Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (Myeloma protein Tro). IV. Isolation and characterization of the chymotryptic peptides of the H-chain (author's transl)]

The preceeding papers dealth with the purification and characterization of IgA immunoglobulin Tro and its H- and L-chains, the separation and characterization of the cyanogen bromide fragments and the isolation of the tryptic splitting products of the H-chain. In this paper the chymotryptic peptides of the H-chain are presented. All chymotryptic peptides were isolated and purified parallel to the isolation of tryptic peptides. On the basis of these overlapping splitting products the arrangement of the tryptic peptides in the protein could be determined. These and other splitting products listed in this paper contributed to the complete sequence determination of of the H-chain.     

Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (myeloma protein Tro), V. The arrangement of the tryptic peptides and a discussion of the complete primary structure of the H-chain (author's transl)]

This communication deals with the sequence work done with tryptic and chymotryptic peptides and some cyanogen bromide splitting products. With these peptides, and if necessary with their splitting peptides, the whole primary structure of the alpha1-H-chain of myeloma protein Tro is established. The position of the amides is determined by electrophoresis and digestion with aminopeptidase M. The alpha1-chain Tro comprises 475 amino acid residues. Because of its specific exchanges and deletions the variable part of alpha1-chain Tro belongs to subgroup III of variable parts of H-chains. The switch from the variable to the constant part occurs at position 119/120 and is analogous to other chains which have been sequenced up to now. The large number of cysteine residues in the alpha-chain which may influence the tertiary structure, especially in the hinge and the subsequent CH2-region, is noteworthy. Furthermore, myeloma protein Tro is compared with the other alpha1-chain Bur[5] sequenced in the meantime, and protein But[6], which is an IgA2 molecule of the allotype A2m(2).     

Primary structure of the hemoglobins from the greater Kudu antelope (Tragelaphus strepsiceros)

The adult greater Kudu antelope has two hemoglobin components, Hb A and Hb B, with one alpha and two beta chains. The complete amino-acid sequences of these three chains are presented. The two beta chains differ only in one residue at position 16 (Gly----Ser) and may be the product of two allelic genes. The primary structure of the chains was determined by sequencing the tryptic peptides after their isolation from the tryptic digest of the chains by high performance liquid chromatography. The alignment of these peptides was deduced from homology with the chains of bovine hemoglobin. Between the Kudu hemoglobins and those of cattle a high degree of homology was found.     

Solution structure of the loops of bacteriorhodopsin closely resembles the crystal structure

Bacteriorhodopsin is one of very few transmembrane proteins for which high resolution structures have been solved. The structure shows a bundle of seven helices connected by six turns. Some turns in proteins are stabilized by short range interactions and can behave as small domains. These observations suggest that peptides containing the sequence of the turns in a membrane protein such as bacteriorhodopsin may form stable turn structures in solution. To test this hypothesis, we determined the solution structure of three peptides each containing the sequence of one of the turns in bacteriorhodopsin. The solution structures of the peptides closely resemble the structures of the corresponding turns in the high resolution structures of the intact protein.     

Pattern recognition of the secondary structure of proteins (alpha-helix and beta-structure)

In this paper, an algorithm for the pattern recognition of secondary structure of proteins is proposed. The procedure simultaneously evaluates the contribution of all the residues of a given peptide to its conformation. By means of the algorithm it is possible to select from a universe of well known proteins the most representative alpha-helix and beta-structure peptides, and to use these peptides, as screening matrices to define the unknown structure of any peptide.     

Two-dimensional structure of beta-amyloid(10-35) fibrils

Beta-amyloid (Abeta) peptides are the main protein component of the pathognomonic plaques found in the brains of patients with Alzheimer's disease. These heterogeneous peptides adopt a highly organized fibril structure both in vivo and in vitro. Here we use solid-state NMR on stable, homogeneous fibrils of Abeta(10-35). Specific interpeptide distance constraints are determined with dipolar recoupling NMR on fibrils prepared from a series of singly labeled peptides containing (13)C-carbonyl-enriched amino acids, and skipping no more that three residues in the sequence. From these studies, we demonstrate that the peptide adopts the structure of an extended parallel beta-sheet in-register at pH 7.4. Analysis of DRAWS data indicates interstrand distances of 5.3 +/- 0.3 A (mean +/- standard deviation) throughout the entire length of the peptide, which is compatible only with a parallel beta-strand in-register. Intrastrand NMR constraints, obtained from peptides containing labels at two adjacent amino acids, confirm the secondary structural findings obtained using DRAWS. Using peptides with (13)C incorporated at the carbonyl position of adjacent amino acids, structural transitions from alpha-helix to beta-sheet were observed at residues 19 and 20, but using similar techniques, no evidence for a turn could be found in the putative turn region comprising residues 25-29. Implications of this extended parallel organization for Abeta(10-35) for overall fibril formation, stability, and morphology based upon specific amino acid contacts are discussed.     

Carnivora: primary structure of the hemoglobins from ratel (Mellivora capensis)

The erythrocytes of adult ratel contain two hemoglobin components, with two alpha- and one beta-chains. In this paper, their complete amino acid sequences are presented. The two alpha-chains differ in one residue at position 34 (Ala----Val) only. The primary structure of the chains was determined by sequencing the N-terminal regions (45 steps) and the tryptic peptides after their isolation from the digests by reversed-phase high-performance liquid chromatography. The alignment of these peptides was deduced from homology with other carnivora globins. The alpha-chains show 21 and the beta-chains 11 exchanges compared with human globin chains. In the alpha-chains, one heme- and two alpha 1/beta 1 contacts are exchanged. In the beta-chains there are three exchanges which involve one alpha 1/beta 1-, one alpha 1/beta 2- and one heme-contact. Between the ratel hemoglobin and those of carnivora a high degree of homology was found.     

The primary structure of the hemoglobin alpha-chain of the arctic ground squirrel

The amino acid sequence of the alpha-chain from the arctic ground squirrel Citellus parryii) is reported. The tryptic peptides prepared from the hemoglobin were isolated by reverse phase HPLC and sequenced. Data from the tryptic peptides were supported by that from cyanogen bromide peptides and acid cleavage peptides which were partially sequenced. Comparison with other rodent alpha-chains shows 15 differences with mouse, 20 with rat, 25 with muskrat, 16 with mole rat, 33 with the guinea-pig and 23 with the hamster. Comparison of arctic ground squirrel hemoglobin alpha-chain with the amino-terminal 25 residues of the marmot shows one amino acid difference at position 13.     

Immunogenic structure of the influenza virus hemagglutinin

We chemically synthesized 20 peptides corresponding to 75% of the HA1 molecule of the influenza virus. Antibodies to the majority (18) of these peptides were capable of reacting with the hemagglutinin molecule. These 18 peptides are not confined to the known antigenic determinants of the hemagglutinin molecule, but rather are scattered throughout its three-dimensional structure. In contrast, antibody raised to intact hemagglutinin did not react with any of the 20 peptides. Taken together these results suggest that the immunogenicity of an intact protein molecule is not the sum of the immunogenicity of its pieces.     

The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.     

Purification and primary structure of low molecular mass peptides from scorpion (Buthus sindicus) venom

The primary structures of four low molecular mass peptides (Bs 6, 8, 10 and 14) from scorpion Buthus sindicus were elucidated via combination of Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. Bs 8 and 14 are cysteine-rich, thermostable peptides composed of 35-36 residues with molecular weights of 3.7 and 3.4 kDa, respectively. These peptides show close sequence homologies (55-78%) with other scorpion chlorotoxin-like short-chain neurotoxins (SCNs) containing four intramolecular disulfide bridges. Despite the sequence variation between these two peptides (37% heterogeneity) their general structural organization is very similar as shown by their clearly related circular dichroism spectra. Furthermore, Bs6 is a minor component, composed of 38 residues (4.1 kDa) containing six half-cystine residues and having close sequence identities (40-80%) with charybdotoxin-like SCNs containing three disulfide bridges. The non-cysteinic, bacic and thermolabile Bs10 is composed of 34 amino acid residues (3.7 kDa), and belongs to a new class of peptides, with no sequence resemblance to any other so far reported sequence isolated from scorpions. Surprisingly, Bs10 shows some limited sequence analogy with oocyte zinc finger proteins. Results of these studies are discussed with respect to their structural similarities within the scorpion LCNs, SCNs and other biologically active peptides.     

Immunological analysis of the polypeptide structure of calf thymus DNA polymerase-primase complex

Five major polypeptides are found in immunoaffinity-purified calf thymus DNA polymerase-DNA primase complex: 185, 160, 68, 55, and 48 kDa. Individual polypeptides purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to produce antibodies in rabbits to aid in identifying the relationships between these polypeptides by immunoblotting and enzyme neutralization procedures. Immunoblot analyses showed that the 160-kDa peptide is derived from the 185-kDa peptide and the 48-kDa peptide is derived from the 68-kDa peptide while antibodies to the 55-kDa peptide do not cross-react with other peptides found in the complex. Direct enzyme neutralization studies demonstrated that antibodies to 185- and 160-kDa peptides inhibit DNA polymerase activity in the complex, confirming earlier suggestions that these peptides are the catalytic peptides for DNA polymerase. DNA primase activity in the complex is inhibited by antibodies to 68-, 55-, and 48-kDa peptides and to a lesser extent by antibodies to the 160-kDa peptide. Free DNA primase isolated from the complex was estimated to have a native molecular weight of about 110,000. The 55- and 48-kDa peptides are found to be associated with the free primase activity. Rabbit antibodies to both 55- and 48-kDa peptides are inhibitory to this primase activity. From these results we suggest that the native calf thymus DNA polymerase-DNA primase complex contains only three unique peptides with the 185-kDa peptide as the catalytic peptide of DNA polymerase and the 55- and 68-kDa peptides constituting the primase peptides. A model illustrating the roles of these peptides in initiation and replication of DNA is presented.     

Rule of antibody structure. Primary structure of a human monoclonal IgAl-immunoglobulin (myeloma protein Tro). VI. Amino acid sequence of the L-chain, lambda-type, subgroup II]

The primary structure of the L-chain of an IgA1-immunoglobulin (Myeloma protein Tro) has been determined by means of cleavage with trypsin and, if necessary, with alpha-chymotrypsin. The tryptic peptides of the variable part were characterized by amino acid analysis, Dansyl-Edman degradation and cleavage with carboxypeptidase; the peptides of the constant part were identified by amino acid analyses and determination of its N- and C-terminal residues. The sequence of the remaining amino acids and the arrangement of the peptides were established in homology to known structures. The protein comprises 216 amino acids. The homology of the variable part clearly characterizes it as belonging to subgroup II of lambda-chains. In positions 27a, b and c, there are the subgroup-specific additional residues and in position 96 is the characteristic deletion. The constant part of the chain is Kern- and Oz- which indicates that it has serine in position 154 and arginine in position 191.     

Primary structure of hemoglobin of the polar bear (Ursus maritimus, Carnivora) and the Asiatic black bear (Ursus tibetanus, Carnivora

The adult Polar and Asiatic Black Bear have one hemoglobin component each. The complete amino-acid sequences of their alpha- and beta-chains are presented. Their primary structures were determined by sequencing the tryptic and prolyl peptides. The alignment of these peptides was deduced from homology to human hemoglobin chains. The hemoglobin sequences of the two species proved to be identical. The evolutionary aspects of this result are discussed. A table of identical hemoglobin sequences from different species is given.     

The primary structure of troponin T and the interaction with tropomyosin.

1. Eight peptides were separated from the CNBr digest of troponin T from rabbit white skeletal muscle and characterized. 2. By study of the amino acid sequence of the methionine-containing peptides isolated after chymotryptic and tryptic digestion and of the N- and C-terminals of the CNBr peptides, six of the latter were shown to be arranged in the sequence CNB1-CNB2-CNB5-CNB6-CNB8-CNB7. The other two peptides, CNB1' and CNB3, have been shown to be partial digestion products. 3. The CNBr peptides CNB1' and CNB2 contained a common sequence and were the only peptides in CNBr digests of troponin T that formed a complex with tropomyosin as judged by viscometric and electrophoretic studies. 4. It is concluded that tropomyosin interacts with the N-terminal half of the troponin T molecule approximately in the region lying between residues 70 and 160. 5. Electrophoretic evidence indicates that tropomyosin and troponin C interact with troponin T. 6. None of the major CNBr peptides of troponin T isolated formed a complex with troponin C on electrophoresis at pH 8.6.     

Isolation and structure elucidation of a novel 5-kDa peptide from neurohaemal lobes of the corpora cardiaca of Locusta migratoria (Insecta, Orthoptera)

Two predominant peptides have been isolated from neurohaemal lobes of corpora cardiaca of 8000 adults of Locusta migratoria. Both peptides have been unambiguously characterized by automated peptide microsequencing and liquid secondary-ion mass spectrometry as a 50-residue peptide (5K peptide) and a 48-residue isologue (5K' peptide). Computer search of sequence data banks did not reveal any significant similarity with other identified proteins. The 5K peptides are remarkably rich in alanine residues (25%) and contain a stretch of five consecutive alanines. This structure suggests that these molecules could correspond to spacer peptides. This assumption is corroborated in the accompanying paper [Lagueux et al. (1990) Eur. J. Biochem. 187, 249-254] on the molecular cloning of the precursor protein which attributes to the 5K peptides a role analogous to that of the C peptides of insulins.     

Carnivora: the primary structure of the hemoglobin from the silver fox (Vulpes vulpes var., Canidae)

The primary structure determination of the hemoglobin alpha- and beta-chains from the silver fox (Vulpes vulpes var., Canidae) is described. The separation of the chains could be achieved directly from the hemoglobin by RP-HPLC as well as by column chromatography of the globin using carboxymethyl-cellulose. Following tryptic digestion of the chains, the peptides were isolated by RP-HPLC. Amino-acid sequences were determined by Edman degradation in liquid and gas phase sequencers. The peptides could be aligned by homology with human and other Carnivora hemoglobins. Compared to human hemoglobin the alpha- and beta-chains of the silver fox exhibit 24 and 13 amino-acid exchanges, respectively. They differ by one alpha- and two beta-chain replacements from the domestic dog and the coyote. The substitutions affecting contact positions are discussed.     

Calcium binding cyclic hexapeptide. Crystal structure of cyclo-(L-prolyl-glycyl)3 calcium complex

The synthetic cyclic hexapeptide (L-prolyl-glycyl)3 forms a 2:1 complex with Ca2+ ion. The cation is sandwiched between the two peptide molecules. The glycyl carbonyls from each of the peptides are octahedrally coordinated to the cation with an average calcium oxygen coordination distance of 2.26A. Both the molecules coordinating to the calcium ion have three fold symmetry, but show significant conformational differences. In one of the peptides of the sandwich, the alternate carbonyls point to the opposite sides of the peptide ring while in the other, all the six carbonyls point to the same side of the ring. Three NH ... O hydrogen bonds between the peptides add to the stability of the sandwich.     

The structure of bovine rhodopsin

We have isolated 16 peptides from a cyanogen bromide digest of rhodopsin. These cyanogen bromide peptides account for the complete composition of the protein. Methionine-containing peptides from other chemical and enzymatic digests of rhodopsin have allowed us to place the cyanogen bromide peptides in order, yielding the sequence of the protein. We have completed the sequence of most of the cyanogen bromide peptides. This information, in conjunction with that from other laboratories, forms the basis for our prediction of the secondary structure of the protein and how it may be arranged in the disk membrane.     

Carnivora: primary structure of the hemoglobin from the spotted hyena (Crocuta crocuta, Hyenidae)

The primary structure of the alpha- and beta-chains of hemoglobin from spotted hyena (Crocuta crocuta, Hyenidae) is presented. The structure-function relationship is discussed. The separation of the chains directly from hemoglobin was performed by RP-HPLC. After tryptic digestion of the chains, the peptides were isolated by RP-HPLC. Amino-acid sequences were determined by Edman degradation in liquid- and gas-phase sequencers. The alignment of the tryptic peptides was made by homology with human and other Carnivora hemoglobins. The hemoglobin from spotted hyena (Crocuta crocuta) exhibits in its alpha- and beta-chains 22 and 20 exchanges, respectively, compared to human hemoglobin. In the alpha-chains, two alpha 1 beta 1-contacts are exchanged. In the beta-chains five exchanges involve one alpha 1 beta 1-contact, one alpha 1 beta 2-contact, one heme contact, and two 2,3-DPG-binding sites.