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B T Chait

Publications and source records attributed to B T Chait.

At least 37 records · Page 2Linked to original sources
PubMed2000

Expression and properties of recombinant HbA2 (alpha2delta2) and hybrids containing delta-beta sequences.

Hemoglobin A2 (alpha2delta2), which is present at low concentration (1-2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O2 binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A2 and systematically studying its functional properties. The construct expressing HbA2 contains only alpha and delta genes so that the extensive purification required to isolate natural HbA2 is circumvented. Although natural hemoglobin A2 is expressed at low levels in vivo, the amount of recombinant alpha2delta2 expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the alpha + beta or the alpha + gamma genes, respectively, are present on the construct. Recombinant HbA2 is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA2 are described.

Amino Acid Sequence↗
PubMed1999

Human TATA-binding protein-related factor-2 (hTRF2) stably associates with hTFIIA in HeLa cells.

The TATA-binding protein (TBP)-related factor TRF1, has been described in Drosophila and a related protein, TRF2, has been found in a variety of higher eukaryotes. We report that human (h)TRF2 is encoded by two mRNAs with common protein coding but distinct 5' nontranslated regions. One mRNA is expressed ubiquitously (hTRF2-mRNA1), whereas the other (hTRF2-mRNA2) shows a restricted expression pattern and is extremely abundant in testis. In addition, we show that hTRF2 forms a stable stoichiometric complex with hTFIIA, but not with TAFs, in HeLa cells stably transfected with flag-tagged hTRF2. Neither recombinant human (rh)TRF2 nor the native flag.hTRF2-TFIIA complex is able to replace TBP or TFIID in basal or activated transcription from various RNA polymerase II promoters. Instead, rhTRF2, but not the flag.hTRF2-TFIIA complex, moderately inhibits basal or activated transcription in the presence of rhTBP or flag.TFIID. This effect is either completely (TBP-mediated transcription) or partially (TFIID-mediated transcription) counteracted by addition of free TFIIA. Neither rhTRF2 nor flag. hTRF2-TFIIA has any effect on the repression of TFIID-mediated transcription by negative cofactor-2 (NC2) and neither substitutes for TBP in RNA polymerase III-mediated transcription.

Amino Acid Sequence↗
PubMed1999

Lysozyme degradation by the bovine multicatalytic proteinase complex (proteasome): evidence for a nonprocessive mode of degradation.

The multicatalytic proteinase complex (MPC, proteasome) is composed of 28 subunits organized into four rings surrounding a water-filled canal. The catalytic centers face the inner canal confining protein substrates to an enclosed space. Experimental findings obtained with MPC from archaebacteria suggest that degradation of proteins by the complex is processive and have led to the proposal that the lengths of the peptides formed during degradation depend on the distances between active sites in the catalytic chamber. To test whether these postulates are valid for the MPC from a higher organism, we examined the size distributions of products formed early versus late in the course of protein degradation using reduced carboxamidomethylated lysozyme (RCM-lysozyme) and MPC from bovine spleen and pituitary. The majority of final degradation products ranged in length from 6 to 20 amino acids without a clear predilection for peptides of a particular, uniform size. Our observations suggest that selection of cleavage sites is governed by the amino acid sequence specificity of the MPC catalytic sites rather than the distances between the active sites. Early in the course of degradation, peptides with masses between 5 and 10 kDa accumulated in more than 80-fold molar excess over the MPC, indicating dissociation of large, partially degraded intermediates. Initial cleavages occurred at distances between 10 and 44 amino acids from the N- or C-terminus of the molecule and often involved removal of a fragment from both the N- and C-termini of RCM-lysozyme. Our data indicate that degradation of proteins by MPCs from higher organisms involves a nonprocessive mechanism comprised of multiple, independent cleavages with dissociation of degradation intermediates. A general model for protein degradation by the MPC is discussed.

Animals↗
PubMed1999

Accurate quantitation of protein expression and site-specific phosphorylation.

A mass spectrometry-based method is described for simultaneous identification and quantitation of individual proteins and for determining changes in the levels of modifications at specific sites on individual proteins. Accurate quantitation is achieved through the use of whole-cell stable isotope labeling. This approach was applied to the detection of abundance differences of proteins present in wild-type versus mutant cell populations and to the identification of in vivo phosphorylation sites in the PAK-related yeast Ste20 protein kinase that depend specifically on the G1 cyclin Cln2. The present method is general and affords a quantitative description of cellular differences at the level of protein expression and modification, thus providing information that is critical to the understanding of complex biological phenomena.

Animals↗
PubMed1999

Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation.

The analysis of pea rbcS-3A promoter sequence showed that BoxII was necessary for the control of rbcS-3A gene expression by light. GT-1, a DNA-binding protein that interacts with BoxII in vitro, is a good candidate for being a light-modulated molecular switch controlling gene expression. However, the relationship between GT-1 activity and light-responsive gene activation still remains hypothetical. Because no marked de novo synthesis was detected after light treatment, light may induce post-translational modifications of GT-1 such as phosphorylation or dephosphorylation. Here, we show that recombinant GT-1 (hGT-1) of Arabidopsis can be phosphorylated by various mammalian kinase activities in vitro. Whereas phosphorylation by casein kinase II had no apparent effect on hGT-1 DNA binding, phosphorylation by calcium/calmodulin kinase II (CaMKII) increased the binding activity 10-20-fold. Mass spectrometry analyses of the phosphorylated hGT-1 showed that amongst the 6 potential phosphorylatable residues (T86, T133, S175, T179, S198 and T278), only T133 and S198 are heavily modified. Analyses of mutants altered at T86, T133, S175, T179, S198 and T278 demonstrated that phosphorylation of T133 can account for most of the stimulation of DNA-binding activity by CaMKII, indicating that this residue plays an important role in hGT-1/BoxII interaction. We further showed that nuclear GT-1 DNA-binding activity to BoxII was reduced by treatment with calf intestine phosphatase in extracts prepared from light-grown plants but not from etiolated plants. Taken together, our results suggest that GT-1 may act as a molecular switch modulated by calcium-dependent phosphorylation and dephosphorylation in response to light signals.

Amino Acid Sequence↗
PubMed1999

Immunoglobulin motif DNA recognition and heterodimerization of the PEBP2/CBF Runt domain.

The polyomavirus enhancer binding protein 2 (PEBP2) or core binding factor (CBF) is a heterodimeric enhancer binding protein that is associated with genetic regulation of hematopoiesis and osteogenesis. Aberrant forms of PEBP2/CBF are implicated in the cause of the acute human leukemias and in a disorder of bone development known as cleidocranial dysplasia. The common denominator in the natural and mutant forms of this protein is a highly conserved domain of PEBP2/CBF alpha, termed the Runt domain (RD), which is responsible for both DNA binding and heterodimerization with the beta subunit of PEBP2/CBF. The three-dimensional structure of the RD bound to DNA has been determined to be an S-type immunoglobulin fold, establishing a structural relationship between the RD and the core DNA binding domains of NF-kappaB, NFAT1, p53 and the STAT proteins. NMR spectroscopy of a 43.6 kD RD-beta-DNA ternary complex identified the surface of the RD in contact with the beta subunit, suggesting a mechanism for the enhancement of RD DNA binding by beta. Analysis of leukemogenic mutants within the RD provides molecular insights into the role of this factor in leukemogenesis and cleidocranial dysplasia.

Cleidocranial Dysplasia↗
PubMed1999

Mutational analysis of sickle haemoglobin (Hb) gelation.

The use of recombinant Hb has provided the advantage that any amino acid substitution can be made at sites not represented by natural mutants or that cannot be modified by chemical procedures. We have recently reported the expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that carries a plasmid containing the human alpha- and beta-globin cDNA sequences; N-terminal nascent protein processing is correct and a soluble correctly folded Hb tetramer is produced. The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle-cell anaemia patients. Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells. The misfolding of Hb reported for the Escherichia coli-expressed protein is not apparent for Hb expressed in yeast by any of the criteria that we have used for characterization. These findings indicate that this system is well suited to the production of HbS mutants to explore those areas of the HbS tetramer whose roles in the gelation process are not yet defined and to measure quantitatively the strength of such interactions at certain inter-tetrameric contact sites in the deoxy-HbS aggregate. This article reviews our studies on a number of sickle Hb mutants, including polymerization-enhancing HbS mutants and polymerization-inhibiting HbS mutants.

Amino Acid Sequence↗
PubMed1998

The N-terminal sequence affects distant helix interactions in hemoglobin. Implications for mutant proteins from studies on recombinant hemoglobin felix.

The N-terminal 18-amino acid sequence of the beta-chain of hemoglobin, as far as the end of the A helix, has been replaced by the corresponding sequence of the gamma-chain of fetal hemoglobin with the remaining sequence of the beta-chain retained (helices B through H). The gamma-beta-chain had the correct mass, and its entire sequence was established by mass spectrometric analysis of its tryptic peptides; the alpha-chain also had the correct mass. This recombinant hemoglobin (named Hb Felix) retains cooperativity and has an oxygen affinity like that of HbA both in the presence and absence of the allosteric regulators, 2,3-diphosphoglycerate or chloride but differs from HbF in its 2,3-diphosphoglycerate response. However, Hb Felix has some features that resemble fetal hemoglobin, i.e. its significantly decreased tetramer-dimer dissociation and its circular dichroism spectrum, which measure the strength of the tetramer-dimer interface in the oxy conformation and its rearrangement to the deoxy conformation, respectively. Even though Hb Felix contains the HbA amino acids at its tetramer-dimer interface, which is located at a distance from the substitution sites, its interface properties resemble those of HbF. Therefore, the N-terminal sequence and not just those amino acids directly involved at the subunit interface contacts with alpha-chains must have a strong influence on this region of the molecule. The results reinforce the concept of fluid long range relationships among various parts of the hemoglobin tetramer (Dumoulin, A., Manning, L. R., Jenkins, W. T., Winslow, R. M., and Manning, J. M. (1997) J. Biol. Chem. 272, 31326-31332) and demonstrate the importance of the N-terminal sequence, especially in some mutant hemoglobins, in influencing its overall structure by affecting the relationship between helices.

Amino Acid Sequence↗
PubMed1998

Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification.

Although mass spectrometric peptide mapping has become an established technique for the rapid identification of proteins isolated by polyacrylamide gel electrophoresis (PAGE), the results of the identification procedure can sometimes be ambiguous. Such ambiguities become increasingly prevalent for proteins isolated as mixtures or when only very small amounts of the proteins are isolated. The quality of the identification procedure can be improved by increasing the number of peptides that are extracted from the gel. Here we show that cysteine alkylation is required to ensure maximal coverage in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mapping of proteins isolated by PAGE. In the described procedure, alkylation was performed prior to electrophoresis to avoid the adventitious formation of acrylamide adducts during electrophoresis. In this way, homogeneous alkylation was obtained with three different alkylating reagents (4-vinylpyridine, iodoacetamide, acrylamide). Cysteine alkylation was also used as a tool for the identification of cysteine-containing peptides. Using a 1:1 mixture of unlabeled acrylamide and deuterium-labeled acrylamide ([2,3,3'-D3]acrylamide), the proteins of interest were alkylated prior to electrophoretic separation. Peptide mixtures produced by trypsin digestion of the resulting protein bands were analyzed by MALDI-TOF MS, and the cysteine content of the peptides was inferred from the isotopic distributions. The cysteine content information was readily obtained and used to improve the protein identification process.

Acrylamide↗
PubMed1998

Ser727-dependent recruitment of MCM5 by Stat1alpha in IFN-gamma-induced transcriptional activation.

Stat1alpha is a latent cytoplasmic transcription factor activated in response to interferon-gamma (IFN-gamma). The C-terminal 38 amino acids of Stat1alpha are required to trigger transcription and therefore may possibly serve as a transcription activation domain (TAD). Here we show that the C-terminus of Stat1alpha is an independent TAD which can interact with a specific group of nuclear proteins. Mutation of the Stat1 Ser727 and Leu724 decreases its transcriptional activity and affinity for the nuclear proteins. One of the interacting proteins was identified as MCM5, a member of the mini-chromosome maintenance (MCM) family involved in DNA replication. Both in vitro and in vivo interaction of Stat1alpha and MCM5 were demonstrated. Furthermore, the in vitro interaction required Ser727 and was enhanced by its phosphorylation. Transient over-expression of MCM5 enhanced transcriptional activation by Stat1alpha in a Ser727-dependent manner. Finally, changes in the level of nuclear localized MCM5 during the cell cycle correlated with the changes in transcriptional response to IFN-gamma acting through Stat1alpha. These results strongly suggest that MCM5 is recruited through interaction with Stat1alpha in a Ser727- and Leu724-dependent manner to play a role in optimal transcriptional activation.

Cell Cycle↗
PubMed1998

Crystal structure and functional analysis of the HERG potassium channel N terminus: a eukaryotic PAS domain.

The HERG voltage-dependent K+ channel plays a role in cardiac electrical excitability, and when defective, it underlies one form of the long QT syndrome. We have determined the crystal structure of the HERG K+ channel N-terminal domain and studied its role as a modifier of gating using electrophysiological methods. The domain is similar in structure to a bacterial light sensor photoactive yellow protein and provides the first three-dimensional model of a eukaryotic PAS domain. Scanning mutagenesis of the domain surface has allowed the identification of a hydrophobic "hot spot" forming a putative interface with the body of the K+ channel to which it tightly binds. The presence of the domain attached to the channel slows the rate of deactivation. Given the roles of PAS domains in biology, we propose that the HERG N-terminal domain has a regulatory function.

Animals↗
PubMed1998

Subunits of the yeast SWI/SNF complex are members of the actin-related protein (ARP) family.

The yeast SWI/SNF chromatin remodeling complex is comprised of 11 tightly associated polypeptides (SWI1, SWI2, SWI3, SNF5, SNF6, SNF11, SWP82, SWP73, SWP59, SWP61, and SWP29). We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify the genes that encode the SWP59 and SWP61 subunits. Surprisingly, we find that SWP59 and SWP61 are encoded by the ARP9 and ARP7 genes, respectively, which encode members of the actin-related protein (ARP) family. Sequence analyses have shown that ARP9 and ARP7 are 24-26% identical (48-51% similar) to yeast actin and that they are likely to maintain the overall actin fold. Deletion of either the ARP9 or ARP7 gene causes typical swi/snf phenotypes, including growth defects on media containing galactose, glycerol, or sucrose as sole carbon sources. ARP9 and ARP7 are also required for expression of an HO-lacZ fusion gene and for full transcriptional enhancement by the GAL4 activator. The identification of two ARP family members as crucial subunits of the SWI/SNF complex suggests that the complex may contain a total of three different ATPase subunits; furthermore, the similarity of ARP7 and ARP9 to the HSP and HSC family of ATPases suggests the possibility that chromatin remodeling by SWI/SNF may involve chaperone-like activities.

Actins↗
PubMed1998

Tissue plasminogen activator binding to the annexin II tail domain. Direct modulation by homocysteine.

Tissue plasminogen activator binds to endothelial cells via the calcium-regulated phospholipid-binding protein annexin II, an interaction that is inhibited by the prothrombotic amino acid homocysteine. We sought to identify the tissue plasminogen activator binding domain of annexin II and to determine the mechanism of its modulation by homocysteine. Tissue plasminogen activator binding to immobilized annexin II was inhibited by intact fluid phase annexin II but not by its "core" fragment (residues 25-339). Two overlapping "tail" peptides specifically blocked 65-75% of binding. Localization of the tissue plasminogen activator binding domain was confirmed upon specific inhibition by the hexapeptide LCKLSL (residues 7-12). Expressed C9G annexin II protein failed to support tissue plasminogen activator binding, while binding to C133G, C262G, and C335G was equivalent to that of wild type annexin II. Upon exposure to homocysteine, annexin II underwent a 135 +/- 4-Da increase in mass localizing specifically to Cys9 and a 60-66% loss in tissue plasminogen activator-binding capacity (I50 = 11 microM). Upon treatment of cultured endothelial cells with [35S]homocysteine, the dithiothreitol-sensitive label was recovered by immunoprecipitation with anti-annexin II IgG. These data provide a potential mechanism for the prothrombotic effect of homocysteine by demonstrating direct blockade of the tissue plasminogen activator binding domain of annexin II.

Amino Acid Sequence↗
PubMed1998

Structural conservation in prokaryotic and eukaryotic potassium channels.

Toxins from scorpion venom interact with potassium channels. Resin-attached, mutant K+ channels from Streptomyces lividans were used to screen venom from Leiurus quinquestriatus hebraeus, and the toxins that interacted with the channel were rapidly identified by mass spectrometry. One of the toxins, agitoxin2, was further studied by mutagenesis and radioligand binding. The results show that a prokaryotic K+ channel has the same pore structure as eukaryotic K+ channels. This structural conservation, through application of techniques presented here, offers a new approach for K+ channel pharmacology.

Amino Acid Sequence↗
PubMed1998

The structure of the potassium channel: molecular basis of K+ conduction and selectivity.

The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.

Amino Acid Sequence↗
PubMed1998

Substrate inhibition of D-amino acid transaminase and protection by salts and by reduced nicotinamide adenine dinucleotide: isolation and initial characterization of a pyridoxo intermediate related to inactivation.

D-Amino acid transaminase, a pyridoxal phosphate (PLP) enzyme, is inactivated by its natural substrate, D-alanine, concomitant with its alpha-decarboxylation [Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., Manning, J. M., Ringe, D., and Soda, K. (1992) Biochemistry 31, 6018-6023; Bhatia, M. B., Martinez del Pozo, A., Ringe, D., Yoshimura, T., Soda, K., and Manning, J. M. (1993) J. Biol. Chem. 268, 17687-17694]. beta-Decarboxylation of d-aspartate to d-alanine leads also to this inactivation [Jones, W. M., van Ophem, P. W., Pospischil, M. A., Ringe, D., Petsko, G., Soda, K., and Manning, J. M. (1996) Protein Sci. 5, 2545-2551]. Using a high-performance liquid chromatography-based method for the determination of pyridoxo cofactors, we detected a new intermediate closely related to the inactivation by d-alanine; its formation occurred at the same rate as the inactivation and upon reactivation it reverted to PLP. Conditions were found under which it was characterized by ultraviolet-visible spectral analysis and mass spectroscopy; it is a pyridoxamine phosphate-like compound with a C2 fragment derived from the substrate attached to the C'-4 of the pyridinium ring and it has a molecular mass of 306 consistent with this structure. In the presence of d-serine, slow accumulation of a quinonoid intermediate is also related to inactivation. The inactivation can be prevented by salts, which possibly stabilize the protonated aldimine coenzyme complex. The reduced cofactor, nicotinamide adenine dinucleotide, prevents D-aspartate-induced inactivation. Both of these events also are related to formation of the novel intermediate.

Alanine↗