Proton Magnetic Resonance Studies of Conformational Changes in Cleaved Halves of Histone F2B

THE amino-acid sequences of the histones F2A1¹ and F2B² are of particular interest in that they have such a high degree of non-uniformity that different regions of the polypeptide chains have quite different characters. The amino-halves of the molecules have a high density of basic residues while the carboxyl-halves have far fewer basic residues and higher proportions of apolar residues and other residues which favour the formation of helical conformations. These properties have led to the suggestions that the regions of high basicity are the primary sites for interaction with DNA^1–4 in chromatin and that the non-basic regions contain any secondary structure that the molecule is capable of forming^1–4 and are the sites for histone–histone interactions^3,4. Evidence in support of this scheme has been obtained from nuclear magnetic resonance and optical spectroscopic studies of conformational changes and interactions in histones F1 and F2A1³ and F2B⁴. In these studies, however, the whole molecule has been examined and the possibility of the formation of some secondary structure in the basic region of the molecule cannot be excluded.     

Specific Conformations and Interactions in Chicken Erythrocyte Histone F2C

THE close association of histones with DNA in the eukaryote chromosome has implicated them in two possible functions; first, a structural role in maintaining and controlling the conformations of the chromosome through the cell cycle and second, involvement in genetic control mechanisms¹. The relatively small number of histones and the rigid conservation of sequence found for histone F2A1 (see ref. 2) and suspected for other histones argue more for a structural role and involvement in the gross repression of inactive genes than for a role in the precise control of active genes. The sequence studies of histones^2–6 have delineated well defined regions of the chain, rich either in basic and helix-destabilizing residues or rich in apolar, acidic and aromatic residues. Physical studies^7–11 have demonstrated that the later regions are the sites of secondary structure and histone-histone interaction, while the former are the sites of DNA interaction. Thus, although particular regions of the histone chains have been correlated with quite different functions, there has been no evidence so far to show that the relatively non-basic segments fold up so specifically as in globular proteins. If, however, histones are involved in controlling the conformation of chromosomes, then specific and reversible interactions are to be expected. The very lysine-rich histones are distinguished from other histones in that microheterogeneity has been found for F1 (refs. 12–14), while F2C is polymorphic¹⁵ and it has been suggested that they have a function additional to structural. Histone F2C is unique to nucleated erythrocytes¹⁶, largely replacing F1 therein and could be involved in the total repression of erythrocyte genes.     

PHYSICO-CHEMICAL CHARACTERISTICS AND REGIONAL DISTRIBUTION STUDIES OF GP-350, A SOLUBLE SIALOGLYCOPROTEIN FROM BRAIN

The apparent molecular weight of GP-350, a sialoglycoprotein from calf and rat brain, has been determined by SDS-polyacrylamide gel electrophoresis. The electrophoretic mobility corresponds to the mobility of a polypeptide with a molecular weight of 11,600 ± 200. On this basis it can be calculated that only one sialic acid residue is present/GP-350 molecule. From isoelectric focusing experiments it appeared that the isoelectric point of GP-350 is about 2. The determination of the amide content of the polypeptide chain showed that out of 22.0 acidic amino acid residues of glutamic acid and aspartic acid, only 4.9 residues are amidated. The total amount of the basic amino acid residues lysine and arginine, is 6.5. So, per molecule GP-350 10.6 acidic amino acid residues are not counteracted by basic amino acid residues. The surplus of the acidic amino acid residues as well as sialic acid result in the pronounced acidic character of GP-350. This fact is supported by the electrophoretic experiments. The carbohydrate-polypeptide linkage type has been studied by alkaline sodium borohydride treatment. Two thirds of all the galactosamine was destroyed, whereas the amount of glucosamine remained the same. Amino acid analysis indicated a decrease in serine and threonine with a concomitant small increase in alanine. These data point to the occurrence of linkages between the carbohydrate chain and the polypeptide core of the galactosamineserine or –threonine type. Per molecule GP-350 about two residues of galactosamine are destroyed, indicating that two carbohydrate chains of this binding type are present. Only one of these chains can be terminated by a sialic acid residue. The other carbohydrate chain may be terminated by fucose. Regional distribution studies showed the presence of GP-350 in all brain areas studied; in relatively large amounts in the regions rich in ganglia such as caudate nucleus, cerebellar grey matter, pons and medulla oblongata, and in relatively small amounts in the regions poor in ganglia such as corpus callosum, cerebral white, cerebral grey and cerebellar white matter. GP-350 is also present in the pituitary gland. In the cerebrospinal fluid a glycoprotein is present with the same electrophoretic mobility as GP-350. However, this glycoprotein gave no precipitin reaction with GP-350 specific antiserum. Moreover, the amino acid composition was quite different from that of GP-350. Subcellular distribution study revealed that GP-350 is present in the soluble cell fraction and in the synaptosomal membrane fraction, whereas it is absent from the purified nuclei, mitochondria, myelin, and also from the microsomal fraction.     

Probing the role of negatively charged amino acid residues in ion permeation of skeletal muscle ryanodine receptor

Sequence comparison suggests that the ryanodine receptors (RyRs) have pore architecture similar to that of the bacterial K+ channel KcsA. The lumenal loop linking the two most C-terminal transmembrane spanning segments in the RyRs has a predicted pore helix and an amino acid motif (GGGIG) similar to the selectivity filter (TVGYG) of KcsA identified by x-ray analysis. The RyRs have many negatively charged amino acid residues in the two regions linking the GGGIG motif and predicted pore helix with the two most C-terminal transmembrane spanning segments. We tested the role of these residues by generating single-site mutants, focusing on amino acid residues conserved among the mammalian RyRs. Replacement of two acidic residues immediately after the GGGIG motif in skeletal muscle ryanodine receptor (RyR1-D4899 and -E4900) with asparagine and glutamine profoundly affected ion permeation and selectivity. By comparison, mutagenesis of aspartate and glutamate residues in the putative linker regions showed a K+ conductance and selectivity for Ca2+ compared to K+ (P(Ca)/P(K)) close to wild-type. The results show that the negatively charged carboxyl oxygens of D4899 and E4900 side chains are major determinants of RyR ion conductance and selectivity.     

Basement membrane (type IV) collagen is a heteropolymer

Type IV collagen was isolated in high yield from bovine kidney cortex. The protein revealed Mr = 380,000 and contained, in a 2:1 ratio, two different disulfide-linked polypeptide chains, C-1 and D-1 (Mr = 125,000). Carboxymethyl-cellulose chromatography before and after reduction proved that the two polypeptide chains are arranged in a single triple helical molecule with the chain composition (C-1)2(D-1). The disulfide bridges appear to be located 180 amino acid residues from the NH2 terminus of the chains.     

Structural studies on rat prostatic binding protein. The primary structure of component C2 from subunit S

The amino acid sequence of component C2, the polypeptide specific for subunit S of prostatic binding protein, the major secretory glycoprotein of the rat ventral prostate, has been determined. Its structure was established using the manual Edman degradation on the most relevant fragments obtained by enzymatic digestion of the S-carboxamidomethylated component C2 and the native subunit S and by chemical cleavage of the remaining undigestible 'cores' with cyanogen bromide. Component C2 contains 92 amino acids corresponding to a molecular weight of 10619. It is a slightly acidic polypeptide in which the acidic and basic residues are unevenly distributed. The N terminus is blocked and three cysteine residues are almost evenly distributed over the peptide chain. A highly polar region is found in position 23-34 and two hydrophobic segments are located in the C-terminal part of the molecule. Component C2 is compared with component C1 of subunit F and their high sequence homology reveals an evolutionary relationship.     

Structural analyses of a monoclonal heterodimeric suppressor factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10

A GAT-specific, MHC-restricted "second-order" suppressor T cell factor (TsF2) from the hybridoma 762 B3.7 was biosynthetically radiolabeled with 35S-methionine and was isolated from cell extracts. The isolation procedure involved two-dimensional nonreducing/reducing SDS-PAGE and electroelution of the reduced off-diagonal polypeptide chains from the gel. Biochemical characterization studies revealed that TsF2 is a disulfide-linked heterodimer composed of a basic and an acidic polypeptide chain, both having m.w. of 30,000. Both chains are glycosylated and contain sialic acid residues. The basic polypeptide reacts with anti-I-J antisera, whereas the acidic chain contains the antigen-binding capacity. Monoclonal antibodies induced by immunizing rats with TsF2 purified from hybridoma supernatants were selected for the ability to block immunosuppression mediated by TsF2 in vitro. These antibodies, but not irrelevant antibodies, immunoprecipitated the 35S-methionine-labeled protein that migrates off the diagonal in two-dimensional gels. Thus, we have verified that the immunosuppressive protein that migrates off the diagonal in two-dimensional gels binds to antibodies that are known to inhibit the biologic activity of unpurified TsF2.     

The interfaces of actin and Acanthamoeba actobindin. Identification of a new actin-binding motif

Actobindin is an 88-amino acid polypeptide, containing two almost identical repeated domains of 33 and 34 residues. Depending on the molar ratios in which they are mixed, actobindin binds either one or two actin molecules. We cross-linked actobindin and actin in the 1:1 complex, using the zero-length cross-linker 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. The cross-linked peptides were purified after consecutive CNBr cleavage and trypsin and Staphylococcus protease V8 digestions, and the cross-linked side chains were identified by amino acid sequencing. Isopeptide linkages were formed between residues Glu-100 of actin and Lys-16 of actobindin. In addition, we found a connection between one or more of the acidic residues 1,2, or 3 of actin and Lys-16 and Lys-52 of actobindin. The cross-linked regions in actobindin contain Leu-Lys-His-Ala-Glu-Thr motifs, similar to sequences observed in several other actin-binding proteins.     

Isolation and complete amino acid sequence of the mitochondrial ATP synthase epsilon-subunit of the yeast Saccharomyces cerevisiae

All five subunits of yeast mitochondrial F1-ATPase have been isolated by reverse-phase high performance liquid chromatography. This procedure allows micro-preparative purification of all the subunits with 60% recoveries. The complete amino acid sequence of the epsilon-subunit has been established. This has been achieved by the sequence analysis of subnanomole amounts of the intact molecule and that of peptides derived by enzymatic digestion with endoproteinase Arg-C and by chemical cleavage with hydroxylamine. Yeast ATP synthase epsilon-subunit is composed of 61 residues with a calculated molecular mass of 6612 Da. This polypeptide is rather basic since it contains 7 basic residues and 3 acidic residues. This study shows a slight similarity with the bovine epsilon-subunit ATP synthase since there are 16 identical residues.     

Isolation and sequencing of cDNAs and genomic DNAs encoding the alpha 4 chain of basement membrane collagen type IV and assignment of the gene to the distal long arm of human chromosome 2.

We cloned three overlapping cDNAs covering 2,452 base pairs encoding a new basement membrane collagen chain, alpha 4(IV), from rabbit corneal endothelial cell RNA. Nucleotide sequence analysis demonstrated that the clones encoded a triple-helical domain of 392 1/3 amino acid residues and a carboxyl non-triple-helical (NC1) domain of 231 residues. We also isolated a genomic DNA fragment for the human alpha 4(IV) chain, which contained two exons encoding from the carboxyl end of the triple-helical domain to the amino end of the NC1 domain. Identification of the clones was based on the amino acid sequence identity between the cDNA-deduced amino acid sequence and the reported amino acid sequence obtained from a fragment of the alpha 4(IV) collagen polypeptide M28+ (Butkowski, R. J., Shen, G.-Q., Wieslander, J., Michael, A. F., and Fish, A. J. (1990) J. Lab. Clin. Med. 115, 365-373). When compared with four other type IV collagen chains, the NC1 domain contained 12 cysteinyl residues in positions identical to those of the residues in those chains. The domain demonstrated 61, 70, 55, and 60% amino acid similarity with human alpha 1, human alpha 2, bovine alpha 3, and human alpha 5 chains, respectively. The human genomic DNA fragment allowed us to map the alpha 4(IV) gene (COL4A4) to the 2q35-2q37.1 region of the human genome.     

Effects of Physical and Chemical Treatments on the Biological Activity of Ricin D

Effects of physical and chemical treatments on the cytoagglutinating activity, toxicity and inhibitory activity of cell-free protein synthesis of ricin D or its constituent polypeptide chains were investigated. The results indicated that the isolated polypeptide chains were much less stable than intact ricin D in acidic pH, heating as well as chemicals, and the Ala chain was more unstable than the lie chain.

Chemical modifications of ricin D with specific reagents revealed that the tryptophan and tyrosine residues as well as the carboxyl groups participated in the phenomena of cyto- agglutination and toxic action of ricin D, whereas arginine residues were considered not to be directly involved. Trinitrophenylation of free amino groups did not result in a loss of cytoagglutinating activity, whereas caused a loss of toxicity, suggesting that free amino groups in the lie chain were involved in the toxic action of ricin D.     

Escherichia coli H+-ATPase: loss of the carboxyl terminal region of the gamma subunit causes defective assembly of the F1 portion

Mutant genes for the gamma subunit of H+-translocating ATPase (H+-ATPase) were cloned from eight different strains of Escherichia coli isolated in this laboratory. Determination of their nucleotide sequences revealed that they are amber nonsense mutations: a Gln codon at position 15, 158, 227, 262, and 270, respectively, was replaced by a termination codon in these strains. As terminal Met is missing in the gamma subunit, these results indicate that these strains are capable of synthesizing fragments of gamma subunits of 13, 156, 225, 260, and 268 amino acid residues, respectively. Studies on the properties of membranes of these strains suggested the importance of the region between Gln 269 and the carboxyl terminus (residue 286) for forming a stable F1 complex with ATPase activity and the region between Gln 226 and Gln 261 for normal interaction of F1 with F0. The sequence from Gln 261 to Gln 269 also seemed to be important for stability of F1 assembly on the membranes. The high frequency of the nonsense mutations suggested that the number of essential residues is limited in this subunit. Comparison of the homologies of the amino acid sequences of the gamma subunits from four different sources confirmed this notion: 19% of amino acid residues are identically conserved in these four strains, and the conserved regions are the amino terminal and carboxyl terminal regions.     

Interaction of Escherichia coli F1-ATPase with dicyclohexylcarbodiimide-binding polypeptide

Antibody raised against the N,N'-dicyclohexylcarbodiimide (DCCD)-binding polypeptide of Escherichia coli bound to the cytoplasmic surface of the cell membrane. A weak reaction was seen with everted vesicles of the thermophile PS3. Rat-liver mitochondrial membranes did not react with the antibody. Reaction of the isolated DCCD-binding polypeptide with the antibody was prevented by oxidation of methionine residues or cleavage of the polypeptide with cyanogen bromide. Modification of the arginine residues of the DCCD-binding polypeptide did not affect interaction with the antibody. Purified F1-ATPase of E. coli bound to the isolated DCCD-binding polypeptide as shown by solid-phase radioimmune assay. Binding involved the alpha and/or beta subunits of F1 and the arginine residues of the polar central region of the DCCD-binding polypeptide. Our results are consistent with a looped arrangement of the DCCD-binding polypeptide in the membrane in which the carboxyl- and amino-terminal regions of the molecule are at the periplasmic surface and the polar central region, interacting with F1, is at the cytoplasmic surface of the cell membrane.     

A processing enzyme for prorenin in mouse submandibular gland. Purification and characterization

Renin is produced from an inactive precursor, prorenin, through proteolytic cleavage at paired basic amino acid residues. In this study, an enzyme which specifically cleaves mouse Ren 2 prorenin at the paired basic residues has been purified from mouse submandibular gland by CM-Toyopearl chromatography, antipain-Sepharose chromatography, and isoelectric focusing. This enzyme, named prorenin converting enzyme, consists of two polypeptide chains of 17 and 10 kDa. The enzyme has an isoelectric point of 9.5-9.8, and its pH optimum is between 7.5 and 8.5. It specifically cleaves the peptide bond on the carboxyl side of the Arg at the Lys-Arg pair of mouse Ren 2 prorenin to yield mature renin but does not cleave mouse Ren 1 and human prorenins. Studies on the effects of inhibitors indicate that this enzyme is a serine protease that differs from the enzymes processing other prohormones at paired basic amino acid residues.     

Molecular cloning of matrin 3. A 125-kilodalton protein of the nuclear matrix contains an extensive acidic domain

We report here the cloning and sequencing of matrin 3, an acidic internal matrix protein, from a rat insuloma cDNA library. The nucleotide sequence has a single open reading frame encoding a polypeptide of 845 amino acids. The Genbank and National Biomedical Research Foundation databases did not contain any sequences similar to that of matrin 3. The primary structure consists of 33% charged residues and is generally hydrophilic. The amino-terminal region (residues 1-120) is positively charged and contains a large number of amino acids with free hydroxyl groups (26 of the first 100 residues) as in the lamins and several non-lamin intermediate filament proteins. A highly acidic domain (approximately 170 amino acids) near the carboxyl terminus, in which 32% of the amino acid residues are acidic (Glu or Asp), is a characteristic found in other nuclear proteins (Earnshaw, W. C. (1987) J. Cell Biol. 105, 1479-1482). A putative nuclear targeting signal sequence (Ser-Lys-Lys-Lys-Leu-Lys-Lys-Val-Glu) is located in the middle of the highly acidic domain. The corresponding human deduced partial amino acid sequence is 96% identical to the rat sequence, indicating that matrin 3 is a highly conserved protein.     

The endoxylanases from family 11: computer analysis of protein sequences reveals important structural and phylogenetic relationships

Eighty-two amino acid sequences of the catalytic domains of mature endoxylanases belonging to family 11 have been aligned using the programs MATCHBOX and CLUSTAL. The sequences range in length from 175 to 233 residues. The two glutamates acting as catalytic residues are conserved in all sequences. A very good correlation is found between the presence (at position 100) of an asparagine in the so-called 'alkaline' xylanases, or an aspartic acid in those with a more acidic pH optimum. Four boxes defining segments of highest similarity were detected; they correspond to regions of defined secondary structure: B5, B6, B8 and the carboxyl end of the alpha helix, respectively. Cysteine residues are not common in these sequences (0.7% of all residues), and disulfide bridges are not important in explaining the stability of several thermophilic xylanases. The alignment allows the classification of the enzymes in groups according to sequence similarity. Fungal and bacterial enzymes were found to form mostly separate clusters of higher similarity.     

Separation of both the Bbeta- and the gamma-polypeptide chains of human fibrinogen into two main types which differ in sialic acid content

The gamma- and Bbeta-polypeptide chains of purified human fibrinogen have each been resolved into two major species: gammaL and gammaR and BbetaL and BbetaR. These molecular variants, separable on CM-cellulose, differ from each other in sialic acid content: approximately 2 residues of sialic acid per molecule of polypeptide chain for the L species to 1 residue of sialic acid per molecule for the R species. The two types of each polypeptide are demonstrable in preparations of fibrinogen from single donors as well as in pooled fibrinogen. The L and R forms of the gamma chains or the Bbeta chains do not differ in their electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels, suggesting that they are similar in molecular weight. They are also indistinguishable in polyacrylamide gels in the presence of urea at pH 2.7. Maps of ninhydrin-positive tryptic peptides of the L and R forms of the gamma chain displayed differences within a small group of peptides which have been shown to contain the sialic acid residues present in the gamma-polypeptides. No differences between the peptide maps of BbetaL and BbetaR chains were obvious. A larger ratio of L/R in the gamma and Bbeta chains of dysfibrinogenemia fibrinogen "Zürich II" than in those of normal fibrinogen explains the higher content of sialic acid measured in the native Zürich II fibrinogen molecule.     

Concerted millisecond timescale dynamics in the intrinsically disordered carboxyl terminus of γ‐tubulin induced by mutation of a conserved tyrosine residue

Tubulins are an ancient family of eukaryotic proteins characterized by an amino‐terminal globular domain and disordered carboxyl terminus. These carboxyl termini play important roles in modulating the behavior of microtubules in living cells. However, the atomic‐level basis of their function is not well understood. These regions contain multiple acidic residues and their overall charges are modulated in vivo by post‐translational modifications, for example, phosphorylation. In this study, we describe an application of NMR and computer Monte Carlo simulations to investigate how the modification of local charge alters the conformational sampling of the γ‐tubulin carboxyl terminus. We compared the dynamics of two 39‐residue polypeptides corresponding to the carboxyl‐terminus of yeast γ‐tubulin. One polypeptide comprised the wild‐type amino acid sequence while the second contained a Y > D mutation at Y11 in the polypeptide (Y445 in the full protein). This mutation introduces additional negative charge at a site that is phosphorylated in vivo and produces a phenotype with perturbed microtubule function. NMR relaxation measurements show that the Y11D mutation produces dramatic changes in the millisecond‐timescale motions of the entire polypeptide. This observation is supported by Monte Carlo simulations that—similar to NMR—predict the WT γ‐CT is largely unstructured and that the substitution of Tyr 11 with Asp causes the sampling of extended conformations that are unique to the Y11D polypeptide.     

The amino acid sequences of chains a, b, and c that form the trimer subunit of the extracellular hemoglobin from Lumbricus terrestris

The extracellular hemoglobin of Lumbricus terrestris comprises four major heme-containing chains, a, b, c, and d in equal proportions. We have determined the amino acid sequences of chains a, b, and c which form a disulfide-linked trimer. Chains a, b, and c have 151, 145, and 153 residues and calculated molecular weights of 17,525, 16,254, and 17,289, respectively. The sequence of chain b, reported previously (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 287, 9005-9015) has been completely redetermined and found to contain 12 fewer residues than originally reported. Chains a and c both contain unusual, highly polar NH2-terminal extensions of 7 residues before the A helix. These segments must be close together because they are joined by a disulfide bond. We suggest that this structure, with seven negatively charged groups, may be part of a functionally important Ca2+-binding site in the trimer. Comparison of the sequences of chains a, b, and c with those of chain d (Shishikura, F., Snow, J. W., Gotoh, T., Vinogradov, S. N., and Walz, D. A. (1987) J. Biol. Chem. 262, 3123-3131) and the four chains of the hemoglobin of Tylorrhynchus heterochaetus (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267) shows that the number and positions of the cysteinyl residues are all conserved. This suggests that the extracellular hemoglobins from both the Oligochaeta and Polychaeta have the same number and configuration of disulfide bonds within the molecule. Phylogenetic analysis suggests that gene duplication first generated an intracellular hemoglobin branch and an extracellular hemoglobin branch. DNA coding for a signal peptide would have been acquired by the extracellular globin gene after this event. At least two further gene duplications are required to account for the present four polypeptide chains.