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B L Iverson

Publications and source records attributed to B L Iverson.

At least 19 recordsLinked to original sources
PubMed2001

(1)H NMR investigation of solvent effects in aromatic stacking interactions.

One of the marquis challenges in modern Organic Chemistry concerns the design and synthesis of abiotic compounds that emulate the exquisite complex structures and/or functions of biological macromolecules. Oligomers possessing the propensity to adopt well-defined compact conformations, or foldamers, have been attained utilizing hydrogen bonding, torsional restriction, and solvophobic interactions.(1) In this laboratory, aromatic electron donor--acceptor interactions have been exploited in the design of aedamers--foldamers that adopt a novel, pleated secondary structure in aqueous solution. Herein is reported detailed (1)H NMR binding studies of aedamer monomers that were carried out in solvents and solvent mixtures covering a broad polarity range. Curve-fitting analysis of the binding data using a model that incorporated the formation of higher order and self-associated complexes yielded a linear free energy relationship between the free energy of complexation and the empirical solvent polarity parameter, E(T)(30). From these studies, the association of electron-rich and electron-deficient aedamer monomers was seen to be driven primarily by hydrophobic interactions in polar solvents. However, the magnitude of these interactions is modulated to a significant extent by the geometry of the donor--acceptor complex, which, in turn, is dictated by the electrostatic complementarity between the electron-deficient and electron-rich aromatic faces of the monomers.

Journal Article↗
PubMed2001

Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones.

Disulfide bonds are normally formed after a polypeptide has been exported from the reducing environment of the cytoplasm into a more oxidizing compartment, such as the bacterial periplasm. Recently, we showed that in Escherichia coli trxB gor mutants, in which the reduction of thioredoxin and glutathione is impaired, the redox potential of the cytoplasm becomes comparable to that of the mammalian endoplasmic reticulum, thus allowing the formation of disulfide bonds in certain complex proteins (P. H. Bessette et al., 1999, Proc. Natl. Acad. Sci. USA 96, 13703-13708]. Here, we investigate the expression of a Fab antibody fragment in the bacterial cytoplasm. The effect of coexpressing cytoplasmic chaperones (GroEL/ES, trigger factor, DnaK/J), as well as signal sequenceless versions of periplasmic chaperones (DsbC and Skp), was examined. Skp coexpression was shown to have the most significant effect (five- to sixfold increase) on the yield of correctly folded Fab. A maximum yield of 0.8 mg Fab/L/OD(600) Fab was obtained, indicating that cytoplasmic expression may be a viable alternative for the preparative production of antibody fragments.

Amino Acid Sequence↗
PubMed2001

An octakis-intercalating molecule.

Herein we report the synthesis and characterization of a polyintercalator with eight potential intercalating l,4,5,8-naphthalenetetracarboxylic diimide (NDI) units linked in a head-to-tail arrangement via a peptide linker. UV spectroscopy and viscometry measurements indicated the molecule binds to double-stranded DNA with all eight NDI units intercalated simultaneously. Competition dialysis and DNAse 1 footprinting studies revealed a preference for GC-rich regions of DNA, and circular dichroism studies revealed significant distortion of B-form DNA upon binding. Our so-called "octamer" represents, to the best of our knowledge, the first intercalator that binds as an octakis-intercalator, capable of spanning at least 16 base pairs of DNA.

Animals↗
PubMed2001

The synthesis and screening of 1,4,5,8-naphthalenetetracarboxylic diimide-peptide conjugates with antibacterial activity.

We have employed an initial combinatorial approach followed by systematic lead optimization to investigate a series of novel molecules that exhibit antimicrobial activity against Gram-negative and Gram-positive bacteria. The new molecules contain various sequences of amino acids, generally L-lysine and glycine, attached to the 1,4,5,8-naphthalenetetracarboxylic diimide aromatic unit. Systematic structure-activity studies found that increasing positive charge enhanced activity and molecules containing one naphthalenetetracarboxylic diimide unit as well as at least seven lysine residues were optimum for antimicrobial activity. The naphthalenetetracarboxylic diimide derivatives were found to be inactive against mammalian cell lines, making them excellent antimicrobial candidates. Our results indicate that combining positive charge with aromatic and/or hydrophobic elements may be an interesting new approach to antimicrobial agents and adds an important new dimension to the field of cationic peptides.

Anti-Bacterial Agents↗
PubMed2001

Peptide bis-intercalator binds DNA via threading mode with sequence specific contacts in the major groove.

BACKGROUND: We previously described a general class of DNA polyintercalators in which 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) intercalating units are connected via peptide linkers, resulting in the first known tetrakis- and octakis-intercalators. We showed further that changes in the composition of the peptide tether result in novel DNA binding site specificities. We now examine in detail the DNA binding mode and sequence specific recognition of Compound 1, an NDI bis-intercalator containing the peptide linker gly-gly-gly-lys. RESULTS: 1H-NMR structural studies of Compound 1 bound to d(CGGTACCG)(2) confirmed a threading mode of intercalation, with four base pairs between the diimide units. The NMR data, combined with DNAse I footprinting of several analogs, suggest that specificity depends on a combination of steric and electrostatic contacts by the peptide linker in the floor of the major groove. CONCLUSIONS: In view of the modular nature and facile synthesis of our NDI-based polyintercalators, such structural knowledge can be used to improve or alter the specificity of the compounds and design longer polyintercalators that recognize correspondingly longer DNA sequences with alternating access to both DNA grooves.

Base Sequence↗
PubMed2001

Models of higher-order structure: foldamers and beyond.

Folding, an attribute common to biological macromolecules such as proteins and nucleic acids, enables the formation of complex three-dimensional structure and thus enables the function of these exquisite molecular machines. Chemists are exploring the folding of natural and artificial systems with increasing enthusiasm and boldness of molecular design. The most recent achievements in the area of artificial folding molecules are described in this review.

Nucleic Acid Conformation↗
PubMed2001

Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS).

Periplasmic expression with cytometric screening (PECS) is a powerful and rapid "display-less" technology for isolating ligand-binding proteins from diverse libraries. Escherichia coli expressing a library of proteins secreted into the periplasmic space are incubated with a fluorescent conjugate of the target ligand. Under the proper conditions, ligands as large as about 10 kDa can equilibrate within the periplasmic space without compromising the cell's integrity or viability. The bacterial cell envelope effectively serves as a dialysis bag to selectively retain receptor-fluorescent probe complexes but not free ligand. Cells displaying increased fluorescence are then isolated by flow cytometry. We demonstrate that scFv antibodies with both very high and low affinity to digoxigenin can be isolated from libraries screened by PECS using a benchtop flow cytometer. We also show that preexisting libraries constructed for display on filamentous bacteriophage can be screened by PECS without the need for subcloning. In fact, PECS was found to select for proteins that could be missed by conventional phage panning and screening methods.

Biochemistry↗
PubMed2000

Noncompetitive immunoassay of small analytes at the femtomolar level by affinity probe capillary electrophoresis: direct analysis of digoxin using a uniform-labeled scFv immunoreagent.

A general method for noncompetitive immunoassay of small analytes using affinity probe capillary electrophoresis (APCE) is demonstrated using digoxin as a model analyte. A uniform immunoreagent was prepared from a single-chain antibody (scFv) gene specific for digoxin. Site-directed mutagenesis introduced a unique cysteine residue for uniform labeling with a thiol-reactive fluorochrome. After expression in E. coli, the scFv was purified by immobilized metal affinity chromatography (IMAC) using an added C-terminal 6-histidine sequence. The protein was renatured and labeled while immobilized on the IMAC resin. After 0.02-microm filtration to remove microaggregates, the resulting reagent was highly uniform and stable at -12 degrees C for at least 1 year. Three formats of APCE using the scFv reagent were explored. A "mix-and-inject" assay optimized for low detection limits demonstrated analysis of 10 pM digoxin in aqueous standard solutions in 10 min. A rapid mix-and-inject format in a short capillary allowed detection of 1 nM digoxin in 1 min. Digoxin samples in serum and urine were injected directly after 10-fold dilution. In combination with solid-phase extraction, 400 fM digoxin was detected in 1 mL of serum. Including solid-phase extraction, reproducibility was within 2.5%, and the linear range was 3 orders of magnitude. The strategy adopted in this paper should be of general use in the low-level analysis of small analytes.

Affinity Labels↗
PubMed2000

Flow cytometric screening of cell-based libraries.

Flow cytometry is a powerful, high-throughput library screening tool in numerous applications including the isolation of bioactive molecules from synthetic combinatorial libraries, the identification of virulence genes in microorganisms, and the study and engineering of protein functions. Using flow cytometry, large libraries of protein mutants expressed in microorganisms can be screened quantitatively for desired functions, including ligand binding, catalysis, expression level, and stability. Rare target cells, occurring at frequencies below 10(-6), can be detected and isolated from heterogeneous library populations using one or more cycles of cell sorting and amplification by growth. Flow cytometry is particularly powerful because it provides the unique opportunity to observe and quantitatively optimize the screening process. However, the ability to isolate cells occurring at such low frequencies within a population requires consideration and optimization of screening parameters. With this aim, an analysis of the various parameters involved in screening cell-based libraries for rare target cells possessing a desired trait is presented.

Cell Separation↗
PubMed2000

Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies.

Random mutagenesis and selection using phage or cell surface display provides an efficient method for affinity maturation of single chain Fv (scFv) antibodies, thereby improving function in various applications. To investigate the effects of mutation frequency on affinity maturation, error-prone PCR was used to generate libraries containing an average (m) of between 1.7 and 22.5 base substitutions per gene in a high affinity scFv antibody that binds to the cardiac glycoside digoxigenin. The scFv antibody libraries were displayed on Escherichia coli, and mutant populations were analyzed by flow cytometry. At low to moderate mutation frequencies with an average mutation rate of m </= 8, the fraction of clones exhibiting binding to a fluorescently labeled conjugate of digoxigenin decreased exponentially (r(2) = 0.99), but the most highly mutated library (m = 22.5) had significantly more active clones than expected relative to this trend. A library with a low error rate (m = 1.7), one with moderate error rate (m = 3.8), and the one with high error rate (m = 22.5) were screened for high affinity clones under conditions of identical stringency using fluorescence-activated cell sorting. After several rounds of enrichment, each of the three libraries yielded clones with improved affinity for the hapten. The moderate and high error rate libraries gave rise to clones exhibiting the greatest affinity improvement. Taken together, our results indicate that (i) functional clones occur at an unexpectedly high frequency in hypermutated libraries, (ii) gain-of-function mutants are well represented in such libraries, and (iii) the majority of the scFv mutations leading to higher affinity correspond to residues distant from the binding site.

Amino Acid Sequence↗
PubMed2000

An investigation of antibody acyl hydrolysis catalysis using a large set of related haptens.

An aspect of catalytic antibody research that receives little attention in the literature involves hapten systems that fail to elicit antibody catalysts despite a high affinity immune response and hapten designs that resemble those known to elicit catalysts. We have investigated a series of 12 phosphate and phosphonate haptens in a total of three animal systems. Dramatic and reproducible differences were observed in the catalytic activities of polyclonal antibodies elicited by the different haptens. A phosphate hapten with a phenyl ring on the side of the hapten opposite the linker elicited reproducibly high levels of polyclonal antibody catalytic activity. The other 11 haptens, most with benzyl groups on the side of the hapten opposite the linker, elicited immune responses in which catalytic activity was significantly weaker in terms of the level of observed catalytic activity, as well as frequency of elicited catalysts. Our results indicate that subtle features of transition state analogue hapten structure can have a dramatic and reproducible influence over the catalytic activity of elicited antibodies in related haptens. Whatever the explanation, subtle changes in mechanistic features due to altered leaving group ability/location or overall hapten flexibility, the comprehensive data presented here indicate that phenyl or 4-nitrophenyl leaving groups located opposite the hapten linker are to be preferred in order to elicit highly active antibody catalysts for acyl hydrolysis reactions.

Animals↗
PubMed2000

Altered sequence specificity identified from a library of DNA-binding small molecules.

BACKGROUND: The ability to target specific DNA sequences using small molecules has major implications for basic research and medicine. Previous studies revealed that a bis-intercalating molecule containing two 1,4,5,8-napthalenetetracarboxylic diimides separated by a lysine-tris-glycine linker binds to DNA cooperatively, in pairs, with a preference for G + C-rich sequences. Here we investigate the binding properties of a library of bis-intercalating molecules that have partially randomized peptide linkers. RESULTS: A library of bis-intercalating derivatives with varied peptide linkers was screened for sequence specificity using DNase I footprinting on a 231 base pair (bp) restriction fragment. The library mixtures produced footprints that were generally similar to the parent bis-intercalator, which bound within a 15 bp G + C-rich repeat above 125 nM. Nevertheless, subtle differences in cleavage enhancement bands followed by library deconvolution revealed a derivative with novel specificity. A lysine-tris-beta-alanine derivative was found to bind preferentially within a 19 bp palindrome, without substantial loss of affinity. CONCLUSIONS: Synthetically simple changes in the bis-intercalating compounds can produce derivatives with novel sequence specificity. The large size and symmetrical nature of the preferred binding sites suggest that cooperativity may be retained despite modified sequence specificity. Such findings, combined with structural data, could be used to develop versatile DNA ligands of modest molecular weight that target relatively long DNA sequences in a selective manner.

Base Sequence↗
PubMed2000

Function-based isolation of novel enzymes from a large library.

Here we describe a high-throughput, quantitative method for the isolation of enzymes with novel substrate specificities from large libraries of protein variants. Protein variants are displayed on the surface of microorganisms and incubated with a synthetic substrate consisting of (1) a fluorescent dye (2) a positively charged moiety (3) the target scissile bond, and (4) a fluorescence resonance energy transfer (FRET) quenching partner. Enzymatic cleavage of the scissile bond results in release of the FRET quenching partner while the fluorescent product is retained on the cell surface, allowing isolation of catalytically active clones by fluorescence-activated cell sorting (FACS). Using a synthetic substrate with these characteristics, we enriched Escherichia coli expressing the serine protease OmpT from cells expressing an inactive OmpT variant by over 5,000-fold in a single round. Screening a library of 6 x 10(5) random OmpT variants by FACS using a FRET peptide substrate with a nonpreferred Arg-Val cleavage sequence resulted in the isolation of variant proteases with catalytic activities enhanced by as much as 60-fold. This approach represents a potentially widely applicable method for high-throughput screening of large libraries on the basis of catalytic turnover.

Boron Compounds↗
PubMed1999

In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site.

For the first time, each specificity determining residue (SDR) in the binding site of an antibody has been replaced with every other possible single amino acid substitution, and the resulting mutants analyzed for binding affinity and specificity. The studies were conducted on a variant of the 26-10 antidigoxin single chain Fv (scFv) using in vitro scanning saturation mutagenesis, a new process that allows the high throughput production and characterization of antibody mutants [Burks,E.A., Chen,G., Georgiou,G. and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412-417]. Single amino acid mutants of 26-10 scFv were identified that modulated specificity in dramatic fashion. The overall plasticity of the antibody binding site with respect to amino acid replacement was also evaluated, revealing that 86% of all mutants retained measurable binding activity. Finally, by analyzing the physical properties of amino acid substitutions with respect to their effect on hapten binding, conclusions were drawn regarding the functional role played by the wild-type residue at each SDR position. The reported results highlight the value of in vitro scanning saturation mutagenesis for engineering antibody binding specificity, for evaluating the plasticity of proteins, and for comprehensive structure-function studies and analysis.

Amino Acids↗
PubMed1999

Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface.

Polypeptide library screening technologies are critically dependent upon the characteristics of the expression system employed. A comparative analysis of the lpp-lac, tet and araBAD promoters was performed to determine the importance of tight regulation and expression level in library screening applications. The surface display of single-chain antibody (scFv) in Escherichia coli as an Lpp-OmpA' fusion was monitored using a fluorescently tagged antigen in conjunction with flow cytometry. In contrast to the lpp-lac promoter, both tet and araBAD promoters could be tightly repressed. Tight regulation was found to be essential for preventing rapid depletion of library clones expressing functional scFv and thus for maintaining the initial library diversity. Induction with subsaturating inducer concentrations yielded mixed populations of uninduced and fully induced cells for both the tet and araBAD expression systems. In contrast, homogeneous expression levels were obtained throughout the population using saturating inducer concentrations and could be adjusted by varying the induction time and plasmid copy number. Under optimal induction conditions for the araBAD system, protein expression did not compromise either cell viability or library diversity. This expression system was used to screen a library of random scFv mutants specific for digoxigenin for clones exhibiting improved hapten dissociation kinetics. Thus, an expression system has been developed which allows library diversity to be preserved and is generally applicable to the screening of E. coli surface displayed libraries.

Antibodies↗
PubMed1998

Anion selectivity of a sapphyrin-modified silica gel HPLC support.

A sapphyrin-modified silica gel support for use in high-performance liquid chromatography was prepared by attaching a sapphyrin monocarboxylic acid to aminopropyl silica gel through an amide bond. The anion retention characteristics of this modified silica gel were tested by exploring the extent to which a specific anion in the mobile phase would act to affect the rate at which AMP was eluted from an HPLC column containing this functionalized stationary phase. In general, it was found that phosphate and arsenate anions were more effective as eluents than carboxylic acids and halides, a result that was interpreted in terms of these former species binding better to sapphyrin (and hence being more effective in terms of displacing AMP) than other anions tested. Support for the contention that phosphate anions will bind to sapphyrin subunits covalently tethered to the silica gel came from solid state 31P NMR spectroscopic analyses. These revealed that the 31P nucleus undergoes a 5 ppm upfield shift, relative to control, when allowed to interact with the sapphyrin-containing support.

Adenosine Monophosphate↗
PubMed1998

Polyclonal antibody catalytic variability.

We have performed a systematic variability study of polyclonal antibody catalysis by using five rabbits immunized with the same hapten. Important results from this work are the following. (1) Similarities were observed in the catalytic polyclonal antibodies derived from all five rabbits. Four of the five rabbits produced polyclonal samples that were nearly the same in terms of catalytic activity, whereas the fifth rabbit, designated as rabbit 2, displayed a somewhat higher level of catalytic activity. The catalytic activities (as kcat/kuncat) of these polyclonal samples were similar to that from the best murine monoclonal antibody that had been previously elicited by the same hapten. (2) Titre was not an accurate indicator of polyclonal antibody catalytic activity. (3) A mathematical analysis to describe a distribution of Michaelis-Menten catalysts was performed to help interpret our results. (4) Kinetic analysis indicated that the binding parameters of the different samples were remarkably homogeneous, because one or two components were all that were required to fit the on-rate and off-rate data satisfactorily. Interestingly, the most active catalytic polyclonal sample, that from rabbit 2, displayed the slowest off-rate (so slow it could not be measured) and thus the highest overall affinity. (5) Catalytic analysis of eluted fractions of antibody from a substrate column indicated that each polyclonal sample was also relatively homogeneous in terms of catalytic parameters. The main conclusion of our study is that for this hapten-animal system, the overall catalytic immune response is relatively consistent at two levels. Consistent catalytic activity was observed between the polyclonal samples elicited in the different animals, and the elicited hapten-specific polyclonal antibodies were relatively homogeneous in terms of binding and catalytic parameters within each immunized animal. The observed similarities of the catalytic activity in the different animals is surprising, because the immune response is based on specific binding of antibodies to hapten. There is no known selective pressure to maintain consistent levels of catalytic activity. Our results can therefore be interpreted as providing evidence that for this hapten there is a fixed relationship between hapten structure and catalytic activity and/or consistent genetic factors that dominate the catalytic immune response.

Animals↗