Linker Composition Research Articles

Red Fluorescent Genetically Encoded Voltage Indicators with Millisecond Responsiveness

Genetically encoded fluorescent indicators typically consist of the sensitive and reporter protein domains connected with the amino acid linkers. The final performance of a particular indicator may depend on the linker length and composition as strong as it depends on the both domains nature. Here we aimed to optimize interdomain linkers in VSD-FR189-188—a recently described red fluorescent protein-based voltage indicator. We have tested 13 shortened linker versions and monitored the dynamic range, response speed and polarity of the corresponding voltage indicator variants. While the new indicators didn’t show a contrast enhancement, some of them carrying very short interdomain linkers responded 25-fold faster than the parental VSD-FR189-188. Also we found the critical linker length at which fluorescence response to voltage shift changes its polarity from negative to positive slope. Our observations thus make an important contribution to the designing principles of the fluorescent protein-derived voltage indicators.

Optimal Linker Length for Small Molecule PROTACs that Selectively Target P38α and P38β for Degradation

We report the rational design of hetero-bifunctional small molecules that selectively target p38a and p38b for degradation These proteolysis targeted chimeras (PROTACs) are based on an ATP competitive inhibitor of p38a and p38b, which is linked to thalidomide analogues to recruit the Cereblon E3 ubiquitin ligase complex. Compound synthesis was facilitated by the use of a copper catalyzed “click” reaction. We show that optimization of the linker length and composition is crucial for the degradation-inducing activity of these PROTACs. We provide evidence that these chemical compounds can induce degradation of p38a and p38b but no other related kinases at nanomolar concentrations in several mammalian cell lines. Moreover, we show that the PROTACs mimic the effect of genetic deletion of p38a, and also induce p38a and p38b degradation upon administration to mice. Our compounds contribute to understanding the rational development of PROTACs, and will be a useful tool to validate functions of the p38 MAPK pathway and its involvement in pathologies.

Intrinsically Disordered Linkers Impart Processivity on Enzymes by Spatial Confinement of Binding Domains.

(1) Background: Processivity is common among enzymes and mechanochemical motors that synthesize, degrade, modify or move along polymeric substrates, such as DNA, RNA, polysaccharides or proteins. Processive enzymes can make multiple rounds of modification without releasing the substrate/partner, making their operation extremely effective and economical. The molecular mechanism of processivity is rather well understood in cases when the enzyme structurally confines the substrate, such as the DNA replication factor PCNA, and also when ATP energy is used to confine the succession of molecular events, such as with mechanochemical motors. Processivity may also result from the kinetic bias of binding imposed by spatial confinement of two binding elements connected by an intrinsically disordered (ID) linker. (2) Method: By statistical physical modeling, we show that this arrangement results in processive systems, in which the linker ensures an optimized effective concentration around novel binding site(s), favoring rebinding over full release of the polymeric partner. (3) Results: By analyzing 12 such proteins, such as cellulase, and RNAse-H, we illustrate that in these proteins linker length and flexibility, and the kinetic parameters of binding elements, are fine-tuned for optimizing processivity. We also report a conservation of structural disorder, special amino acid composition of linkers, and the correlation of their length with step size. (4) Conclusion: These observations suggest a unique type of entropic chain function of ID proteins, that may impart functional advantages on diverse enzymes in a variety of biological contexts.

Factors contributing to variability of glycan microarray binding profiles.

Protein-carbohydrate interactions play significant roles in a wide variety of biological systems. Glycan microarrays are commonly utilized to interrogate the selectivity, sensitivity, and breadth of these complex protein-carbohydrate interactions. During the past two decades, numerous distinct glycan microarray platforms have been developed, each assembled from a variety of slide-surface chemistries, glycan-attachment chemistries, glycan presentations, linkers, and glycan densities. Comparative analyses of glycan microarray data have shown that while many protein-carbohydrate interactions behave predictably across microarrays, there are instances when various array formats produce different results. For optimal construction and use of this technology, it is important to understand sources of variances across array platforms. In this study, we performed a systematic comparison of microarray data from 8 lectins across a range of concentrations on the CFG and neoglycoprotein array platforms. While there was good general agreement on the binding specificity of the lectins on the two arrays, there were some cases of large discrepancies. Differences in glycan density and linker composition contributed significantly to variability. The results provide insights for interpreting microarray data and designing future glycan microarrays.

Differential Conformational Dynamics Encoded by the Linker between Quasi RNA Recognition Motifs of Heterogeneous Nuclear Ribonucleoprotein H.

Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) F/H family are multipurpose RNA binding proteins that participate in most stages of RNA metabolism. Despite having similar RNA sequence preferences, hnRNP F/H proteins function in overlapping and, in some cases, distinct cellular processes. The domain organization of hnRNP F/H proteins is modular, consisting of N-terminal tandem quasi-RNA recognition motifs (F/HqRRM1,2) and a third C-terminal qRRM3 embedded between glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker, with several amino acids strictly conserved between hnRNP H and F. A significant difference occurs at position 105 of the linker, where hnRNP H contains a proline and hnRNP F an alanine. To investigate the influence of P105 on the conformational properties of hnRNP H, we probed the structural dynamics of its HqRRM1,2 domain with X-ray crystallography, NMR spectroscopy, and small-angle X-ray scattering. The collective results best describe that HqRRM1,2 exists in a conformational equilibrium between compact and extended structures. The compact structure displays an electropositive surface formed at the qRRM1–qRRM2 interface. Comparison of NMR relaxation parameters, including Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion, between HqRRM1,2 and FqRRM1,2 indicates that FqRRM1,2 primarily adopts a more extended and flexible conformation. Introducing the P105A mutation into HqRRM1,2 alters its conformational dynamics to favor an extended structure. Thus, our work demonstrates that the linker compositions confer different structural properties between hnRNP F/H family members that might contribute to their functional diversity.

Linker engineering in anti-TAG-72 antibody fragments optimizes biophysical properties, serum half-life, and high-specificity tumor imaging

Antibody (Ab) fragments have great clinical potential as cancer therapeutics and diagnostics. Their small size allows for fast clearance from blood, low immunoreactivity, better tumor penetration, and simpler engineering and production. The smallest fragment derived from a full-length IgG that retains binding to its antigen, the single-chain variable fragment (scFV), is engineered by fusing the variable light and variable heavy domains with a peptide linker. Along with switching the domain orientation, altering the length and amino acid sequence of the linker can significantly affect scFV binding, stability, quaternary structure, and other biophysical properties. Comprehensive studies of these attributes in a single scaffold have not been reported, making design and optimization of Ab fragments challenging. Here, we constructed libraries of 3E8, an Ab specific to tumor-associated glycoprotein 72 (TAG-72), a mucinous glycoprotein overexpressed in 80% of adenocarcinomas. We cloned, expressed, and characterized scFVs, diabodies, and higher-order multimer constructs with varying linker compositions, linker lengths, and domain orientations. These constructs dramatically differed in their oligomeric states and stabilities, not only because of linker and orientation but also related to the purification method. For example, protein L-purified constructs tended to have broader distributions and higher oligomeric states than has been reported previously. From this library, we selected an optimal construct, 3E8.G4S, for biodistribution and pharmacokinetic studies and in vivo xenograft mouse PET imaging. These studies revealed significant tumor targeting of 3E8.G4S with a tumor-to-background ratio of 29:1. These analyses validated 3E8.G4S as a fast, accurate, and specific tumor-imaging agent.

Acid Gas Stability of Zeolitic Imidazolate Frameworks: Generalized Kinetic and Thermodynamic Characteristics

Acid gases such as SO2 and CO2 are present in many environments in which the use of nanoporous metal–organic frameworks (MOFs) is envisaged. Among metal–organic frameworks, zeolitic imidazolate frameworks (ZIFs) have been extensively explored as membranes or adsorbents. However, there is little systematic knowledge of the effects of acid gas exposure on the structure of ZIFs, in particular the mechanistic aspects of ZIF degradation by acid gases as well as the effects of ZIF crystal topology and linker composition on their stability. Here we present a generalized and quantitative investigation of the kinetic and thermodynamic acid gas stability of a diverse range of ZIF materials. The stability of 16 ZIFs (of SOD, RHO, ANA, and GME topologies) under different environments—humid air, liquid water, and acid gases CO2 and SO2 (dry, humid, and aqueous)—is investigated by a suite of experimental and computational methods. The kinetics of ZIF degradation under exposure to humid SO2 is studied in detail, and eff...

Effect of peptide linker length and composition on immobilization and catalysis of leucine zipper‐enzyme fusion proteins

Linkers are critical components of fusion proteins, as they physically separate individual domains to enable each to fold and retain function. The role of peptide linker properties was investigated for fusions of a leucine zipper immobilization domain (ZE) to a chimeric amine dehydrogenase (AmDH) or a formate dehydrogenase (cbFDH). A linker library was developed, which varied in length, orientation, and proline content, as a way to vary stiffness. Fusion proteins were characterized by melting temperature, immobilization ability, cofactor binding, and kinetic activity. The best linker candidate for each enzyme was tested in a dual‐functionality assay, where enzymatic activity of fusions immobilized in protein‐inorganic supraparticles was greater than 80% after washing. The best linker for AmDH was completely different than that for cbFDH. This work highlights the need to experimentally assess linker properties in the design of new fusion proteins and provides a linker library for this purpose. © 2018 American Institute of Chemical Engineers AIChE J, 64: 2934–2946, 2018

Structure and Conformational Dynamics of the Splicing Factor hnRNP H

Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) H/F family are multi purpose RNA binding proteins that participate in most stages of RNA metabolism. Despite having similar RNA sequence preferences, hnRNP H/F proteins function in overlapping but distinct cellular processes. The domain organization of hnRNP H/F proteins is modular consisting of N-terminal tandem quasi-RNA Recognition Motifs (H/FqRRM12) and a third C-terminal qRRM3 embedded within glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker with most of the amino acids strictly conserved between hnRNP H and F. A significant difference occurs at position 99 of the linker where hnRNP H contains a proline and hnRNP F an alanine. To investigate the influence of P99 on the conformational properties of hnRNP H, we probed the structural dynamics of its HqRRM12 domain with x-ray crystallography, NMR spectroscopy, and Small Angle X-ray Scattering (SAXS). We observed in the HqRRM12 contains multiple structures in solution by SAXS. These exchangeable conformations that located on the linker region and RNA recognition sites reach certain equilibrium in different temperature by NMR relaxation dispersion. The collective results best describe that HqRRM12 exists in a conformational equilibrium between compact and extended structures. The compact structure displays an electropositive surface formed at the qRRM1-qRRM2 interface; the surface is abrogated within the extended structure. Comparison of NMR relaxation parameters between HqRRM12 and FqRRM12 indicate that FqRRM12 primarily adopts an extended conformation. Introducing the P99A mutation into HqRRM12 alters its conformational dynamics to favor the more extended structure. Thus, our work demonstrates that the linker compositions confer different structural properties between hnRNP H/F family members that might contribute to their functional diversity.

Impact of Linker Length and Composition on Fragment Binding and Cell Permeation: Story of a Bisbenzimidazole Dye Fragment.

Small molecules that modulate biological functions are targets of modern day drug discovery efforts. In a common platform fragment-based drug discovery, two fragments that bind to adjacent sites on a target are identified and are then linked together using different linkers to identify the linkage for optimum activity. What are not known from these studies are the effects these linkers, which typically contain C, H, and O atoms, have on the properties of the individual fragment. Herein, we investigate such effects in a bisbenzimidazole fragment whose derivatives have a wide range of therapeutic applications in nucleic acid recognition, sensing, and photodynamic therapy and as cellular probes. We report a dramatic effect of linker length and composition of alkynyl (clickable) Hoechst 33258 derivatives in target binding and cell uptake. We show that the binding of Hoechst 33258-modeled bisbenzimidazoles (1-9) that contain linkers of varying lengths (3-21 atoms) display length- and composition-dependent variation in B-DNA stabilization using a variety of spectroscopic methods. For a dodecamer DNA duplex, the thermal stabilization varied from 0.3 to 9.0 °C as the linker length increased from 3 to 21 atoms, respectively. Compounds with linker lengths of ≤11 atoms (such as compounds 1 and 5) are localized in the nucleus, while compounds with long linkers (such as compounds 8 and 9) are distributed in the extranuclear space, as well, with possible interactions with extranuclear targets. These findings provide insights into future drug design by revealing how linkers can influence the biophysical and cellular properties of individual drug fragments.

Abstract 3234: Development of potent and selective antibody-drug conjugates with pyrrole-based KSP inhibitors as novel payload class

Abstract The number of cytotoxic payload classes with different modes-of-action which have been successfully employed in antibody-drug conjugates (ADC) is still rather limited. So far, only ADCs with microtubule inhibitors, DNA binding payloads or topoisomerase I inhibitors have been advanced into clinical testing. To this end, the identification of ADC payload classes with a novel mode of action will increase therapeutic options and potentially help to overcome resistance. Inhibitors of kinesin spindle protein (KSP/Eg5) have generated interest due to their high antitumor potency. However, transferring the preclinical potency of small molecule KSP inhibitors (KSPis) into highly efficient clinical regimens with a sufficient therapeutic window has remained challenging. We have investigated a new pyrrole subclass of KSPis which showed subnanomolar potency against a large panel of tumor cell lines for their utility as a novel payload class in ADCs. Towards this goal different attachment sites for linkers have been explored in the KSPi molecule which were found compatible with cleavable and/or non-cleavable linkers. Subnanomolar potency and selectivity of ADCs with antibodies targeting either HER2, EGFR or TWEAKR could be demonstrated in vitro. For selected ADCs, the intracellular trafficking and metabolite formation was investigated and KSP inhibition was confirmed as the ADC mode of action. Depending on the linker composition differential profiles of the ADC metabolites with regard to efflux, cellular permeation, and bystander effect have been achieved. Moreover, specific accumulation in the tumor versus other tissues was demonstrated in biodistribution studies in vivo. In conclusion, KSP inhibitors have been established as a versatile new payload class for the generation of highly potent and selective ADCs. Citation Format: Hans-Georg Lerchen, Sven Wittrock, Nils Griebenow, Mario Lobell, Anne-Sophie Rebstock, Yolanda Cancho-Grande, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Anette Sommer, Sandra Berndt, Carsten Terjung, Heiner Apeler, Bertolt Kreft, Rolf Jautelat. Development of potent and selective antibody-drug conjugates with pyrrole-based KSP inhibitors as novel payload class [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3234. doi:10.1158/1538-7445.AM2017-3234

Tobacco etch virus protease mediating cleavage of the cellulose-binding module tagged colored proteins immobilized on the regenerated amorphous cellulose.

In this study, four fusion proteins were designed, in which the N-terminal cellulose-binding module as the affinity tag was immobilized on the regenerated amorphous cellulose (RAC), and the release of the C-terminal colored proteins was detected easily and rapidly after on-resin cleavage using the free tobacco etch virus protease (TEVp) variant, or the immobilized cognate protease with a binding capacity of up to 220mg protein per gram of RAC. The enhanced stability and repetitive use of the immobilized TEVp compensated slight loss of the catalytic efficiency toward the soluble protein substrate. On-resin cleavage and purity of the released target proteins are related to the context of the fusion tag, the incorporated linker composition, and the colored protein. Owing to low cost and high binding capacity of the RAC, the TEVp immobilized on the resin is an ideal alternative for removing fusion tag. The colored proteins are easily monitored in the on-resin process of fusion proteins, and rapid separation from RAC.

A "Click Chemistry Platform" for the Rapid Synthesis of Bispecific Molecules for Inducing Protein Degradation.

Proteolysis targeting chimeras (PROTACs) are bispecific molecules containing a target protein binder and an ubiquitin ligase binder connected by a linker. By recruiting an ubiquitin ligase to a target protein, PROTACs promote ubiquitination and proteasomal degradation of the target protein. The generation of effective PROTACs depends on the nature of the protein/ligase ligand pair, linkage site, linker length, and linker composition, all of which have been difficult to address in a systematic way. Herein, we describe a "click chemistry" approach for the synthesis of PROTACs. We demonstrate the utility of this approach with the bromodomain and extraterminal domain-4 (BRD4) ligand JQ-1 (3) and ligase binders targeting cereblon (CRBN) and Von Hippel-Lindau (VHL) proteins. An AlphaScreen proximity assay was used to determine the ability of PROTACs to form the ternary ligase-PROTAC-target protein complex and a MSD assay to measure cellular degradation of the target protein promoted by PROTACs.

Primer-Aided Truncation for the Creation of Hybrid Proteins.

Proteins frequently display modular architecture with several domains and segments connected by linkers. Proper protein functionality hinges on finely orchestrated interactions among these constituent elements. The underlying modularity lends itself to the engineering of hybrid proteins via modular rewiring; novel properties can thus be obtained, provided the linkers connecting the individual elements are conducive to productive interactions. As a corollary, the process of protein engineering often encompasses the generation and screening of multiple linker variants. To aid these steps, we devised the PATCHY method (primer-aided truncation for the creation of hybrid proteins) to readily generate hybrid gene libraries of predefined composition. We applied PATCHY to the mechanistic characterization of hybrid receptors that possess blue-light-regulated histidine kinase activity. Comprehensive sampling of linker composition revealed that catalytic activity and response to light are primarily functions of linker length. Variants with linkers of 7n residues mostly have light-repressed activity but those with 7n + 1 residues mostly have inverted, light-induced activity. We further probed linker length in the context of single residue exchanges that also lead to an inversion of the signal response. As in the original context, activity is only observed for certain periodic linker lengths. Taken together, these results provide mechanistic insight into signaling strategies employed by sensory photoreceptors and sensor histidine kinases. PATCHY represents an adequate and facile method to efficiently generate and probe hybrid gene libraries and to thereby identify key determinants for proper function.

Transposon for protein engineering.

Protein insertional fusion and circular permutation are 2 promising protein engineering techniques for creating integrated functionalities and sequence diversity of a protein, respectively. Finding insertion locations for protein insertional fusion and new termini for circular permutation through a rational approach is not always straightforward, especially, for proteins without detailed structural knowledge. On the contrary, a combinatorial approach facilitates a comprehensive search to evaluate all potential insertion sites and new termini locations. Conventional methods used to create random insertional fusion libraries generate sub-optimal inter-domain linker length and composition between fused proteins. There are also methods available for construction of random circular permutation libraries. However, these methods too, impose many drawbacks, such as significant sequence modification at the new termini of circular permutants and additionally, require re-design of transposons for tailored expression of circular permutants. Furthermore, these conventional methods employ relatively inefficient blunt-end ligation during library construction. In this commentary, we present a concise overview and key findings of engineered Mu transposons, which have recently been developed in our group as a facile and efficient tool to alleviate limitations realized from conventional methods and to construct high quality libraries for random insertional fusion and random circular permutation.

Synthesis, characterization, and tunable adsorption and diffusion properties of hybrid ZIF‐7‐90 frameworks

Hybrid zeolitic imidazolate frameworks (ZIFs), containing more than one type of imidazolate linker, can allow highly tunable molecular sieving and adsorption. Their crystallization becomes more challenging when the end‐member (single‐linker) ZIFs crystallize in different crystal systems. We demonstrate the controlled synthesis and detailed characterization of hybrid ZIF‐7‐90 frameworks containing linkers of ZIF‐7 (rhombohedral) and ZIF‐90 (cubic). ZIF‐7‐90 materials with SOD‐type topology are obtained in three crystalline phases depending on the linker composition and synthesis technique. The effect of synthesis conditions on the activation‐induced phase transition from rhombohedral to other topologies is studied. Nitrogen physisorption at 77 K and CO2 physisorption at 273 K shows the tunability of the pore‐size distribution and the framework flexibility as a function of framework composition. Measurements of water adsorption and butane isomer diffusion illustrate the tunability of diffusivity over seven orders of magnitude and control of hydrophobic to hydrophilic adsorption behavior. © 2015 American Institute of Chemical Engineers AIChE J, 62: 525–537, 2016

Influence of linker length and composition on enzymatic activity and ribosomal binding of neomycin dimers.

The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA.

Stabilization of resveratrol in blood circulation by conjugation to mPEG and mPEG-PLA polymers: investigation of conjugate linker and polymer composition on stability, metabolism, antioxidant activity and pharmacokinetic profile.

Resveratrol is naturally occurring phytochemical with diverse biological activities such as chemoprevention, anti-inflammatory, anti-cancer, anti-oxidant. But undergoes rapid metabolism in the body (half life 0.13h). Hence Polymer conjugation utilizing different chemical linkers and polymer compositions was investigated for enhanced pharmacokinetic profile of resveratrol. Ester conjugates such as α-methoxy-ω-carboxylic acid poly(ethylene glycol) succinylamide resveratrol (MeO-PEGN-Succ-RSV) (2 and 20 kDa); MeO-PEG succinyl ester resveratrol (MeO-PEGO-Succ-RSV) (2 kDa); α-methoxy poly(ethylene glycol)-co-polylactide succinyl ester resveratrol (MeO-PEG-PLAO-Succ-RSV) (2 and 6.6kDa) were prepared by carbodiimide coupling reactions. Resveratrol-PEG ethers (2 and 5 kDa) were synthesized by alkali-mediated etherification. All polymer conjugates were fully characterized in vitro and the pharmacokinetic profile of selected conjugates was characterized in rats. Buffer and plasma stability of conjugates was dependent on polymer hydrophobicity, aggregation behavior and PEG corona, with MeO-PEG-PLAO-Succ-RSV (2 kDa) showing a 3h half-life in rat plasma in vitro. Polymer conjugates irrespective of linker chemistry protected resveratrol against metabolism in vitro. MeO-PEG-PLAO-Succ-RSV (2 kDa), Resveratrol-PEG ether (2 and 5 kDa) displayed improved pharmacokinetic profiles with significantly higher plasma area under curve (AUC), slower clearance and smaller volume of distribution, compared to resveratrol.

Linker length and fusion site composition improve the optical signal of genetically encoded fluorescent voltage sensors.

Several genetically encoded fluorescent sensors of voltage were created by systematically truncating the length of the linker sequence between the voltage-sensing domain and the position of the fluorescent protein, Super Ecliptic A227D. In addition to varying the length, the amino acid composition at the fusion site for the fluorescent protein was modified. Both linker length and amino acid composition affected the size and voltage sensitivity of the optical signal. The truncation mutants revealed a potential structural periodicity with a maximum signal three amino acids from the voltage-sensing domain and another maximum 11 amino acids from the voltage-sensing domain. These results confirm that the linker length and composition can fine tune the size and voltage range of the sensor. The potential periodicity suggests that the orientation of the fluorescent protein could be important for improving the signal size implicating dimerization of the fluorescent protein.

Modulation of domain–domain interaction and protein function by a charged linker: A case study of mycobacteriophage D29 endolysin

Phage-encoded cell wall peptidoglycan hydrolyzing enzymes, called endolysins, are essential for efficient release of virions from bacteria, and show species-specific killing of the host. We have demonstrated previously that the interaction between N-terminal catalytic and C-terminal cell wall binding domains of mycobacteriophage D29 endolysin makes the enzyme inactive in Escherichiacoli. Here, we demonstrate that such interaction occurs intramolecularly and is facilitated by a charged linker that connects the two domains. We also show that linker composition is crucial for the inactivation of PG hydrolase in E. coli. Such knowledge will immensely help in bioengineering of endolysins with narrow or broad spectrum antimicrobial activity.