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B T Chait

Publications and source records attributed to B T Chait.

At least 73 records · Page 4Linked to original sources
PubMed1996

A recombinant sickle hemoglobin triple mutant with independent inhibitory effects on polymerization.

As part of a comprehensive effort to map the most important regions of sickle hemoglobin that are involved in polymerization, we have determined whether two sites previously shown to be involved, Leu-88(beta) and Lys-95(beta), had additive effects when substituted. The former site is part of the hydrophobic pocket that binds Val-6(beta), the natural mutation of HbS, and the latter site is a prominent part of the hemoglobin exterior. A sickle hemoglobin triple mutant with three amino acid substitutions on the beta-chain, E6V/L88A/K95I, has been expressed in yeast and characterized extensively. Its oxygen binding curve, cooperativity, response to allosteric effectors, and the alkaline Bohr effect showed that it was completely functional. The polymer solubility of the deoxy triple mutant, measured by a new micromethod requiring reduced amounts of hemoglobin, was identical to that of the E6V(beta)/K95I(beta) mutant, i.e. when the K95I(beta) substitution was present on the same tetramer together with the naturally occurring E6V(beta) substitution, the L88A(beta) replacement had no additive effect on polymer inhibition. The results suggest that Lys-95(beta) on the surface of the tetramer and its complementary binding region on the adjoining tetramer are potential targets for the design of an effective antisickling agent.

Allosteric Site↗
PubMed1996

Matrix-assisted laser desorption ion trap mass spectrometry: efficient trapping and ejection of ions.

The present paper explores the coupling of a matrix-assisted laser desorption/ionization (MALDI) ion source with an ion trap mass analyzer, with particular emphasis on the development of methods for improving the efficiency of ion trapping and ejection. A technique is described for directly measuring, for the first time, the trapping efficiency of peptide ions produced in a remote external MALDI ion source. The technique was used to devise an improved scheme for trapping, which yielded efficiencies as high as 39%. An improved understanding of the resonant ejection process led us to a new resonant ejection parameter set that increased the ejection efficiency by 1 order of magnitude over more conventionally used parameter sets and allowed for marked improvements in the mass resolution of ions with m/z > 2500. The presently described improvements in the efficiencies of ion trapping and ejection together with improved methods for isolating and fragmenting ions (Qin, J.; Chait, B.T.Anal. Chem., following article in this tissue) lay the foundation for highly sensitive MALDI ion trap mass spectrometry of proteins (Qin, J.; et al. Anal. Chem. 1996, 68, 1784-1791).

Amino Acid Sequence↗
PubMed1996

Matrix-assisted laser desorption ion trap mass spectrometry: efficient isolation and effective fragmentation of peptide ions.

Effective analysis of the sequence of peptides using matrix-assisted laser desorption/ionization (MALDI) tandem ion trap mass spectrometry requires efficient mass isolation and the ability to induce extensive sequence-specific fragmentation. The present paper describes a new excitation scheme, which we term red-shifted off-resonance large-amplitude excitation (RSORLAE), that can deposit higher amounts of internal energy in ions than is feasible with conventional resonant excitation. The new method provides an effective means for inducing fragmentation of MALDI-produced peptide ions with m/z values up to 3500. Prior to excitation, it is necessary to isolate ions of interest with high efficiency. We demonstrate that isolation efficiencies of > 95% can be achieved by careful design of the rf scan functions used during ion isolation. In particular, sudden transitions in the amplitude of the rf field (from low to high amplitudes) must be avoided. The combined improvements in the efficiency for ion isolation and the efficacy of ion activation make MALDI tandem ion trap mass spectrometry a practical tool for the characterization of proteins with high sensitivity.

Amino Acid Sequence↗
PubMed1996

Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain.

The crystal structure of the conserved core of HIV-1 Nef has been determined in complex with the SH3 domain of a mutant Fyn tyrosine kinase (a single amino acid substitution, Arg-96 to isoleucine), to which Nef binds tightly. The conserved PxxP sequence motif of Nef, known to be important for optimal viral replication, is part of a polyproline type II helix that engages the SH3 domain in a manner resembling closely the interaction of isolated peptides with SH3 domains. The Nef-SH3 structure also reveals how high affinity and specificity in the SH3 interaction is achieved by the presentation of the PxxP motif within the context of the folded structure of Nef.

Amino Acid Sequence↗
PubMed1996

A practical ion trap mass spectrometer for the analysis of peptides by matrix-assisted laser desorption/ionization.

The present paper describes the performance of a newly configured matrix-assisted laser desorption/ionization quadrupole ion trap mass spectrometer (MALDI-ITMS), designed for biological applications that require the determination of the primary structures of proteins, e.g., the rapid identification of proteins and the elucidation of posttranslational modifications. The strategy used for solving problems of this type involves enzymatic digestion of the protein, followed by MALDI ion trap mass spectrometric analysis of the components of the resulting complex mixture of peptide ions. The new instrument is demonstrated to be a highly practical tool for analyzing proteins. In particular, mixtures containing as many as 30 peptide components can be rapidly and sensitively analyzed without prior chromatographic separation of the components. Informative tandem mass spectra can be obtained from the peptide components with m/z values up to 3500. A single subpicomole sample loading of a complex peptide mixture is more than sufficient for a complete set of experiments that includes both low- and high-resolution molecular mass determinations as well as a complete MS/MS study of the various components present in the sample. Extensive use is made of improved methods for trapping, isolating, fragmenting, and detecting ions in the ITMS (details are to be provided in two future papers).

Amino Acid Sequence↗
PubMed1996

Mapping protein-protein interactions by affinity-directed mass spectrometry.

A precise and rapid method for identifying sites of interaction between proteins was demonstrated; the basis of the method is direct mass spectrometric readout from the complex to determine the specific components of the proteins that interact--a method termed affinity-directed mass spectrometry. The strategy was used to define the region of interaction of a protein growth factor with a monoclonal antibody. A combination of proteolytic digestion and affinity-directed mass spectrometry was used to rapidly determine the approximate location of a continuous binding epitope within the growth factor. The precise boundaries of the binding epitope were determined by affinity-directed mass spectrometric analysis of sets of synthetic peptide ladders that span the approximate binding region. In addition to the mapping of such linear epitopes, affinity-directed mass spectrometry can be applied to the mapping of other types of molecule-molecule contacts, including ligand-receptor and protein-oligonucleotide interactions.

Amino Acid Sequence↗
PubMed1996

Structural similarity between TAFs and the heterotetrameric core of the histone octamer.

A complex of two TFIID TATA box-binding protein-associated factors (TA FIIs) is described at 2.0A resolution. The amino-terminal portions of dTAFII42 and dTAFII62 from Drosophila adopt the canonical histone fold, consisting of two short alpha-helices flanking a long central alpha-helix. Like histones H3 and H4, dTAFII42 and dTAFII62 form an intimate heterodimer by extensive hydrophobic contacts between the paired molecules. In solution and in the crystalline state, the dTAFII42/dTAFII62 complex exists as a heterotetramer, resembling the (H3/H4)2 heterotetrameric core of the histone octamer, suggesting that TFIID contains a histone octamer-like substructure.

Amino Acid Sequence↗
PubMed1996

Influence of matrix solution conditions on the MALDI-MS analysis of peptides and proteins.

Sample-matrix preparation procedures are shown to greatly influence the quality of the matrix-assisted laser desorption/ionization (MALDI) mass spectra of peptides and proteins. In particular, dramatic mass discrimination effects are observed when the matrix 4-hydroxy-alpha-cyanocinnamic acid is used for analyzing complex mixtures of peptides and proteins. The discrimination effects are found to be strongly dependent on the sample-matrix solution composition, pH, and the rates at which the sample-matrix cocrystals are grown. These findings demonstrate the need to exercise great care in performing and interpreting the MALDI analysis of biological samples. The results also indicate that there is a reverse-phase chromatographic-like dimension in the sample-matrix preparation procedures that can be exploited to optimize the analysis. The present work describes the conditions under which the majority of components of a complex mixture of peptides and proteins can be successfully measured.

Amino Acid Sequence↗
PubMed1996

Expression of human chorionic gonadotropin uniformly labeled with NMR isotopes in Chinese hamster ovary cells: an advance toward rapid determination of glycoprotein structures.

Most secreted eukaryotic proteins are modified by glycosylation, and it has been difficult to solve their structures by crystallographic or NMR techniques because of problems posed by the presence of the carbohydrate. The structure of a chemically deglycosylated form of the human pregnancy hormone, human chorionic gonadotropin (hCG), has been solved by crystallographic methods. Since chemical deglycosylation may have induced changes in the structure, and since it is known that deglycosylated hCG is biologically inactive, the crystallographic structure confirmation by NMR techniques. Also, it has not been possible to determine the structures of the isolated subunits, nor the nature of interactions between the carbohydrate side chains and the protein backbone by crystallographic methods. Structural information via NMR techniques can be obtained from proteins in solution if they can be uniformly labeled with 13C and 15N isotopes. We report the first such uniform labeling of a glycoprotein using a universal 13C- and 15N-labeling medium to express 13C, 15N-labeled hCG, suitable for solving the structure in solution of the native, biologically active form of hCG as well as that of its free subunits. The 13C, 15N-labeled recombinant hCG and its separated subunits are shown to be nearly identical to urinary hCG reference preparations on the basis of protein chemical studies, immunochemistry, biological activity, and the capability of isolated hormone subunits to recombine to form biologically active hormone. Mass spectrometric analysis and preliminary NMR studies indicate that the isotopic labeling is uniform and greater than 90% after only two growth passages in the labeling media. One unexpected finding during subunit purification was that lyophilization of glycoproteins from trifluoroacetic acid HPLC buffers may result in the loss of a significant portion of sialic acid.

Animals↗
PubMed1996

Nociceptin in vitro biotransformation in human blood.

In vitro biotransformation of a newly sequenced neuropeptide of 17 amino acid residues, named nociceptin and orphanin FQ by two separate research groups, was studied in human blood using matrix-assisted laser desorption/ionization mass spectrometry. Processing was carried out in freshly drawn blood incubated at 37 degrees C for various time periods. It was found that cleavage at peptide linkage Phe1-Gly2 was the predominant biotransformation pathway. Nociceptin (2-17) was the major biotransformation product. Further processing also occurred with the formation of a variety of minor biotransformation products. Cleavages at basic amino acid residues were observed, although these were not major biotransformation pathways found under these in vitro experimental conditions. Biotransformation of nociceptin followed a similar pattern to that of another neuropeptide, the endogenous opioid dynorphin A(1-17), but it appeared that nociceptin was more resistant to biotransformation in human blood in vitro than dynorphin A(1-17).

Adult↗
PubMed1996

Differential biotransformation of dynorphin A (1-17) and dynorphin A (1-13) peptides in human blood, ex vivo.

The biotransformation in human blood in vitro of three dynorphin A (Dyn A) peptides was studied by matrix assisted laser desorption mass spectrometry to determine whether the natural peptide, Dyn A(1-17), is biotransformed differently from Dyn A (1-13), the natural sequence shortened form used in numerous neurobiological and pharmacological studies. In addition to studies of Dyn A(1-17), a natural product from prodynorphin and Dyn A(1-13), a natural sequence truncation of Dyn A(1-17), Dyn A(1-10)amide, a synthetic analogue of Dyn A(1-17) presumed to be protected from rapid biotransformation was also studied Synthetic Dyn A peptides were incubated in freshly drawn blood for various periods of time prior to mass spectrometric analysis. Several peptide products were identified from each precursor; the time profiles of appearance and disappearance of the major products were followed. Substantial differences in products and especially in the rate of biotransformation were observed between the processing of Dyn A(1-17) and the two shorter Dyn A peptides, Dyn A(1-13) and Dyn A(1-10)amide. Significant amounts of the natural Dyn A(1-17) survived 4 h of incubation (half-life 3 h). Dyn A (2-17), a major processed product of Dyn A(1-17) in blood, continued to accumulate during the 4-h incubation period. By contrast, both Dyn A(1-13) and Dyn A(1-10) amide were biotransformed very rapidly with half-lives of < 1 min and 10 min, respectively. Most of the products from these two peptide precursors were also further processed rapidly, with the exception of Dyn A(4-12) and Dyn A(4-10)amide, which were detected for over 2 h. Dyn A(1-6) was found as a minor biotransformation product from all three precursor peptides. These findings suggest that an important function of the four C-terminal amino acid residues of the natural form, Dyn A(1-17) [compared to Dyn A(1-13)], is to stabilize or protect the peptide from biotransformation by enzymes, by preserving a natural hairpin structure possibly near the carboxyl-terminus.

Adult↗
PubMed1996

Umbilical cord transforming growth factor-beta 3: isolation, comparison with recombinant TGF-beta 3 and cellular localization.

The transforming growth factor beta (TGF-beta) family of growth modulators play critical roles in tissue development and maintenance. Recent data suggest that individual TGF-beta isoforms (TGF-beta 1, -beta 2 and -beta 3) have overlapping yet distinct biological actions and target cell specificities, both in developing and adult tissues. The TGF-beta 3 isoform was purified to homogeneity from both natural and recombinant sources and characterized by laser desorption mass spectrometry, by protein sequencing, by amino acid analysis and by biological activity. TGF-beta 3 was the major TGF-beta isoform in umbilical cord (230 ng/g), and was physically and biologically indistinguishable from recombinant TGF-beta 3 and from the tumor growth inhibitory (TGI) protein found in umbilical cord. Immunohistochemistry using antipeptide TGF-beta 3 specific antibody showed TGF-beta 3 localization in perivascular smooth muscle.

Amino Acid Sequence↗