Structural analysis of the peptides derived from specific acid-catalyzed hydrolysis at aspartylprolyl peptide bonds in human J chain.

Human J chain from IgM has been selectively cleaved at three aspartylprolyl peptide bonds to yield four fragments containing 62, 20, 25, and 22 amino acids, respectively. The amino acid sequence of each peptide has been partially determined, (59 of a total of 129 residues) and its position in the J chain ascertained. There were no obvious similarities to known sequences in other immunoglobulin polypeptide chains.     

Terminal restriction fragment length polymorphism monitoring of genes amplified directly from bacterial communities in soils and sediments

Terminal Restriction Fragment Length Polymorphism (T-RFLP) or Fluorescent Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (FluRFLP) have made a significant impact on the way in which PCR products amplified from mixed community DNA extracts have been assessed. Technically, these approaches are essentially the same. PCR products are generated that contain at one 5′ end label, typically a fluorescent moiety, that will be detected by a DNA sequencing machine. Upon digestion using a specific restriction endonuclease, labeled and unlabeled fragments are generated. This restriction endonuclease is chosen such that following this digestion, each labeled fragment corresponds to a different sequence variant. During electrophoretic separation, the DNA sequencing machine detects only these labeled fragments and therefore detects only the sequence variants. The aim of this article is to describe the protocois and demonstrate that this profiling can be performed using different DNA sequencing machines. The analysis and applications of this approach are also discussed.     

A code governing specific binding of regulatory proteins to DNA and structure of stereospecific sites of regulatory proteins]

A model is proposed for the structure of stereospecific sites in regulatory proteins. On its basis a possible code is suggested that governs the binding of regulatory proteins at specific control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel beta-sheet, with single-stranded regions at the ends of the beta-structure. The model predicts that binding reaction between a regulatory protein and double-helical DNA is a cooperative phenomenon and is accompanied by significant structural alteration at the stereospecific site of the protein. Half of hydrogen bonds normally existing in beta-structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. In a stereospecific site, one chain (t-chain) is attached through hydrogen bonds to the carbonyl oxygens of pyramides and N3 adenines lying in one DNA strand, while the second polypeptide chain (g chain) is hydrogen bonded to the 2-amino groups of guanine residues lying in the opposite DNA strand. The amide groups serve as specific reaction sites being hydrogen bond acceptors in g-chain and hydrogen bond donors in t-chain. The single-stranded portions of t- and g-chains lying in neighbouring subunits of regulatory protein interact with each other forming deformed beta-sheets. The recognition of regulatory sequences by proteins is based on the structural complementarity between stereospecific sites of regulatory proteins and base pairs sequences at the control sites. An essential feature of these sequences is the asymmetrical distribution of guanine residues between the two DNA strands. The code predicts that there are six fundamental amino acid residues (serine, threonine, asparagine, histidine, glutamine and cysteine) whose sequence in stereospecific site determines the base pair sequence to which a given regulatory protein would bind preferentially. The code states a correspondence between four amino acid residues at the stereospecific site of regulatory protein with the two residues being in t- and g-segments, respectively, and AT(GC) base pair at the control site. It is thus possible to determine which amino acid residues in the repressor and which base pairs in the operator DNA are involved in specific interactions with each other, as exemplified by lac repressor binding to lac operator.     

Free thiols of platelet thrombospondin. Evidence for disulfide isomerization

The free thiols of platelet thrombospondin (TSP) were derivatized with labeled N-ethylmaleimide (NEM) or iodoacetamide (IAM). When Ca2+ was chelated with EDTA, 2.9 mol of NEM or 2.6 mol of IAM reacted/mol of native TSP. No additional thiols were found after denaturation with urea. Since TSP has three apparently identical polypeptide chains, this suggests one free thiol/polypeptide chain. Ca2+ protected all of the thiols from reaction with IAM. In Ca2+ about half the thiols reacted normally with NEM and the others were unreactive, indicating that the thiols of TSP are not identical. The number of reactive thiols as a function of [Ca2+] revealed a sigmoidal curve with a transition midpoint of 207 microM. The ability of analogs of NEM to compete for derivatization of the thiols with labeled NEM was greater with larger, more hydrophobic agents. Gel electrophoretic separation of labeled TSP that had been partially digested with thrombin and trypsin indicated that some of the label was in the C-terminal tryptic fragment but that most was in the adjacent trypsin-sensitive region. After cyanogen bromide cleavage of the labeled and reduced protein, four labeled fractions were obtained from a gel filtration column. With subsequent combinations of tryptic digestion and reversed-phase high performance liquid chromatography, labeled peptides were purified from these four fractions, and the amino acid sequences were determined. Twelve labeled cysteines were identified, each with a specific radioactivity less than that of the thiol labeling reagent, indicating that only a fraction of that cysteine in a population of TSP molecules was a free thiol at the time of derivatization. While 2 labeled cysteines are in the non-repeating C-terminal portion of the molecule, the other 10 labeled cysteines are in the adjacent trypsin-sensitive type 3 repeats proposed (Lawler, J., and Hynes, R. O. (1986) J. Cell. Biol. 103, 1635-1648) as the calcium-binding region of the molecule. The disulfide bonds most sensitive to reduction by dithioerythritol were also stabilized by Ca2+, implying location in the Ca2(+)-sensitive part of the molecule. It is proposed that one equivalent of free thiol/polypeptide chain is distributed among 12 different cysteine residues through an intramolecular thioldisulfide isomerization.     

Location of a collagen-binding domain in fibronectin

Preferential labeling of COOH-terminal sequences in newly synthesized fibronectin was achieved by short term incorporation of radiolabeled amino acids in the presence of pactamycin, an inhibitor of polypeptide chain initiation. The labeled fibronectin was then cleaved with cathepsin D under conditions that yield a large (137,000-dalton) fragment that lacks collagen-binding properties, and a smaller (72,000-dalton) fragment that retains the ability of fibronectin to bind to collagen. Determination of the relative specific radioactivities of the two fragments leads us to conclude that the collagen-binding domain in fibronectin is located in the NH2-terminal third of the polypeptide chain and not in a COOH-terminal region as previously indicated by other structural studies.     

A code controlling specific binding of regulatory proteins to DNA

A possible code is suggested that describes a correspondence between amino acid sequences in stereospecific sites of regulatory proteins and nucleotide sequences at the control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel -sheet with single-stranded regions at the ends of the -structure. The binding reaction between regulatory protein and double-helical DNA is accompanied by significant structural alterations at stereospecific sites of the protein and DNA. Half of the hydrogen bonds normally existing in -structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. The code states a correspondence between four amino acid residues at a stereospecific site of the regulatory protein and an AT (GC) base pair at the control site. It predicts that there are six fundamental amino acid residues (serine, threonine, histidine, asparagine, glutamine and cysteine) whose arrangement in the stereospecific site determines the base pair sequence to which a given regulatory protein would bind preferentially.     

In vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes

We have studied the in vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes. The major 32P-labeled polypeptides of clathrin-coated vesicles isolated from metabolically labeled cells were the the 165-, 100-110-, and 50-kDa polypeptides of the assembly protein, the clathrin beta-light chain, and to a lesser extent the clathrin alpha-light chain. The phosphorylation of the assembled (particulate) and unassembled (soluble) pools of clathrin and assembly protein was compared by immunoprecipitating the respective protein complexes from particulate and soluble cell fractions. Although all the phosphorylated polypeptides were present in both fractions, the extent of labeling was protein and fraction specific: the apparent specific activities of the assembly protein 50-kDa polypeptide and clathrin light chain were higher in the unassembled pool, whereas those of the 100-110-kDa polypeptides were higher in the assembled pool. The amino acids and polypeptide fragments labeled in vivo appeared similar to those labeled in vitro.     

Factor X activator from Vipera lebetina venom is synthesized from different genes

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.     

Limited proteolysis of N-(5′-phosphoribosyl)anthranilate isomerase: Indoleglycerol phosphate synthase from Escherichia coli yields two different enzymically active,functional domains

The native enzyme must be denatured either by sodium dodecyl sulfate or by urea before limited proteolysis can occur. Under these conditions only one or two peptide bonds are hydrolyzed by each of the following proteases: Staphylococcal V8 protease, trypsin and elastase. The amino-terminal amino acid sequences were determined to identify the cleavage sites. The new sequences comprise approximately 20% of the entire polypeptide chain, and show good agreement with the nucleotide sequence of the trpC gene. Both V8 protease² and elastase yield large carboxy-terminal fragments, about two thirds of the size of the parent enzyme, and corresponding small amino-terminal fragments. Trypsin cleaves a single peptide bond in the last one third of the polypeptide chain. After separation of the fragments, removal of dodecyl sulfate and renaturation, only the large fragments fold to stable structures. The small fragments precipitate. The large amino-terminal fragment catalyzes only the synthesis of indoleglycerol phosphate and precipitates when solutions are frozen and thawed. The large carboxy-terminal fragment catalyzes only the isomerization of N-(5′-phosphoribosyl)anthranilate and is stable towards freezing and thawing. These studies prove that the intact bifunctional enzyme consists of two autonomously folding, functional domains. They also support the notion that the bifunctional enzyme may have arisen by the fusion of separate ancestral genes, and that stabilization of the intrinsically labile indoleglycerol phosphate synthase domain by interdomain interactions is functionally advantageous.     

Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction

We report a rapid and sensitive method for the detection of base changes in given sequences of genomic DNA. This technique is based on the facts that specific regions of genomic sequences can be efficently labeled and amplified simultaneously by using labeled substrates in the polymerase chain reaction and that in nondenaturing polyacrylamide gels, the electrophoretic mobility of single-stranded nucleic acid depends not only on its size but also on its sequence. The process does not involve restriction enzyme digestion, blotting, or hybridization to probes. We found that most single base changes in up to 200-base fragments could be detected as mobility shifts. RAS oncogene activation was detected by this technique. We also show that the interspersed repetitive sequences of human, Alu repeats are highly polymorphic.     

Analysis of the membrane-interacting domain of the sea urchin sperm adhesive protein bindin

We have investigated the domain of the bindin polypeptide that selectively associates with gel-phase phospholipid vesicles. We found that small trypsin fragments of bindin retain the ability to selectively associate with gel-phase vesicles. The primary amino acid sequence of bindin suggests that these peptides are derived from the central portion of the polypeptide between residues 77 and 126, which is the most hydrophobic region of bindin. We have also employed 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID) and novel, radioiodinated, photoactivatable derivatives of the polar head group of phosphatidylethanolamine (ASD-PE and ASA-PE) to identify membrane-associated polypeptide segments after the transfer of radiolabel from the probe to the bindin polypeptide. After photolysis, bindin was selectively labeled only from probes incorporated in gel-phase vesicles. The labeling of bindin was much more efficient from the head group probes ASA-PE and ASD-PE (8 and 2% of the total label, respectively) in comparison to the hydrophobic probe TID (less than 0.02% of the total label), suggesting that bindin is localized within the polar part of the bilayer. Protease mapping experiments with V8 protease, trypsin, and endoprotease Lys-C suggest that some of the probe label is distributed along the amino-terminal portion of bindin between residues 1 and 76 and the rest of the label is restricted to the segments between residues 77 and 126 which also selectively bind to gel-phase vesicles. The carboxyl-terminal portion of bindin between residues 127 and 236 is not labeled.     

The amino acid sequence of pancreatic spasmolytic polypeptide

The sequence of porcine pancreatic spasmolytic polypeptide has been established by a variety of techniques including manual as well as automatic sequencing of fragments resulting from the cleavage of reduced and S-carboxymethylated pancreatic spasmolytic polypeptide with trypsin, chymotrypsin, clostripain, cyanogen bromide and formic acid. The N- and C-terminal sequences were established using pyroglutamate amino-peptidase and carboxypeptidase A, respectively. Pancreatic spasmolytic polypeptide contains 106 amino acid residues in a single chain with seven S-S bridges and a pyroglutamyl blocked N-terminal. The alignment of the sequences representing amino acids 14-49 and 63-98 shows pair-wise identical amino acid residues in 18 out of 36 positions, indicating that these two "domains" have been derived from a common gene.     

Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzymes

We have developed a novel technique for specific amplification of rare methylated DNA fragments in a high background of unmethylated sequences that avoids the need of bisulphite conversion. The methylation-dependent restriction enzyme GlaI is used to selectively cut methylated DNA. Then targeted fragments are tagged using specially designed ‘helper’ oligonucleotides that are also used to maintain selection in subsequent amplification cycles in a process called ‘helper-dependent chain reaction’. The process uses disabled primers called ‘drivers’ that can only prime on each cycle if the helpers recognize specific sequences within the target amplicon. In this way, selection for the sequence of interest is maintained throughout the amplification, preventing amplification of unwanted sequences. Here we show how the method can be applied to methylated Septin 9, a promising biomarker for early diagnosis of colorectal cancer. The GlaI digestion and subsequent amplification can all be done in a single tube. A detection sensitivity of 0.1% methylated DNA in a background of unmethylated DNA was achieved, which was similar to the well-established Heavy Methyl method that requires bisulphite-treated DNA.     

Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail

We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity.     

Amino acid sequence of human erythrocyte carbonic anhydrase C

Human carbonic anhydrase C is the high-specific-activity form of the enzyme found in human red cells. A proposal for the primary structure of this enzyme is presented. Trypsin-catalyzed hydrolysis was restricted to the arginyl bonds of the protein by blocking the lysyl side chains by amidination. The arginine fragments were isolated and their amino acid sequences determined. The sequence contains 259 amino acid residues in a single polypeptide chain devoid of disulfide bonds. The structure obtained should be adequate to permit a detailed interpretation of the 2.0 Å X-ray crystallographic model of the enzyme previously determined. The primary structures of the human B and C enzymes have about 60% identities.     

The primary structure of a halorhodopsin from Natronobacterium pharaonis. Structural, functional and evolutionary implications for bacterial rhodopsins and halorhodopsins

We cloned and sequenced the gene coding for the polypeptide of a halorhodopsin in Natronobacterium pharaonis (named here pharaonis halorhodopsin). Peptide sequencing of cyanogen bromide fragments, and immunoreactions of the protein and synthetic peptides derived from the COOH-terminal gene sequence, confirmed that the open reading frame is the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences, as well as those for other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences (mutations/nucleotide at codon positions which do not result in amino acid changes) were calculated. These indicate very considerable evolutionary distance between each pair of genes. In spite of this, the three protein sequences show extensive similarities, indicating strong selective pressures. Conserved and conservatively replaced amino acid residues in all three proteins identify general features essential for ion-motive bacterial rhodopsins, responsible for overall structure and chromophore properties. Comparison of the bacteriorhodopsin sequence with those of the two halorhodopsins, on the other hand, identifies features involved in their specific (proton and chloride ion) transport functions.     

Amino acid sequence analysis of the beta- and gamma-subunits of eukaryotic initiation factor eIF-2. Identification of regions interacting with GTP

By affinity labelling using two different GTP photoaffinity analogues we previously demonstrated that both the beta- and gamma-subunits of eukaryotic initiation factor eIF-2 are involved in GTP binding (Bommer, U.-A. and Kurzchalia, T.V. (1989) FEBS Lett. 244, 323-327). We have now applied the same method in combination with CNBr cleavage and microsequence analysis in order investigate which part of the polypeptide chain of eIF-2 beta is in close contact to the bound GTP. From the three main CNBr fragments of eIF-2 beta, the C-terminal one was found to be labelled by the applied GTP photoaffinity analogue, Guo(2',3'-TDBH)ppp. Because the cDNA sequence of the gamma-subunit of eIF-2 has not yet been published and because cDNA sequence analysis of eIF-2 beta revealed only two out of three consensus sequence elements of a GTP-binding domain, we also sequenced the CNBr fragments of eIF-2 gamma. In this way, sequences containing about 50 amino acid residues were obtained. Taken together with the recently published N-terminal sequences of tryptic peptides of eIF-2 gamma from pig liver (Suzuki et al. 1990, J. Biochem. 108, 635-641), about 30% of the total sequence is now known. One of the CNBr fragments from rabbit eIF-2 gamma contains a sequence (AXXAXXGK) which in several respects resembles that of the consensus sequence element absent from the beta-subunit.