Biomedical subjects

Alan M Friedman

Publications and source records attributed to Alan M Friedman.

7 recordsLinked to original sources

PubMed2006

Site-directed combinatorial construction of chimaeric genes: general method for optimizing assembly of gene fragments.

Site-directed construction of chimaeric genes by in vitro recombination "mixes-and-matches" precise building blocks from multiple parent proteins, generating libraries of hybrids to be tested for structure-function relationships and/or screened for favorable properties and novel enzymatic activities. A direct annealing and ligation method can construct chimaeric genes without requiring sequence identity between parents, except for the short (approximately 3 nt) sequences of the fragment overhangs used for specific ligation. Careful planning of the assembly process is necessary, though, in order to ensure effective construction of desired fragment assemblies and to avoid undesired assemblies (e.g., repetition of fragments, fragments out of order). We develop algorithms for specific planned ligation of short overhangs (SPLISO) that efficiently explore possible assembly plans, varying the fragment overhangs and the order of ligation steps in the assembly pathway. While there is a combinatorial explosion in the number of possible assembly plans as the number of breakpoints and parent genes increases, we employ a dynamic programming approach to find globally optimal ones in low-order polynomial time (in practice, taking only seconds for basic assembly plans). We demonstrate the effectiveness of our algorithms in planning the assembly of hybrid libraries, under a variety of experimental options and restrictions, including flexibility in the position and amino acid sequence of breakpoints. Our method promises to enable more effective application of site-directed recombination to protein investigation and engineering.

Algorithms↗

PubMed2005

The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A.

Replacement of a cis-proline by glycine at position 114 in ribonuclease A leads to a large decrease in thermal stability and simplifies the refolding kinetics. A crystallographic approach was used to determine whether the decrease in thermal stability results from the presence of a cis glycine peptide bond, or from a localized structural rearrangement caused by the isomerization of the mutated cis 114 peptide bond. The structure was solved at 2.0 A resolution and refined to an R-factor of 19.5% and an R(free) of 21.9%. The overall conformation of the protein was similar to that of wild-type ribonuclease A; however, there was a large localized rearrangement of the mutated loop (residues 110-117-a 9.3 A shift of the Calpha atom of residue 114). The peptide bond before Gly114 is in the trans configuration. Interestingly, a large anomalous difference density was found near residue 114, and was attributed to a bound cesium ion present in the crystallization experiment. The trans isomeric configuration of the peptide bond in the folded state of this mutant is consistent with the refolding kinetics previously reported, and the associated protein conformational change provides an explanation for the decreased thermal stability.

Amino Acid Substitution↗

PubMed2004

High sensitivity EndoV mutation scanning through real-time ligase proofreading.

The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 5' terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool.

Artifacts↗

PubMed2004

Probabilistic cross-link analysis and experiment planning for high-throughput elucidation of protein structure.

Emerging high-throughput techniques for the characterization of protein and protein-complex structures yield noisy data with sparse information content, placing a significant burden on computation to properly interpret the experimental data. One such technique uses cross-linking (chemical or by cysteine oxidation) to confirm or select among proposed structural models (e.g., from fold recognition, ab initio prediction, or docking) by testing the consistency between cross-linking data and model geometry. This paper develops a probabilistic framework for analyzing the information content in cross-linking experiments, accounting for anticipated experimental error. This framework supports a mechanism for planning experiments to optimize the information gained. We evaluate potential experiment plans using explicit trade-offs among key properties of practical importance: discriminability, coverage, balance, ambiguity, and cost. We devise a greedy algorithm that considers those properties and, from a large number of combinatorial possibilities, rapidly selects sets of experiments expected to discriminate pairs of models efficiently. In an application to residue-specific chemical cross-linking, we demonstrate the ability of our approach to plan experiments effectively involving combinations of cross-linkers and introduced mutations. We also describe an experiment plan for the bacteriophage lambda Tfa chaperone protein in which we plan dicysteine mutants for discriminating threading models by disulfide formation. Preliminary results from a subset of the planned experiments are consistent and demonstrate the practicality of planning. Our methods provide the experimenter with a valuable tool (available from the authors) for understanding and optimizing cross-linking experiments.

Algorithms↗

PubMed2003

Catalytic implications from the Drosophila protein L-isoaspartyl methyltransferase structure and site-directed mutagenesis.

Protein L-isoaspartyl methyltransferases (PIMT; EC 2.1.1.77) catalyze the S-adenosylmethionine-dependent methylation of L-isoaspartyl residues that arise spontaneously in proteins with age, thereby initiating a repair process that restores the normal backbone configuration to the damaged polypeptide. In Drosophila melanogaster, overexpression of PIMT in transgenic flies extends the normal life span, suggesting that protein damage can be a limiting factor in longevity. To understand structural features of the Drosophila PIMT (dPIMT) important for catalysis, the crystal structure of dPIMT was determined at a resolution of 2.2 A, and site-directed mutagenesis was used to identify the role of Ser-60 in catalysis. The core structure of dPIMT is similar to the modified nucleotide-binding fold observed in PIMTs from extreme thermophiles and humans. A striking difference of the dPIMT structure is the rotation of the C-terminal residues by 90 degrees relative to the homologous structures. Effectively, this displacement generates a more open conformation that allows greater solvent access to S-adenosylhomocysteine, which is almost completely buried in other PIMT structures. The enzyme may alternate between the open conformation found for dPIMT and the more closed conformations described for other PIMTs during its catalytic cycle, thereby allowing the exchange of substrates and products. Catalysis by dPIMT requires the side chain of the conserved, active site residue Ser-60, since substitution of this residue with Thr, Gln, or Ala reduces or abolishes the methylation of both protein and isoaspartyl peptide substrates.

Amino Acid Sequence↗

PubMed2003

Isolation of BsoBI restriction endonuclease variants with altered substrate specificity.

BsoBI is a thermophilic restriction endonuclease that cleaves the degenerate DNA sequence C/PyCGPuG (where/=the cleavage site and Py=C or T, Pu=A or G). In the BsoBI-DNA co-crystal structure the D246 residue makes a water-mediated hydrogen bond to N6 of the degenerate base adenine and was proposed to make a complementary bond to O6 of the alternative guanine residue. To investigate the substrate specificity conferred by D246 and to potentially alter BsoBI specificity, the D246 residue was changed to the other 19 amino acids. Variants D246A, D246C, D246E, D246R, D246S, D246T, and D246Y were purified and their cleavage activity determined. Variants D246A, D246S, and D246T display 0.2% to 0.7% of the wild-type cleavage activity. However, the substrate specificity of the three variants is altered significantly. D246A, D246S, and D246T cleave CTCGAG sites poorly. In filter binding assays using oligonucleotides, wild-type BsoBI shows almost equal affinity for CTCGAG and CCCGGG sites. In contrast, the D246A variant shows 70-fold greater binding affinity for the CCCGGG substrate. Recycled mutagenesis was carried out on the D246A variant, and revertants with enhanced activity were isolated by their dark blue phenotype on a dinD Colon, two colons lacZ DNA damage indicator strain. Most of the amino acid substitutions present within the revertants were located outside the DNA-protein interface. This study demonstrates that endonuclease mutants with altered specificity and non-lethal activity can be evolved towards more active variants using a laboratory evolution strategy.

Amino Acid Substitution↗

PubMed2002

Crystal structure of human L-isoaspartyl-O-methyl-transferase with S-adenosyl homocysteine at 1.6-A resolution and modeling of an isoaspartyl-containing peptide at the active site.

Spontaneous formation of isoaspartyl residues (isoAsp) disrupts the structure and function of many normal proteins. Protein isoaspartyl methyltransferase (PIMT) reverts many isoAsp residues to aspartate as a protein repair process. We have determined the crystal structure of human protein isoaspartyl methyltransferase (HPIMT) complexed with adenosyl homocysteine (AdoHcy) to 1.6-A resolution. The core structure has a nucleotide binding domain motif, which is structurally homologous with the N-terminal domain of the bacterial Thermotoga maritima PIMT. Highly conserved residues in PIMTs among different phyla are placed at positions critical to AdoHcy binding and orienting the isoAsp residue substrate for methylation. The AdoHcy is completely enclosed within the HPIMT and a conformational change must occur to allow exchange with adenosyl methionine (AdoMet). An ordered sequential enzyme mechanism is supported because C-terminal residues involved with AdoHcy binding also form the isoAsp peptide binding site, and a change of conformation to allow AdoHcy to escape would preclude peptide binding. Modeling experiments indicated isoAsp groups observed in some known protein crystal structures could bind to the HPIMT active site.

Amino Acid Sequence↗