Showing papers by "Peter G. Schultz published in 1997"
TL;DR: The results suggest that the binding potential of the primary antibody repertoire may be significantly expanded by the ability of germline antibodies to adopt more than one combining-site configuration, with both antigen binding and somatic mutation stabilizing the configuration with optimal hapten complementarity.
Abstract: The crystal structures of a germline antibody Fab fragment and its complex with hapten have been solved at 2.1 A resolution. These structures are compared with the corresponding crystal structures of the affinity-matured antibody, 48G7, which has a 30,000 times higher affinity for hapten as a result of nine replacement somatic mutations. Significant changes in the configuration of the combining site occur upon binding of hapten to the germline antibody, whereas hapten binds to the mature antibody by a lock-and-key fit mechanism. The reorganization of the combining site that was nucleated by hapten binding is further optimized by somatic mutations that occur up to 15 A from bound hapten. These results suggest that the binding potential of the primary antibody repertoire may be significantly expanded by the ability of germline antibodies to adopt more than one combining-site configuration, with both antigen binding and somatic mutation stabilizing the configuration with optimal hapten complementarity.
565 citations
TL;DR: Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro.
Abstract: In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an “orthogonal” suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)–tRNA2Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase–tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein–tRNA interface and at sites further away, is discussed.
229 citations
TL;DR: CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation and a brief intraluminal exposure to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation.
153 citations
TL;DR: In this article, the spectroscopic and kinetic properties of wild-type catalase-peroxidase KatG from Mycobacterium tuberculosis as well as a specific mutant (R463L) frequently found in isoniazid-resistant strains have been overexpressed in Escherichia coli, allowing purification of sufficient quantities of enzyme for physical and kinetic characterization.
148 citations
136 citations
113 citations
TL;DR: In this article, the authors used a scanning-tip near-field microwave microscope to image the periodic ferroelectric domain boundaries at submicrometer resolution and showed a growth-instability-induced defect of the periodic domain structure.
Abstract: Variations in dielectric constant and patterns of microwave loss have been imaged in a yttrium-doped LiNbO 3 crystal with periodic ferroelectric domains with the use of a scanning-tip near-field microwave microscope. Periodic profiles of dielectric constant and images of ferroelectric domain boundaries were observed at submicrometer resolution. The combination of these images showed a growth-instability–induced defect of periodic domain structure. Evidence of a lattice-edge dislocation has also been observed through a stress-induced variation in dielectric constant.
102 citations
TL;DR: Comparison of the thermal stabilities of the amide- and ester-containing proteins shows that the ester substitution has a similar thermodynamic effect at all three positions, and introduction of theEster linkage in the middle of the helix, which alters two hydrogen-bonding interactions, destabilizes the protein by 1.7 kcal/mol.
Abstract: The contribution of backbone hydrogen bonds in α-helices to the overall stability of a protein has been examined experimentally by replacing several backbone amide linkages in α-helix 39−50 of T4 lysozyme with ester linkages. T4 lysozyme variants wherein the backbone amide bonds between residues Ser38 and Leu39, Lys43 and Leu44, or Ala49 and Ile50 are replaced with ester bonds were generated by incorporating α-hydroxy acids at positions 39, 44, or 50, respectively, using unnatural amino acid mutagenesis. The stabilities of the proteins were determined from their thermal denaturation curves as monitored by circular dichroism. Comparison of the thermal stabilities of the amide- and ester-containing proteins shows that the ester substitution has a similar thermodynamic effect at all three positions. At the N- and C-terminal positions, where only one hydrogen-bonding interaction is perturbed, the ester substitution is destabilizing by 0.9 and 0.7 kcal/mol, respectively. Introduction of the ester linkage in th...
102 citations
TL;DR: The combinatorial diversity of the immune system provided both an efficient catalyst for a reaction where no enzyme is known, as well as an opportunity to explore the mechanisms and evolution of biological catalysis.
Abstract: Antibody catalysis1 provides an opportunity to examine the evolution of binding energy and its relation to catalytic function in a system that has many parallels with natural enzymes. Here we report such a study involving an antibody AZ-28 that catalyses an oxy-Cope rearrangement, a pericyclic reaction that belongs to a well studied and widely used class of reactions in organic chemistry2. Immunization with transition state analogue 1 results in a germline-encoded antibody that catalyses the rearrangement of hexadiene 2 to aldehyde 3 with a rate approaching that of a related pericyclic reaction catalysed by the enzyme chorismate mutase3. Affinity maturation gives antibody AZ-28, which has six amino acid substitutions, one of which results in a decrease in catalytic rate. To understand the relationship between binding and catalytic rate in this system we characterized a series of active-site mutants and determined the three-dimensional crystal structure of the complex of AZ-28 with the transition state analogue. This analysis indicates that the activation energy depends on a complex balance of several stereoelectronic effects which are controlled by an extensive network of binding interactions in the active site. Thus in this instance the combinatorial diversity of the immune system provided both an efficient catalyst for a reaction where no enzyme is known, as well as an opportunity to explore the mechanisms and evolution of biological catalysis.
83 citations
TL;DR: A method for the combinatorial synthesis of 2,9-substituted purines using a Mitsunobu reaction to alkylate the N-9 position and an amination reaction to install amines at the C-2 position has been developed as mentioned in this paper.
82 citations
TL;DR: Artificially aminoacylated suppressor tRNAs were used to introduce photoreactive amino acids into model mitochondrial precursor proteins to probe the environment along the protein import pathway.
Abstract: Artificially aminoacylated suppressor tRNAs were used to introduce photoreactive amino acids into model mitochondrial precursor proteins to probe the environment along the protein import pathway. Amino acids with benzophenone side chains of various lengths [dl-2-amino-3-(p-benzoylphenyl)propanoic acid (1) and dl-2-amino-5-(p-benzoylphenyl)pentanoic acid (2)] were incorporated at specific sites throughout the cytochrome b2-dihydrofolate reductase fusion proteins, pb2(220)-DHFR and pb2Δ19(220)-DHFR, which were destined for the intermembrane space and the matrix in mitochondria, respectively. In vitro import of pb2(220)-DHFR and pb2Δ19(220)-DHFR bearing 1 or 2 into isolated yeast mitochondria was arrested so that the N terminus reached the intermembrane space or the matrix, respectively, while the DHFR domain remained at the mitochondrial surface. The matrix-targeted pb2Δ19(220)-DHFR was photocrosslinked to Tom40 in the outer membrane, Tim44 in the inner membrane, and Ssc1p in the matrix, suggesting that the protein has an extended conformation in the import channels. On the other hand, incorporation of 2 at various positions in the 50-residue segment of intermembrane-space-targeted pb2(220)-DHFR gave photocrosslinks only to Tom40, suggesting that the segment is not in an extended conformation, but localized near Tom40. The N-terminal portion of pb2(220)-DHFR, but not pb2Δ19(220)-DHFR, was photocrosslinked to an as-yet-unidentified mitochondrial component to generate a 95-kDa crosslinked product.
TL;DR: The crystal structure of 48G7-hapten was determined in the presence of hapten at 2.0 A resolution and in the absence of hapten at 1.7 A resolution.
TL;DR: The characterization of the properties of the resulting tRNAs and their suitability to severe as an orthogonal suppressor are reported, and the observed correlation between the effects of mutations at very distinct regions of the GlnRS-tRNA and possibly the ribosomal/tRNA complexes may contribute in part to the fidelity of protein biosynthesis.
TL;DR: This work has developed the strategyies and technologies for the combinatorial synthesis and evaluation of functional materials, in which large collections of different materials are rapidly synthesized, processed and screened for specific properties of interest.
Abstract: In order to increase the rate of materials discovery and optimization, as well as increase our understanding of increasingly complex modern materials, we have developed the strategyies and technologies for the combinatorial synthesis and evaluation of functional materials. In this approach large collections (libraries) of different materials are rapidly synthesized, processed and screened for specific properties of interest.
TL;DR: This mutant enzyme exhibits kCat/values that are near to those of wild type enzyme; however, the kcat/vs pH profile is much sharper with similar pKas of approximately 7.5 for both the ascending and descending limbs.
Abstract: The primary role of Tyr225 in the aspartate aminotransferase mechanism is to provide a hydrogen bond to stabilize the 3'O- functionality of bound pyridoxal phosphate. The strength of this hydrogen bond is perturbed by replacement of Tyr225 with 3-fluoro-L-tyrosine (FlTyr) by in vitro transcription/translation. This mutant enzyme exhibits kcat/values that are near to those of wild type enzyme; however, the kcat/vs pH profile is much sharper with similar pKas of approximately 7.5 for both the ascending and descending limbs. The pKas are assigned to the endocyclic proton of the internal aldimine and to the bridging hydrogen bond, respectively. The pKas in the kcat vs pH profile of 7.2 and 8.7 are assigned to the epsilon-NH3+ of lysine 258 and to the endocyclic protons of the ketimine complex, respectively. Arginine 292 forms a salt bridge with the beta-COOH of the substrate, aspartate. An improvement on the earlier attempt to invert the substrate charge specificity via R292D mutation-induced arginine transaminase activity [Cronin, C. N., & Kirsch, J. F. (1988) Biochemistry 27, 4572-4579] is described. Here Arg292 is replaced with homoglutamate (R292hoGlu). This construct exhibits 6.8 x 10(4)-fold greater activity for the cationic substrate D,L-[Calpha-3H]-alpha-amino-beta-guanidinopropionic acid (D,L-[Calpha-3H]AGPA) than does wild type enzyme. The gain in selectivity for this substrate is at least 4500-fold greater than that achieved in the 1988 experiment, i.e., [(kcat/KM)R292hoGlu/(kcat/KM)WT (D,L-[Calpha-3H]AGPA)] >/= 4500 x [(kcat/KM)R292D/(kcat/KM)WT (L-arginine)]. The value of (kcat/KM)R292D is 0.43 M-1 s-1 with L-Arg while (kcat/KM)R292hoGlu is 29 M-1 s-1 with D,L-[Calpha-3H]AGPA (it is assumed that the D-enantiomer is unreactive). The latter value is the lower limit because of the uncertain value of 3H kinetic isotope effect.
TL;DR: In this article, an iterative protocol for the solution-phase elaboration of O,S-thioformacetal oligomers is described, and a set of four O S-THI donor monomers with diverse chiral side-chains were coupled using NIS/TfOH activation to afford three model tetramers.
Patent•
22 Apr 1997TL;DR: In this article, the authors present methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon, and provide methods for the parallel synthesis and analysis of novel materials having useful properties.
Abstract: Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials or, alternatively, allowing the components to interact to form at least two different materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, nonbiological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.
TL;DR: In vitro immunization using a 5-membered ring iminocyclitol afforded antibodies that catalyse the hydrolysis of p-nitrophenyl glucopyranoside and p- nitrogen-containing compounds with rate enhancements of greater than 104.
TL;DR: The isolation of two additional Igk-V9 germline sequences are reported and evidence for the existence of several other, yet unidentified members of this gene family in different strains of mice is presented.
Abstract: While the human immunoglobulin loci have been fully mapped and sequenced (Zachau 1993; Matsuda et al. 1993; Cook and Tomlinson 1995; Frippiat et al. 1995; Williams et al. 1996), the mouse germline repertoire is considerably more complex, and the total number of variable (V) region genes is still unknown. In particular, the light chain kappa (Igk) locus has been characterized as heterogeneous, comprising several haplotypes, and germline genes seem to be forming a continuum rather than a set of distinct, nonoverlapping families (Kofler et al. 1989). The identification of germline V genes in mice is of particular interest because the correlation of mature immunoglobulins with their ancestors allows the reconstruction of specific pathways of somatic hypermutation and affinity maturation, as demonstrated in the case of a number of well-defined immune responses (Berek et al. 1985; Bothwell et al. 1981; Crews et al. 1981). These analyses can in turn provide insight into the diversification of the mouse immune repertoire in general (Rajewsky 1996; French et al. 1989). We have characterized the germline ancestors of a number of catalytic antibodies, in order to correlate their affinity maturation with the evolution of their catalytic activities (Patten et al. 1996; Ulrich et al. 1997; Ulrich and Schultz, unpublished results). Here we focus on the IgkV9, or MOPC41 family, as related sequences were found in the Vk regions of four of our catalytic antibodies. The size of the Igk-V9 germline family has been estimated as 7±11 members (Kofler et al. 1989), but only two distinct germline genes have yet been isolated, the myeloma-derived IgkMOPC41 (Seidman et al. 1979), and Igk-MOPC173B (Max et al. 1980), an actively transcribed, but non-functional light chain gene, presumably due to replacement of the conserved Trp 41 with Leu in the resulting protein, which prevents correct association with a heavy chain (Bernard et al. 1981). Its 59 flanking region was reported to differ from that of Igk-MOPC41 by several point mutations as well as seven nucleotide (nt) deletions (Max et al. 1980). Here we report the isolation of two additional Igk-V9 germline sequences and present evidence for the existence of several other, yet unidentified members of this gene family in different strains of mice. The Vk regions of the monoclonal antibodies AZ-12, AZ-14, and AZ-42, as well as 18R.136, which were raised against two unrelated transition state analogue haptens, had previously been cloned and identified as members of the Igk-V9 family (Ulrich et al. 1995; Ulrich and Schultz 1997). Particular germline genes can often be isolated by means of their 59 flanking regions, due to the confined nature of somatic hypermutation. The upstream boundary of somatic mutations has been localized between the cap site and the leader intron (Rothenfluh et al. 1993; Rada et al. 1994; Rogerson 1994); moreover, the non-coding regions often show considerably more diversity than the coding regions, including insertions and deletions that point to recombination events (Rothenfluh et al. 1995). The unmutated 59 flanking region can therefore serve as a marker for the germline origin of a particular V segment. Thus, in order to match the V genes of antibodies AZ-12, AZ-14, AZ-42, and 18R.136 to their germline precursors, their 59 flanking regions were isolated by polymerase chain reaction (PCR) from genomic hybridoma DNA using a primer based on the upstream regions of Igk-MOPC41 and Igk-MOPC173B in combination with a J region-specific primer. From these sequences (Fig. 1) we conclude that VAZ-12, VAZ-14, and VAZ-42 The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession numbers AF003291 (V18R.136), AF003292 (V18R.136 59), AF003293 (Igk-MOPC41), AF003294 (Igk-V9a), AF003295 (Igk-V9b), AF003296 (VAZ-12 59), AF003297 (VAZ-14 59), AF003298 (VAZ-42 59), AF003299 (VAZ-12), AF003300 (VAZ-14), AF003301 (VAZ-42)
TL;DR: A building block containing the 1,4-benzodiazepine-2,5-diazepine pharmacophore has been synthesized for use in constructing both peptide and carbamate combinatorial libraries as discussed by the authors.
Abstract: A building block containing the 1,4-benzodiazepine-2,5-diazepine pharmacophore has been synthesized for use in constructing both peptide and carbamate combinatorial libraries.