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B Chait

Publications and source records attributed to B Chait.

9 recordsLinked to original sources
PubMed1997

Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization.

Interaction of the adenovirus penton base protein with alpha v integrins promotes virus entry into host cells. The location of the integrin binding sequence Arg-Gly-Asp (RGD) on human type 2 adenovirus (Ad2) was visualized by cryo-electron microscopy (cryo-EM) and image reconstruction using a mAb (DAV-1) which recognizes a linear epitope, IRGDTFATR. The sites for DAV-1 binding corresponded to the weak density above each of the five 22 A protrusions on the adenovirus penton base protein. Modeling of a Fab fragment crystal structure into the adenovirus-Fab cryo-EM density indicated a large amplitude of motion for the Fab and the RGD epitope. An unexpected finding was that Fab fragments, but not IgG antibody molecules, inhibited adenovirus infection. Steric hindrance from the adenovirus fiber and a few bound IgG molecules, as well as epitope mobility, most likely prevent binding of IgG antibodies to all five RGD sites on the penton base protein within the intact virus. These studies indicate that the structure of the adenovirus particle facilitates interaction with cell integrins, whilst restricting binding of potentially neutralizing antibodies.

Adenoviridae↗
PubMed1995

Participation and strength of interaction of lysine 95(beta) in the polymerization of hemoglobin S as determined by its site-directed substitution by isoleucine.

The role of Lys-95(beta), which is on the exterior of the hemoglobin (HbS) tetramer, in the aggregation process has been addressed because there is a lack of agreement on its importance. The early studies on the aggregation of HbS in the presence of other mutant hemoglobins are consistent with the subsequent electron microscopic studies in demonstrating the participation of Lys-95(beta) in gelation; the results of the crystal structure do not agree with these conclusions. Therefore, with the objective of clarifying its role we have carried out site-directed substitution of Lys-95(beta) to an isoleucine residue. The mutation was introduced by polymerase chain reaction recombination methodology, and the absence of other mutations in the beta-globin gene was established by sequencing the gene in its entirety. The recombinant mutant hemoglobin was expressed in yeast and characterized by peptide mapping and sequencing, which demonstrated that the only different tryptic peptide had the Ile substitution at position 95(beta). The recombinant hemoglobin had the correct amino acid composition and molecular weight by mass spectrometric analysis. It was also pure as judged by isoelectric focusing. It was fully functional because it had an average Hill coefficient of 3.1 and responded normally to the allosteric regulators, chloride, 2,3-diphosphoglycerate, and inositol hexaphosphate. Of particular interest was the finding that this hemoglobin mutant aggregated at a concentration of about 40 g/dl, nearly twice that at which HbS itself aggregated (24 g/dl). Therefore, Lys-95(beta) has a very important role in the aggregation process and is a good candidate site for the design of a therapeutic agent for sickle cell anemia.

Amino Acid Sequence↗
PubMed1994

Location, structure, and function of the target of a transcriptional activator protein.

We have isolated and characterized single-amino-acid substitution mutants of RNA polymerase alpha subunit defective in CAP-dependent transcription at the lac promoter but not defective in CAP-independent transcription. Our results establish that (1) amino acids 258-265 of alpha constitute an "activation target" essential for CAP-dependent transcription at the lac promoter but not essential for CAP-independent transcription, (2) amino acid 261 is the most critical amino acid of the activation target, (3) amino acid 261 is distinct from the determinants for alpha-DNA interaction, and (4) the activation target may fold as a surface amphipathic alpha-helix. We propose a model for transcriptional activation at the lac promoter that integrates these and other recent results regarding transcriptional activation and RNA polymerase structure and function.

Amino Acid Sequence↗
PubMed1991

Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease.

Hemoglobin is an important nutrient source for intraerythrocytic malaria organisms. Its catabolism occurs in an acidic digestive vacuole. Our previous studies suggested that an aspartic protease plays a key role in the degradative process. We have now isolated this enzyme and defined its role in the hemoglobinolytic pathway. Laser desorption mass spectrometry was used to analyze the proteolytic action of the purified protease. The enzyme has a remarkably stringent specificity towards native hemoglobin, making a single cleavage between alpha 33Phe and 34Leu. This scission is in the hemoglobin hinge region, unraveling the molecule and exposing other sites for proteolysis. The protease is inhibited by pepstatin and has NH2-terminal homology to mammalian aspartic proteases. Isolated digestive vacuoles make a pepstatin-inhibitable cleavage identical to that of the purified enzyme. The pivotal role of this aspartic hemoglobinase in initiating hemoglobin degradation in the malaria parasite digestive vacuoles is demonstrated.

Amino Acid Sequence↗
PubMed1989

Complete assignment of neurophysin disulfides indicates pairing in two separate domains.

The pairing of the 14 half-cystine residues of bovine neurophysin was established by sequential proteolytic digestion. Purified released peptides and the residual disulfide-linked core were monitored at each step by use of amino acid analysis, gas-phase sequencing, and mass spectrometry. The approach included application of gas-phase sequencing to assign disulfide pairs in peptides containing multiple disulfides. The results demonstrate that neurophysin disulfides are paired in two distinct domains--an NH2 domain (residues 10-54) containing four disulfides and a COOH domain (residues 61-85) containing three disulfides. The specific disulfide bridges are Cys-10 to Cys-54, Cys-13 to Cys-27, Cys-21 to Cys-44, Cys-28 to Cys-34, Cys-61 to Cys-73, Cys-74 to Cys-79, and Cys-67 to Cys-85. The results place the internally duplicated segments of neurophysin (residues 12-31 and 60-77) in separate domains. Disulfide-pairing patterns within each domain are homologous with the exception of the Cys-10 to Cys-54 bond, which is unique to the NH2 domain and which links the two ends of this domain together. The potential role of the Cys-10 to Cys-54 bond in organizing the hormone-binding site is discussed.

Amino Acid Sequence↗