Biomedical subjects

Peter G Schultz

Publications and source records attributed to Peter G Schultz.

At least 55 records · Page 3Linked to original sources

PubMed2005

A genome-wide overexpression screen in yeast for small-molecule target identification.

We describe a multicopy gene suppression screen of drug sensitivity in Saccharomyces cerevisiae that facilitates the identification of cellular targets of small molecules. An array of yeast transformants harboring a multicopy yeast genomic library was screened for resistance to growth inhibitors. Comparison of array growth patterns for several such inhibitors allowed the differentiation of general and molecule-specific genetic suppressors. Specific resistance to phenylaminopyrimidine (1), an inhibitor identified from a kinase-directed library, was associated with the overexpression of Pkc1 and a subset of downstream kinases. Components of two other pathways (pheromone response/filamentous growth and Pho85 kinase) that genetically interact with the PKC1 MAPK signaling cascade were also identified. Consistent with the suppression screen, inhibitor 1 bound to Pkc1 in yeast cell lysate and inhibited its activity in vitro. These results demonstrate the utility of this approach for the rapid deconvolution of small-molecule targets.

Drug Evaluation, Preclinical↗

PubMed2005

An expanding genetic code.

A general method was recently developed that makes it possible to genetically encode unnatural amino acids (UAAs) with diverse physical, chemical or biological properties in Escherichia coli, yeast, and mammalian cells. Over 30 UAAs have been cotranslationally incorporated into proteins with high fidelity and efficiency by means of a unique codon and corresponding tRNA-synthetase pair. A key feature of this methodology is the orthogonality between the new translational components and their endogenous host counterparts. Specifically, the codon for the UAA should not encode a common amino acid; neither the new tRNA nor cognate aminoacyl tRNA synthetase should cross-react with any endogenous tRNA-synthetase pairs; and the new synthetase should recognize only the UAA and not any of the 20 common amino acids. This methodology provides a powerful tool for exploring protein structure and function both in vitro and in vivo, as well as generating proteins with new or enhanced properties.

Amino Acids↗

PubMed2005

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine.

The Methanococcus jannaschii tRNA(Tyr)/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-L-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 A, respectively, for comparison with the published structure of TyrRS complexed with tRNA(Tyr) and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257-263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through pi-stacking and hydrogen bonding interactions. Loop 133-143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNA(Tyr). Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133-143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over L-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids.

Crystallography, X-Ray↗

PubMed2005

Small molecules and future regenerative medicine.

Recent advances in stem cell biology may make possible new approaches for the treatment of a number of diseases including cardiovascular disease, neurodegenerative disease, musculoskeletal disease, diabetes and cancer. These approaches could involve cell replacement therapy and/or drug treatment to stimulate the body's own regenerative capabilities by promoting survival, migration/homing, proliferation, and differentiation of endogenous stem/progenitor cells. However, such approaches will require identification of renewable cell sources of engraftable functional cells, an improved ability to manipulate their proliferation and differentiation, as well as a better understanding of the signaling pathways that control their fate. Cell-based phenotypic and pathway-specific screens of synthetic small molecules and natural products have historically provided useful chemical ligands to modulate and/or study complex cellular processes, and recently provided a number of small molecules that can be used to selectively regulate stem cell fate and developmental signaling pathways. Such molecules will likely provide new insights into stem cell biology, and may ultimately contribute to effective medicines for tissue repair and regeneration.

Animals↗

PubMed2004

Porphyrin distortion during affinity maturation of a ferrochelatase antibody, monitored by Resonance Raman spectroscopy.

Resonance Raman (RR) spectra are reported for mesoporphyrin IX bound to the Fab fragment of the ferrochelatase antibody 7G12. Binding induces activation of a Raman band at 680 cm(-1), which is assigned to an out-of-plane porphyrin vibration, gamma15. This is exactly the predicted effect of distorting mesoporphyrin to the geometry of N-methylmesoporphyrin IX, the 7G12 hapten, based on DFT/CIS modeling of the RR spectrum. The modeling also shows that the pyrrole ring that is tilted out of the porphyrin plane bears a nitrogen lone pair, which is therefore available for coordination by an incoming metal ion. The 680 cm(-1) band intensity is approximately 3 times higher for the affinity-matured antibody than for the germline precursor antibody, while intermediate values are found for variants in which germline residues are mutated to mature residues or vice versa. Thus, RR spectroscopy reveals an evolution from weak substrate distortion in the germline antibody to strong substrate distortion in the affinity-matured antibody, and supports the view that catalysis involves a substrate strain mechanism.

Computer Simulation↗

PubMed2004

Expanding the genetic code.

Although chemists can synthesize virtually any small organic molecule, our ability to rationally manipulate the structures of proteins is quite limited, despite their involvement in virtually every life process. For most proteins, modifications are largely restricted to substitutions among the common 20 amino acids. Herein we describe recent advances that make it possible to add new building blocks to the genetic codes of both prokaryotic and eukaryotic organisms. Over 30 novel amino acids have been genetically encoded in response to unique triplet and quadruplet codons including fluorescent, photoreactive, and redox-active amino acids, glycosylated amino acids, and amino acids with keto, azido, acetylenic, and heavy-atom-containing side chains. By removing the limitations imposed by the existing 20 amino acid code, it should be possible to generate proteins and perhaps entire organisms with new or enhanced properties.

Amino Acids↗

PubMed2004

A phage display system with unnatural amino acids.

We report a general method to display peptide-containing unnatural amino acids on filamentous M13 phage. Five distinct unnatural amino acids were site-specifically incorporated at the N-terminal of the M13 phage minor coat protein pIII. Phages that contain p-azidophenylalanine can undergo a highly specific azide-alkyne [3 + 2] cycloaddition reaction with an alkyne-derivatized fluorophore. The generalization of phage display to include unnatural amino acids should significantly increase the scope of phage display technology.

Amino Acids↗

PubMed2004

Site-specific incorporation of the mucin-type N-acetylgalactosamine-alpha-O-threonine into protein in Escherichia coli.

Glycosylation is a prevalent posttranslational process capable of augmenting and modulating protein function. Efficient synthesis of high-purity, homogeneous glycoproteins is essential for the study of unique protein glycoforms and for the manufacture of therapeutically relevant forms. A promising new strategy for controlled in vivo synthesis of glycoproteins was recently established using suppressor tRNA technology. Using an evolved tRNA aminoacyl synthetase-tRNA pair from Methanococcus jannaschii, the glycosyl amino acid, N-acetylglucosamine-beta-O-serine (GlcNAc-beta-Ser), was site-specifically introduced into proteins cotranslationally in Escherichia coli. Herein, we report the evolution of a new tRNA aminoacyl synthetase-tRNA pair that has expanded the repertoire of glycoproteins that can be expressed in E. coli to contain the other major O-linked glycan, N-acetylgalactosamine-alpha-O-threonine (GalNAc-a-Thr).

Acetylgalactosamine↗

PubMed2004

Site-specific PEGylation of proteins containing unnatural amino acids.

Here, we report a generally applicable PEGylation methodology based on the site-specific incorporation of para-azidophenylalanine into proteins in yeast. The azido group was used in a mild [3+2] cycloaddition reaction with an alkyne derivatized PEG reagent to afford selectively PEGylated protein. This strategy should be useful for the generation of selectively PEGylated proteins for therapeutic applications.

Amino Acids↗

PubMed2004

Development of a functionalized xenon biosensor.

NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of "remote" amplified detection. Here, we present a detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin. Binding of "functionalized" xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of ligand-target interaction. A control experiment in which the biotin-binding site of avidin was blocked with native biotin showed no such spectral changes, confirming that only specific binding, rather than nonspecific contact, between avidin and functionalized xenon leads to the effects on the xenon NMR spectrum. The exchange rate of xenon (between solution and cage) and the xenon spin-lattice relaxation rate were not changed significantly upon binding. We describe two methods for enhancing the signal from functionalized xenon by exploiting the laser-polarized xenon magnetization reservoir. We also show that the xenon chemical shifts are distinct for xenon encapsulated in different diastereomeric cage molecules. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor.

Avidin↗

PubMed2004

A genetically encoded photocaged amino acid.

We have developed a second orthogonal tRNA/synthetase pair for use in yeast based on the Escherichia coli tRNALeu/leucyl tRNA-synthetase pair. Using a novel genetic selection, we have identified a series of synthetase mutants that selectively charge the amber suppresor tRNA with the C8 amino acid, alpha-aminocaprylic acid, and the photocaged amino acid, o-nitrobenzyl cysteine, allowing them to be inserted into proteins in yeast in response to the amber nonsense codon, TAG.

Base Sequence↗

PubMed2004

The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination.

A recently developed method makes it possible to genetically encode unnatural amino acids with diverse physical, chemical or biological properties in Escherichia coli and yeast. We now show that this technology can be used to efficiently and site-specifically incorporate p-iodo-L-phenylalanine (iodoPhe) into proteins in response to an amber TAG codon. The selective introduction of the anomalously scattering iodine atom into proteins should facilitate single-wavelength anomalous dispersion experiments on in-house X-ray sources. To illustrate this, we generated a Phe153 --> iodoPhe mutant of bacteriophage T4 lysozyme and determined its crystal structure using considerably less data than are needed for the equivalent experiment with cysteine and methionine. The iodoPhe residue, although present in the hydrophobic core of the protein, did not perturb the protein structure in any meaningful way. The ability to selectively introduce this and other heavy atom-containing amino acids into proteins should facilitate the structural study of proteins.

Amino Acid Substitution↗

PubMed2004

Enthalpy arrays.

We report the fabrication of enthalpy arrays and their use to detect molecular interactions, including protein-ligand binding, enzymatic turnover, and mitochondrial respiration. Enthalpy arrays provide a universal assay methodology with no need for specific assay development such as fluorescent labeling or immobilization of reagents, which can adversely affect the interaction. Microscale technology enables the fabrication of 96-detector enthalpy arrays on large substrates. The reduction in scale results in large decreases in both the sample quantity and the measurement time compared with conventional microcalorimetry. We demonstrate the utility of the enthalpy arrays by showing measurements for two protein-ligand binding interactions (RNase A + cytidine 2'-monophosphate and streptavidin + biotin), phosphorylation of glucose by hexokinase, and respiration of mitochondria in the presence of 2,4-dinitrophenol uncoupler.

2,4-Dinitrophenol↗

PubMed2004

Substrate recognition by the AAA+ chaperone ClpB.

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.

Adenosine Triphosphatases↗

PubMed2004

Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells.

An orthogonal tryptophanyl-transfer RNA (tRNA) synthetase (TrpRS)-mutant opal suppressor tRNA(Trp) (mutRNA(UCA)(Trp)) pair was generated for use in mammalian cells. The anticodon loop of the Bacillus subtilis tRNA(Trp) was mutated to UCA, three positions in the D arm were mutated to generate an internal promoter sequence, and the mutRNA(UCA)(Trp) gene was inserted between the 5' and 3' flanking sequences of the tRNA(Trp-1) gene from Arabidopsis to enhance its expression in mammalian cells. In vitro aminoacylation assays and in vivo opal suppression assays showed that B. subtilis TrpRS (BsTrpRS) charges only the cognate mutRNA(UCA)(Trp) and no endogenous mammalian tRNAs. Similarly, the mutRNA(UCA)(Trp) is specifically charged by B. subtilis TrpRS and not by endogenous synthetases in mammalian cells. Site-directed mutagenesis was then used to alter the specificity of BsTrpRS to uniquely charge 5-hydoxy-l-tryptophan. The resulting mutant BsTrpRS-mutRNA(UCA)(Trp) pair allows the efficient and selective incorporation of 5-hydroxy-l-tryptophan into mammalian proteins in response to the codon, TGA. This amino acid can be used as a fluorescence probe and also undergoes electrochemical oxidation in situ to generate an efficient protein crosslinking.

5-Hydroxytryptophan↗

PubMed2004

An expanded genetic code with a functional quadruplet codon.

With few exceptions the genetic codes of all known organisms encode the same 20 amino acids, yet all that is required to add a new building block are a unique tRNA/aminoacyl-tRNA synthetase pair, a source of the amino acid, and a unique codon that specifies the amino acid. For example, the amber nonsense codon, TAG, together with orthogonal Methanococcus jannaschii or Escherichia coli tRNA/synthetase pairs have been used to genetically encode a variety of unnatural amino acids in E. coli and yeast, respectively. However, the availability of noncoding triplet codons ultimately limits the number of amino acids encoded by any organism. Here, we report the design and generation of an orthogonal synthetase/tRNA pair derived from archaeal tRNA(Lys) sequences that efficiently and selectively incorporates an unnatural amino acid into proteins in response to the quadruplet codon, AGGA. Frameshift suppression with L-homoglutamine (hGln) does not significantly affect protein yields or cell growth rates and is mutually orthogonal with amber suppression, permitting the simultaneous incorporation of two unnatural amino acids, hGln and O-methyl-L-tyrosine, at distinct positions within myoglobin. This work suggests that neither the number of available triplet codons nor the translational machinery itself represents a significant barrier to further expansion of the genetic code.

Amino Acids↗

PubMed2004

A catalysis-based selection for peroxidase antibodies with increased activity.

A biotin-tyramine conjugate (1) was found to covalently cross-link with peroxidase antibody 7G12 upon the catalytic oxidation of the tyramine moiety in the presence of hydrogen peroxide (H2O2). On the basis of this observation, a novel strategy was developed to select mutants of 7G12 Fab with enhanced peroxidase activity from a library of phage displayed antibodies. In such a selection, tyramine is oxidized by hydrogen peroxide in a process catalyzed by peroxidase antibodies displayed on phage. Antibodies with higher peroxidase activity are preferentially labeled with biotin through irreversible adduct formation between oxidized biotin-linked tyramine molecules and phenolic side chains of the antibody. The corresponding phage particles can then be selected via biotin-streptavidin interactions. Using this strategy, phage displayed libraries of antibody 7G12 were selected for higher peroxidase activity. As a result, mutations of antibody 7G12 that led to 10 to 20-fold increases in the peroxidase activity (kcat/Km) were identified, suggesting the validity of this method for the evolution of peroxidase antibodies based directly on catalytic turnover.

Antibodies, Catalytic↗

PubMed2004

Identification of p53 regulators by genome-wide functional analysis.

The p53 tumor-suppressor protein is a critical mediator of cellular growth arrest and the induction of apoptosis. To identify proteins involved in the modulation of p53 transcriptional activity, a gain-of-function cellular screen was carried out with an arrayed matrix of approximately 20,000 cDNAs. Nine genes previously unknown to be involved in regulating p53 activity were identified. Overexpression of seven of these genes (Hey1, Hes1, TFAP4, Osr1, NR2F2, SFRS10, and FLJ11339) resulted in up-regulation of p53 activity; overexpression of two genes (M17S2 and cathepsin B) resulted in down-regulation of p53 activity in mammalian cells. HES1, HEY1, and TFAP4, which are members of the basic helix-loop-helix transcription family, and OSR1 were shown to activate p53 through repression of HDM2 transcription. Ectopic expression of these basic helix-loop-helix transcription factors in both zebrafish and avian developmental systems activated p53 and induced apoptosis in vivo, resulting in a phenotype similar to that of p53 overexpression. Furthermore, ras- and myc-mediated transformation of mouse embryonic fibroblasts was abrogated by expression of HEY1 in a p53-dependent manner. These results suggest that these transcription factors are members of an evolutionarily conserved network that governs p53 function.

Animals↗