Hemoglobinopathic erythrocytosis due to a new electrophoretically silent variant, hemoglobin San Diego (beta109 (G11)val--met).

Examination of 13 members of a Filipino family revealed that 6 had erythrocytosis inherited as a simple autosomal dominant trait. Application of several electrophoretic and chromatographic tests failed to reveal the presence of an abnormal hemoglobin in hemolysates from affected individuals. However, measurements of oxygen dissociation curves using whole bloods, dialyzed hemolysates, and 2,3-diphosphoglyceric acid-stripped hemolysates clearly showed that affected persons had an abnormal hemoglobin characterized by a high affinity for oxygen. Compositional analyses of all tryptic peptides from the beta-chains of the proband revealed a valyl-methionyl ambiguity in betaT12a. Blockage of lysyl residues and subsequent tryptic hydrolysis at arginyl residues permitted the isolation of fragments containing residues 105 through 146. Automatic sequence analysis of the fragments demonstrated the presence of both valine and methionine in nearly equal proportions at position beta109. This new hemoglobin variant is designated Hb San Diego (beta109(G11) Val-->Met).

The active-site and amino-terminal amino acid sequence of bovine intestinal alkaline phosphatase.

The active site of bovine intestinal alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) was labeled with [32P]Pi, a radioactive CNBr peptide was isolated and the amino acid sequence was determined. The sequence of the active-site peptide has limited homology (26%) with the active-site sequence of Escherichia coli alkaline phosphatase except for the ten residues immediately flanking the active-site serine (70%). A possible amino acid sequence deduced from the amino acid composition of an active-site tryptic peptide from human placental alkaline phosphatase is very similar to the bovine intestinal active-site sequence. The amino-terminal sequence of bovine intestinal alkaline phosphatase is homologous (69%) with the human placental enzyme but not with the E. coli phosphatase.

Production of HMG-3 by limited trypsin digestion of purified high-mobility-group nonhistone chromatin proteins.

Three isolated nonhistone proteins (HMG-1, HMG-2 and HMG-E) have been purified from chicken erythrocyte chromatin without exposure to overt denaturing conditions, and subjected to limited proteolysis. When treated with trypsin, the three proteins exhibited similar patterns of degradation, as judged by SDS and acid/urea gel electrophoresis. In particular, the first product, P1 (a relatively stable intermediate in each digestion), was a protein analogous to HMG-3, a principal degradation product in preparations of calf thymus high-mobility-group proteins. At least in the case of HMG-E, the products formed by tryptic attack on P1 are the two individual DNA binding domains of HMG-E. P1 derived from HMG-E and one of the individual DNA binding domains of HMG-E were purified by chromatography on columns containing DNA-cellulose or phosphocellulose. The properties of these two portions of HMG-E are consistent with our recently postulated three-domain structure for HMG-1 and its homologs (Reeck, G.R., Isackson, P.J. and Teller, D.C. (1982) Nature 300, 76-78). Thus, P1 consists of two DNA-binding domains of approximately equal molecular weight covalently linked together. From chromatography on DNA-cellulose columns, it is clear that P1 binds to DNA more tightly than does HMG-E. The highly acidic C-terminal domain of HMG-E (which is removed by trypsin in generating P1) thus counteracts the DNA binding of the two other domains of HMG-E (at least in the protein's interaction with purified DNA).

Structural and functional analyses of the repressor, RbsR, of the ribose operon of Escherichia coli.

The DNA sequence encoding the rbs repressor protein, RbsR, has been determined. Amino acid sequence analyses of the product of an rbsR-lacZ fusion and of affinity-purified RbsR demonstrate that translation begins at an unusual codon, TTG, and that the initial amino acid is removed during maturation of the protein. DNA-binding assays indicate that RbsR binds to a region of perfect dyad symmetry spanning the rbs operon transcriptional start site and that the affinity for the rbs operator is reduced by addition of ribose, consistent with ribose being the inducer of the operon. RbsR is a member of a family of homologous repressor proteins having very similar DNA-binding sites and binding to similar operator sequences.

Structural homology between rbs repressor and ribose binding protein implies functional similarity.

The deduced amino acid sequence of the rbs repressor, RbsR, of Escherichia coli is homologous over its C-terminal 272 residues to the entire sequence of the periplasmic ribose binding protein. RbsR is also homologous to a family of bacterial repressor proteins including LacI. This implies that the structure of the repressor consists of a two-domain binding protein portion attached to a DNA-binding domain having the four-helix structure of the LacI headpiece. The implications of these relationships to the mechanism of this class of repressors are discussed.

The proteins encoded by the rbs operon of Escherichia coli: II. Use of chimeric protein constructs to isolate and characterize RbsC.

Chimeric genes encoding full-length copies of rbsA and rbsC connected by segments coding for short bridge peptides were constructed and expressed in Escherichia coli. Surprisingly, the chimeric genes complemented the strain in which rbsA and rbsC were deleted. The chimeric proteins were overproduced, and the products were purified by affinity chromatography. In order to obtain highly purified protein, a poly-His leader peptide was incorporated so that Ni-chelate affinity chromatography could be employed. The leader peptide and the bridge peptide were designed with factor Xa-cleavable sites to permit recovery of the individual RbsA and RbsC protein. A rbsC gene encoding a poly-His leader was also constructed and expressed. Both the chimeric RbsA-C species and the poly-HisRbsC were produced at levels that permitted isolation of the equivalent of milligram quantities of RbsC per liter of culture. This is a substantial increase in amounts from any previous RbsC production vectors. All proteins from the rbs operon have now been overproduced and substantially purified.

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.

The proteins encoded by the rbs operon of Escherichia coli: I. Overproduction, purification, characterization, and functional analysis of RbsA.

The nucleotide-binding component of the high-affinity ribose transport system of Escherichia coli, RbsA, was overproduced from a T7-7 expression vector, and the protein was purified. Biochemical analyses of the purified protein indicated that the ATP analogues, 5'-FSBA and 8-azido ATP, covalently labeled the protein, a reaction that was inhibited by ATP, but not by GTP or CTP. The pure protein exhibited low-level ATPase activity with a K(m) of about 140 microM. Analyses of bacterial strains carrying chromosomal deletions of rbsA and other rbs genes suggested that RbsA is important for the chemotaxis function, a surprising result that was not anticipated from previous studies. However, an inconsistency between the several results from deletion strains raises questions regarding the interpretations of the in vivo data.

A new assignment of the disulfide linkage in stellacyanin.

Which cysteine of apostellacyanine reacts with 4-vinylpyridine in 6 M guanidine-HC1 depends upon the pH. We infer that a disulfide switch occurs at higher pH and that in the native protein the disulfide bridge occurs between Cys-59 and Cys-93. Thus Cys-87, which is homologous to the cysteine ligands of azurin and plastocyanin, is available to bind copper.

High-yield cleavage of tryptophanyl peptide bonds by o-iodosobenzoic acid.

A new procedure to cleave tryptophanyl peptide bonds in high yield is reported. The method involves treatment of the S-alkylated protein with o-iodosobenzoic acid. The procedure is highly selective for tryptophan and does not modify tyrosine or histidine, but may convert methionine to its sulfoxide derivative. The yields in the cleavage are 70--100%. Tryptophanyl bonds to alanine, glycine, serine, threonine, glutamine, arginine, and S-(pyridylethyl)cysteine are split in nearly quantitative yield, while those preceding isoleucine or valine are split in approximately 70% yield in the proteins examined in this work. The chemical mechanism for tryptophanyl bond cleavage has not been defined, but it is likely that oxidation of the indole ring occurs during the reaction with o-iodosobenzoic acid. Some problems with the quality of commercial preparations of the reagent are discussed.

Separation of large denatured peptides by reverse phase high performance liquid chromatography. Trifluoroacetic acid as a peptide solvent.

A method employing reverse phase high performance liquid chromatography has been developed for the purification of large denatured peptides. The cyanogen bromide fragments of hemoglobin alpha, beta, and gamma chains (13 to 91 residues in length) could be separated and recovered in 71 to 95% yields using LiChrosorb C-8 columns and a binary solvent system consisting of 0.013 M trifluoroacetic acid in water and a limiting organic solvent. This procedure permits the nondestructive detection of peptides at low wavelength (210 nm) on as little as 50 ng of sample. The peptides are recovered directly by evaporation or lyophilization. An elutropic solvent series for the separation of large and small peptides on reverse phase supports is also described.

Amino acid sequence of the L-arabinose-binding protein from Escherichia coli B/r.

The amino acid sequence of the L-arabinose-binding protein of Escherichia coli B/r was determined by sequenator analyses of reduced and S-pyridylethylated L-arabinose-binding protein and fragments derived by chemical and enzymatic cleavage of the native protein. The fragments were the products of cleavage by cyanogen bromide. BNPS-skatole, hydroxylamine, mild acid hydrolysis, limited trypsin digestion, chymotrypsin subdigestion, and subdigestion with Staphylococcus aureus protease V8. The COOH-terminal sequence was determined using bovine carboxypeptidases A and B and amino acid analyses. The L-arabinose-binding protein was determined to contain 306 amino acid residues, the sequence of which is presented below.

Isolation, crystallization, and primary amino acid sequence of human platelet factor 4.

Human platelet factor 4 was purified by a method employing affinity chromatography on heparin/agarose. The amino acid sequence of the protein was determined by automatic Edman degradations and carboxypeptidase Y digestion. There are 70 amino acids in the protein with 5 of the 8 negatively charged residues clustered near the NH2 terminus and 10 of the 13 positively charged residues in clusters of 3 and 4 elsewhere in the protein. Small crystals have been obtained from ammonium sulfate solutions which give a promising preliminary x-ray diffraction pattern.

Amino acid sequence of a carboxypeptidase inhibitor from tomato fruit.

The amino acid sequence of a 37 residue carboxypeptidase inhibitor from tomato fruit has been determined. The amino terminus was shown to be 2-oxopyrrolidine-5-carboxylic acid by digestion of reduced and S-carboxymethylated inhibitor with pyroglutamate aminopeptidase. The remainder of the sequence was assigned by analysis of peptides which had been generated by specific cleavage at the Asp4-Pro5 bond under acid conditions and by treatment with trypsin. The amino acid sequence of this inhibitor is identical with that of an analogous inhibitor from potatoes in 26 positions, and two of the replacements are highly conservative. The identification of the nonconservative replacements has been used to better define regions of the inhibitor which are not believed to contribute significantly to the free energy of association of the enzyme-inhibitor complex.

Caulobacter crescentus pilin. Purification, chemical characterization, and NH2-terminal amino acid sequence of a structural protein regulated during development.

The pili of Caulobacter crescentus are assembled during swarmer cell development at the differentiating cell pole. Under specific growth conditions, it was found that C. crescentus CB15 will produce insoluble pigmented granules which entrap pili lost during cell growth along with other extracellular proteins. This provided a strategy for protein purification. Pilin was purified from these granules by gel filtration in the presence of high levels of detergent. A protein of the appropriate molecular weight for pilin was isolated by this procedure and demonstrated to be pilin by specific labeling of the intact pilus with ferritin-coupled antibodies. CB15 pilin has an apparent molecular weight of 8,000, is rich in hydrophobic amino acids (greater than 70%), and has a broad isoelectric focusing range centered about a pI of 6.6. Periodic acid-Schiff reagent staining did not demonstrate carbohydrate modification of the monomer. The pilins of both CB15 and a related strain CB13 cross-react with anti-CB15 pilin antibody. Both strains also demonstrate similar RNA bacteriophage sensitivity, although there were significant differences in the amino acid composition, molecular weight, and other physical properties of the pilins from these two related strains. The NH2-terminal amino acid sequence of about 40% of the CB15 pilin molecular has been determined and bears some resemblance to that of some common pili; however, the sequences are not homologous and there is no indications of an unusual NH2-terminal amino acid in Caulobacter pilin.