA sequence. PNA templating, in general, allows for various kinds of covalent ligation reactions. As a proof of principle, a native chemical ligation strategy was elaborated. This PNA‐templated ligation includes easy on‐resin procedures to couple linkers and PNA to the respective peptides, and a traceless photocleavage of the linker/PNA oligomer after the ligation step. A 4,5‐dimethoxy‐2‐nitrobenzaldehyde‐based linker that allowed the photocleavable linkage of two bio‐oligomers was developed.     

Guided molecular missiles for tumor-targeting chemotherapy--case studies using the second-generation taxoids as warheads

A long-standing problem in cancer chemotherapy is the lack of tumor-specific treatments. Traditional chemotherapy relies on the premise that rapidly proliferating cancer cells are more likely to be killed by a cytotoxic agent. In reality, however, cytotoxic agents have very little or no specificity, which leads to systemic toxicity, causing undesirable severe side effects. Therefore, the development of innovative and efficacious tumor-specific drug delivery protocols or systems is urgently needed. A rapidly growing tumor requires various nutrients and vitamins. Thus, tumor cells overexpress many tumor-specific receptors, which can be used as targets to deliver cytotoxic agents into tumors. This Account presents our research program on the discovery and development of novel and efficient drug delivery systems, possessing tumor-targeting ability and efficacy against various cancer types, especially multidrug-resistant tumors. In general, a tumor-targeting drug delivery system consists of a tumor recognition moiety and a cytotoxic warhead connected directly or through a suitable linker to form a conjugate. The conjugate, which can be regarded as a "guided molecular missile", should be systemically nontoxic, that is, the linker must be stable in blood circulation, but upon internalization into the cancer cell, the conjugate should be readily cleaved to regenerate the active cytotoxic warhead. These novel "guided molecular missiles" are conjugates of the highly potent second-generation taxoid anticancer agents with tumor-targeting molecules through mechanism-based cleavable linkers. These conjugates are specifically delivered to tumors and internalized into tumor cells, and the potent taxoid anticancer agents are released from the linker into the cytoplasm. We have successfully used omega-3 polyunsaturated fatty acids, in particular DHA, and monoclonal antibodies (for EGFR) as tumor-targeting molecules for the conjugates, which exhibited remarkable efficacy against human tumor xenografts in animal models. We have developed self-immolative disulfide linkers wherein the glutathione-triggered cascade drug release takes place to generate the original anticancer agent. The use of disulfide linkers is attractive beacuse it takes into account the fact that the concentration of glutathione is much higher (>1000 times) in tumor cells than in blood plasma. In order to monitor and elucidate the mechanism of tumor-targeting, internalization, and drug release, several fluorescent and fluorogenic probes using biotin as the tumor-targeting module were developed and used. Then, the progressive occurrence of the designed receptor-mediated endocytosis, drug release, and drug binding to the target protein (microtubules) has been successfully observed and confirmed by means of confocal fluorescence microscopy. These "guided molecular missiles" provide bright prospects for the development of highly efficacious new generation drugs for cancer chemotherapy.     

Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli

The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamideacetyltransferase subunits (E2p) of the Escherichia coli pyruvatedehydrogenase complex are connected by linker sequences whichare characteristically rich in alanine and proline residues.By facilitating domain movement these linkers are thought topromote interactions between the three types of active sitethat participate in the catalytic cycle of the complex. To investigatefunctional constraints associated with linker composition andsequence, the natural linker of an E2p subunit containing onelipoyl domain was replaced by shorter sequences containing:mixtures of alanine plus proline residues; mainly alanine; mainlyproline; and mainly charged residues. Each artificial linkerpossessed a central histidine residue for assessing linker flexibilityby ¹H-NMR spectroscopy. The resultant complexes exhibited 181%(proline), 74–79% (alanine plus proline), 63% (alanine)and 7% (charged residues) of parental activity compared witha value of 75% expected for a complex with a comparably shortenedlinker. The ¹H-NMR spectra showed that the alanine plus prolinelinkers are flexible but the alanine linker and the prolinelinker are relatively inflexible. Substantial variations inlinker sequence and composition were tolerated without lossof function, and the enhanced activity conferred by the prolinelinker was attributed to the combined effects of length andrelative inflexibility.     

Flexibility of C3h‐Symmetrical Linkers in Tris‐oligonucleotide‐Based Tetrahedral Scaffolds

Flexibility of tris‐oligonucleotides is determined by the length of their connecting hydrocarbon chains. Tris‐oligonucleotides are branched DNA building blocks with three oligonucleotide arms attached to a C_3h‐symmetrical linker core at these chains. Four tris‐oligonucleotides hybridise into a tetrahedral nanocage by sequence‐determined self‐assembly. The influence of methylene, ethylene and propylene chains was studied by synthesising sets of tris‐oligonucleotides and analysing the relative stability of the hybridisation products against digestion by mung bean nuclease by using gel electrophoresis. Linkers with ethylene chains showed sufficient flexibility, whereas methylene‐chain linkers were too rigid. Tris‐oligonucleotides based on the latter still formed tetrahedral scaffolds in intermixing experiments with linkers of higher flexibility. Thus, a new generation of versatile isocyanurate‐based linkers was established.     

Immobilization of gamma globulins and polyclonal antibodies of class IgG onto carbon-encapsulated iron nanoparticles functionalized with various surface linkers

The immobilization of gamma-globulins from human and bovine blood and human polyclonal antibody (IgG) onto carbon-encapsulated iron nanoparticles (CEINs) is reported. First, the CEINs were functionalized to introduce the surface acidic linkers with a different length. The shorter linker was introduced by a typical oxidation treatment in concentrated acids. The longer linker was anchored by a free radical addition route, in which the radicals were generated by the thermal decomposition of succinic acid acyl peroxide. Both functionalization routes caused a partial perforation of the carbon coating. This structural degradation was determined quantitatively by evaluation the Fe content in the functionalized CEINs. Gamma-globulins and human polyclonal antibody (IgG) were immobilized onto the linker-functionalized surface using the carbodiimide–amine type reaction. The success of immobilization was confirmed by infrared spectroscopy, thermogravimetry and protein assay, respectively. The immune reactivity of the immobilized primary human polyclonal antibody (IgG) was also confirmed in a reaction with a FITC-labeled goat secondary antibody (anti-IgG) recognizing the primary human antibody in CEINs. It has been found that the immobilization yield is primarily dependent on the length of the acidic linker, whilst the content of carboxylic groups is of secondary importance.     

The Charged Linker Modulates the Conformations and Molecular Interactions of Hsp90

The molecular chaperone Hsp90 supports the functional activity of specific substrate proteins (clients). For client processing, the Hsp90 dimer undergoes a series of ATP-driven conformational rearrangements. Flexible linkers connecting the three domains of Hsp90 are crucial to enable dynamic arrangements. The long charged linker connecting the N-terminal (NTD) and middle (MD) domains exhibits additional functions in vitro and in vivo. The structural basis for these functions remains unclear. Here, we characterize the conformation and dynamics of the linker and NTD−MD domain interactions by NMR spectroscopy. Our results reveal two regions in the linker that are dynamic and exhibit secondary structure conformation. We show that these regions mediate transient interactions with strand β8 of the NTD. As a consequence, this strand detaches and exposes a hydrophobic surface patch, which enables binding to the p53 client. We propose that the charged linker plays an important regulatory role by coupling the Hsp90 NTD−MD arrangement with the accessibility of a client binding site on the NTD.     

Fluorescent analysis of translesion DNA synthesis by using a novel, non-natural nucleotide analogue

The replication of damaged DNA is a promutagenic process that can lead to disease development. This report evaluates the dynamics of nucleotide incorporation opposite an abasic site, a commonly formed DNA lesion, by using two fluorescent nucleotide analogues, 2-aminopurine deoxyribose triphosphate (2-APTP) and 5-phenylindole deoxyribose triphosphate (5-PhITP). In both cases, the kinetics of incorporation were compared by using a 32P-radiolabel extension assay versus a fluorescence-quenching assay. Although 2-APTP is efficiently incorporated opposite a templating nucleobase (thymine), the kinetics for incorporation opposite an abasic site are significantly slower. The lower catalytic efficiency hinders its use as a probe to study translesion DNA synthesis. In contrast, the rate constant for the incorporation of 5-PhITP opposite the DNA lesion is 100-fold faster than that for 2-APTP. Nearly identical kinetic parameters are obtained from fluorescence quenching or the 32P-radiolabel assay. Surprisingly, distinct differences in the kinetics of 5-PhITP incorporation opposite the DNA lesion are detected when using either bacteriophage T4 DNA polymerase or the Escherichia coli Klenow fragment. These differences suggest that the dynamics of nucleotide incorporation opposite an abasic site are polymerase-dependent. Collectively, these data indicate that 5-PhITP can be used to perform real-time analyses of translesion DNA synthesis as well as to functionally probe differences in polymerase function.     

Further Investigations into Rigidity-Activity Relationships in BisQAC Amphiphilic Antiseptics

Thirty-six biscationic quaternary ammonium compounds were efficiently synthesized in one step to examine the effect of molecular geometry of two-carbon linkers on antimicrobial activity. The synthesized compounds showed strong antimicrobial activity against a panel of both Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). While the linker geometry showed only a modest correlation with antimicrobial activity, several of the synthesized bisQACs are promising potential antiseptics due to good antimicrobial activity (MIC≤2 μM) and their higher therapeutic indices compared to previously reported QACs.     

Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris

Fusion proteins composed of a cellulose-binding domain fromNeocallimastix patriciarum cellulase A and Candida antarcticalipase B were constructed using different linker peptides. Theaim was to create proteolytically stable linkers that were ableto join the functional modules without disrupting their function.Six fusion variants containing linkers of 4–44 residueswere expressed in Pichia pastoris and analysed. Three variantswere found to be stable throughout 7-day cultivations. The cellulose-bindingcapacities of fusion proteins containing short linkers wereslightly lower compared with those containing long linkers.The lipase-specific activities of all variants, in solutionor immobilized on to cellulose, were equal to that of the wild-typelipase.