Structural properties of carnation mottle virus p7 movement protein and its RNA-binding domain

Plant viral movement proteins (MPs) participate actively in the intra- and intercellular movement of RNA plant viruses to such an extent that MP dysfunction impairs viral infection. However, the molecular mechanism(s) of their interaction with cognate nucleic acids are not well understood, partly due to the lack of structural information. In this work, a protein dissection approach was used to gain information on the structural and RNA-binding properties of this class of proteins, as exemplified by the 61-amino acid residue p7 MP from carnation mottle virus (CarMV). Circular dichroism spectroscopy showed that CarMV p7 is an alpha/beta RNA-binding soluble protein. Using synthetic peptides derived from the p7 sequence, we have identified three distinct putative domains within the protein. EMSA showed that the central region, from residue 17 to 35 (represented by peptide p7(17-35)), is responsible for the RNA binding properties of CarMV p7. This binding peptide populates a nascent alpha-helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroethanol and monomeric SDS, or RNA (which also changes its conformation upon binding to the peptide). Thus, the RNA recognition appears to occur via an "adaptive binding" mechanism. Interestingly, the amino acid sequence and structural properties of the RNA-binding domain of p7 seem to be conserved among carmoviruses and some other RNA-binding proteins and peptides. The low conserved N terminus of p7 (peptide p7(1-16)) is unstructured in solution. In contrast, the highly conserved C terminus motif (peptide p7(40-61)) adopts a beta-sheet conformation in aqueous solution. Alanine scanning mutagenesis of the RNA-binding motif showed how selected positive charged amino acids are more relevant than others in the RNA binding process and how hydrophobic amino acid side chains would participate in the stabilization of the protein-RNA complex.     

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis.     

A single-stranded nucleic acid binding sequence common to the heterogeneous nuclear ribonucleoparticle protein A1 and murine recombinant virus gp70.

We have used an antisynthetic peptide antiserum to a murine recombinant virus gp70 to probe normal mouse tissues for immunologically related proteins. In addition to cognate gp70s, this antiserum reacts with the heterogeneous nuclear ribonucleoparticle protein A1 by virtue of a 5-amino acid epitope, PRNQG. Further structural similarity is evident both 5' and 3' of this epitope. Since the function of the heterogeneous nuclear ribonucleoprotein particles in the cell is to aid in the stabilization and processing of newly synthesized RNA, we have investigated whether this retroviral sequence exhibits any nucleic acid-binding properties by the same criteria established for the identification of heterogeneous nuclear ribonucleoprotein particles. Analysis of the peptide in a poly(eA) binding assay shows this retroviral sequence to bind with high affinity to single-stranded nucleic acid. This binding occurs in a salt-sensitive manner characteristic of single-stranded nucleic acid-binding proteins. Flanking peptides not containing this sequence generated from either the A1 or gp70 show no ability to bind single-stranded nucleic acids by this assay.     

Identification of a novel plasmin(ogen)-binding motif in surface displayed alpha-enolase of Streptococcus pneumoniae

The interaction of Streptococcus pneumoniae with human plasmin(ogen) represents a mechanism to enhance bacterial virulence by capturing surface-associated proteolytic activity in the infected host. Plasminogen binds to surface displayed pneumococcal alpha-enolase (Eno) and is subsequently activated to the serine protease plasmin by host-derived tissue plasminogen activator (tPA) or urokinase (uPA). The C-terminal lysyl residues of Eno at position 433 and 434 were identified as a binding site for the kringle motifs of plasmin(ogen) which contain lysine binding sites. In this report we have identified a novel internal plamin(ogen)-binding site of Eno by investigating the protein-protein interaction. Plasmin(ogen)-binding activity of C-terminal mutated Eno proteins used in binding assays as well as surface plasmon resonance studies suggested that an additional binding motif of Eno is involved in the Eno-plasmin(ogen) complex formation. The analysis of spot synthesized synthetic peptides representing Eno sequences identified a peptide of nine amino acids located between amino acids 248-256 as the minimal second binding epitope mediating binding of plasminogen to Eno. Binding of radiolabelled plasminogen to viable pneumococci was competitively inhibited by a synthetic peptide FYDKERKVYD representing the novel internal plasmin(ogen)-binding motif of Eno. In contrast, a synthetic peptide with amino acid substitutions at critical positions in the internal binding motif identified by systematic mutational analysis did not inhibit binding of plasminogen to pneumococci. Pneumococcal mutants expressing alpha-enolase with amino acid substitutions in the internal binding motif showed a substantially reduced plasminogen-binding activity. The virulence of these mutants was also attenuated in a mouse model of intranasal infection indicating the significance of the novel plasminogen-binding motif in the pathogenesis of pneumococcal diseases.     

A procedure to verify an amino acid sequence which has been derived from a nucleotide sequence: application to the 26S RNA of Semliki Forest virus.

We describe a peptide sequencing procedure which can be used to verify an amino acid sequence which is derived from a nucleotide sequence. One first labels the protein with a 3H- and a 14C-labelled amino acid and then cleaves the protein into a set of peptides using a cleavage reaction specific for a particular amino acid residue. Finally one performs Edman degradations on the whole mixture of peptides. The released amino acids reflect the combined aminoterminal amino acid sequences of all the peptides that have been formed by the cleavage reaction. The data can therefore be used to check a deduced sequence simultaneously at several regions of the polypeptide chain. We have applied this sequencing procedure to verify the amino acid sequence deduced from the 26S RNA of Semliki Forest virus.     

Substrate recognition determinants for rhodopsin kinase: studies with synthetic peptides, polyanions, and polycations

Rhodopsin kinase phosphorylates serine- and threonine-containing peptides from bovine rhodopsin's carboxyl-terminal sequence. Km's for the peptides decrease as the length of the peptide is increased over the range 12-31 amino acids, reaching 1.7 mM for peptide 318-348 from the rhodopsin sequence. The Km for phosphorylation of rhodopsin is about 10(3) lower than that for the peptides, which suggests that binding of rhodopsin kinase to its substrate, photolyzed rhodopsin, involves more than just binding to the carboxyl-terminal peptide region that is to be phosphorylated. A synthetic peptide from the rhodopsin sequence that contains both serines and threonines is improved as a substrate by substitution of serines for the threonines, suggesting that serine residues are preferred as substrates. Analogous 25 amino acid peptides from the human red or green cone visual pigment, a beta-adrenergic receptor, or M1 muscarinic acetylcholine receptors are better substrates for bovine rhodopsin kinase than is the peptide from bovine rhodopsin. An acidic serine-containing peptide from a non-receptor protein, alpha s1B-casein, is also a good substrate for rhodopsin kinase. However, many basic peptides that are substrates for other protein kinases--histone IIA, histone IIS, clupeine, salmine, and a neurofilament peptide--are not phosphorylated by rhodopsin kinase. Polycations such as spermine or spermidine are nonessential activators of phosphorylation of rhodopsin or its synthetic peptide 324-348. Polyanions such as poly(aspartic acid), dextran sulfate, or poly(adenylic acid) inhibit the kinase. Poly(L-aspartic acid) is a competitive inhibitor with respect to rhodopsin (KI = 300 microM) and shows mixed type inhibition with respect to ATP.     

Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA

In order to develop a novel molecule that recognizes a specific structure of RNA, we have attempted to design peptides having L-alpha-amino acids with a nucleobase at the side chain (nucleobase amino acid (NBA)), expecting that the function of a nucleobase which can specifically recognize a base in RNA is regulated in a peptide conformation. In this study, to demonstrate the applicability of the NBA units in the peptide to RNA recognition, we designed and synthesized a variety of NBA-conjugated peptides, derived from HIV-1 Rev. Circular dichroism study revealed that the conjugation of the Rev peptide with an NBA unit did not disturb the peptide conformation. RNA-binding affinities of the designed peptides with RRE IIB RNA were dependent on the structure of the nucleobase moieties in the peptides. The peptide having the cytosine NBA at the position of the Asn40 site in the Rev showed a higher binding ability for RRE IIB RNA, despite the diminishing the Asn40 function. Furthermore, the peptide having the guanine NBA at the position of the Arg44 site, which is the most important residue for the RNA binding in the Rev, bound to RRE IIB RNA in an ability similar to Rev34-50 with native sequence. These results demonstrate that an appropriate NBA unit in the peptide plays an important role in the RNA binding with a specific contact such as hydrogen bonding, and the interaction between the nucleobase in the peptide and the base in the RNA can enhance the RNA-binding affinity and specificity.     

Synthesis and nucleic acids binding properties of diastereomeric aminoethylprolyl peptide nucleic acids (aepPNA)

A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation. Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested that the "cis-" homothymine aepPNA decamers with (2'R,4'R) and (2'S,4'S) configurations can bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the complementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA decamers with (2'R,4'S) and (2'S,4'R) configurations failed to form stable hybrid with poly(adenylic acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base (2'R,4'R)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations.     

Complete amino acid sequence of the ubiquinone binding protein (QP-C), a protein similar to the 14,000-dalton subunit of the yeast ubiquinol-cytochrome c reductase complex

The amino acid sequence of the ubiquinone binding protein (QP-C) in the cytochrome bc1 region of the mitochondrial electron transfer chain was determined by analysis of peptides obtained by cyanogen bromide cleavage and staphylococcal protease digestion of succinylated derivatives. It was found to consist of 110 amino acid residues and its amino terminus to be blocked by an acetyl group, as determined by mass spectrometry of the amino-terminal peptide and a comparison with peptides chemically synthesized on high-performance liquid chromatography. The molecular weight of this ubiquinone binding protein including the acetyl group was calculated to be 13,389. The predicted secondary structure of QP-C has alpha-helical content of about 50% and QP-C was classified as an "all-alpha" or "alpha + beta" protein. This is the first report describing the amino acid sequence of the ubiquinone binding protein. A comparison of this sequence with that of the 14-kDa subunit of the yeast ubiquinol-cytochrome c reductase complex from the nucleotide sequence showed these two sequences to be quite similar.     

Arsenite binding to synthetic peptides: the effect of increasing length between two cysteines

We utilized radioactive 73As-labeled arsenite and vacuum filtration methodology to determine the binding affinity of arsenite to eight synthetic peptides ranging from 13 to 24 amino acids long and containing one or two cysteines separated by 0-17 intervening amino acids. Six of the eight peptides were highly similar in amino acid sequence and were based on cysteine containing regions of the hormone-binding site of the human estrogen receptor-alpha (e.g., the sequence of peptide 28 is LEGAWCGKGVEGTEHLYSMKCKNV). The peptides with 0-14 intervening amino acids between two cysteines bound arsenite with Kd values of 2.7-20.1 uM and with Bmax values from 36 to 103 nmol/mg protein (from 0.083 to 0.19 nmol/nmol of protein). Thus, increasing the number of intervening amino acids from 0 to 14 made very little difference in the observed Kd values for arsenite, a surprising finding. Therefore, these peptides are flexible in solution and effectively contain a dithiol high affinity binding site for arsenite. Peptide 17 with two C separated by 19 amino acids bound arsenite with a Kd of 123 uM and a Bmax of 41.8 nmol/mg. The monothiol peptide 19 bound arsenite with a Kd of 124 uM and a Bmax of 26 nmol/mg protein. All experimental binding curves fit well to a one site binding model.     

Characterization of bacteriophage T4 regA protein-nucleic acid interactions

The bacteriophage T4 regA protein is a translational repressor of a group of T4 early mRNAs. We have characterized the binding of regA protein to polynucleotides and to specific RNAs. Binding to nucleic acids was monitored by the quenching of the intrinsic tryptophan fluorescence of regA protein. regA protein exhibited differential affinities for the polynucleotides examined, with the order of affinity being poly(rU) greater than poly(dT) greater than poly(dU) = poly(rG) greater than poly(rC) = poly(rA). The binding site size calculated for regA protein binding to poly(rU) was n = 9 +/- 1 nucleotides. Cooperativity was observed in binding to multiple-site oligonucleotides, with a cooperativity parameter (omega) value of 10-22. To study the specific interaction between regA protein and T4 gene 44 mRNA, the affinity of regA protein for synthetic gene 44 RNA fragments was measured. The association constant (Ka) for regA protein binding to gene 44 RNA fragments was 100-fold higher than for binding to nontarget RNA. Study of variant gene 44 RNA fragments indicated that the nucleotides required for specific binding are contained within a 12-nucleotide sequence spanning -12 to -1, relative to the AUG codon. The bases of five nucleotides (indicated in upper case type) are critical for specific regA protein interaction with the gene 44 recognition element, 5'-aaUGAGgAaauu-3'. These studies further showed that formation of a regA protein-RNA complex involves a maximum of 2-3 ionic interactions and is primarily an enthalpy-driven process.     

A synthetic peptide corresponding to the phosphorylcholine (PC)-binding region of human C-reactive protein possesses the TEPC-15 myeloma PC-idiotype.

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. CRP molecules from all species display Ca2(+)-dependent binding to phosphorylcholine (PC). The conserved PC-binding region of CRP corresponds to amino acids 51-66 within the human CRP sequence. A synthetic peptide composed of residues 47-63 of human CRP was previously shown to possess PC binding activity. The charged amino acids at positions 57, 58, 60, and 62 of this synthetic peptide were critical for PC-binding based on lower binding activity of synthetic peptides containing uncharged residues at these positions. The PC-binding peptide was used to generate mouse mAb that were tested for reactivity with intact CRP and with the TEPC-15 (T-15) mouse myeloma protein that also binds PC. The PC-binding peptide of CRP was recognized by two mAb specific for the T-15 Id. One of the mAb generated against the PC-binding peptide of CRP (IID6.2) recognized an epitope on the T-15 protein that was also recognized by the near-binding site-specific mAb (F6) to the T-15 PC-Id. Binding of IID6.2 to T-15 myeloma protein was not inhibited by PC and did not require Ca2+; however, binding was inhibited by the synthetic PC-binding peptide itself. Recognition of synthetic peptides containing uncharged amino acid substitutions by mAb F6 and IID6.2 was greatly reduced indicating that the shared epitope on T-15 and CRP was composed of similar charged residues. Therefore, CRP displays the same idiotope as an antibody that shares its specificity for the hapten, PC.     

Monoclonal antibodies distinguish synthetic peptides that differ in one chemical group

By using human calcitonin (hCT), human calcitonin-gene-related peptide (hCGRP), and a synthetic peptide with a sequence analogous to the 34 C-terminal amino acids of human preprocalcitonin (designated as PQN-34) as haptens in the generation of monoclonal antibodies, we assessed the role of amido and amino groups in paratope-epitope binding. By using peptide inhibition experiments and solid-phase immunoassays, monoclonal anti-hCT antibody CT07 and monoclonal anti-hCGRP antibody CGR01 were found to bind to an antigenic determinant located in the C-terminal segment of the hormones. These epitopes comprise the seven C-terminal amino acids of the hormones, and the presence of the hormone-ending carboxamide group was found to be essential for antibody binding. The corresponding heptapeptides, either bearing a carboxyl group or else linked to a glycine residue at their C-terminal part, failed to react with the antibodies. Moreover, these monoclonal antibodies did not bind to synthetic peptides analogous to the C-terminal region of the hormone precursor molecules that comprised the epitope site flanked by a peptide sequence. In an attempt to assess whether amido groups when present on the side-chain of amino acids may also modulate antibody binding, a monoclonal antibody referred to as QPO1 was produced and was found to recognize an antigenic determinant localized in the N-terminal region of the PQN-34 peptide bearing a glutamine residue as the N-terminal amino acid. The epitope was found to correspond to a topographic assembled site, and binding of QPO1 was found to be substantially dependent on the presence of the free amino and the side-chain amido groups borne by the N-terminal glutamine residue of this peptide PQN-34. In contrast to these findings, an antigenic determinant located in the internal sequence of calcitonin and recognized by monoclonal anti-hCT antibody CT08 was found to be expressed on the mature form of the hormone, as well as on synthetic peptides with sequence mimicking that of preprocalcitonin. These data should guide the choice of synthetic peptide haptens for the production of anti-protein antibodies.     

RNA-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp

Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His(6)-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (-) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met(11) to Arg(60). Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M(11)PVRRPLPPQPPRNARLI(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.     

Augmentation of the affinity of HLA class I-binding peptides lacking primary anchor residues by manipulation of the secondary anchor residues

A direct binding assay has been used to investigate the effect of the secondary anchor residues on peptide binding to class I proteins of the major histocompatibility complex. Based on predictions from a previous chemometric approach, synthetic peptide analogues containing unnatural amino acids were synthesized and tested for B*2705 binding. Hydrophobic unnatural amino acids such as α-naphthyl- and cyclohexyl-alanine were found to be excellent substituents in the P3 secondary anchor position giving peptides with very high B*2705-binding affinity. The binding to B*2705 of peptides optimized for their secondary anchor residues, but lacking one of the P2 or P9 primary anchor residues was also investigated. Most such peptides did not bind, but one peptide, lacking the P2 Arg residue generally considered essential for binding to all B27 subtypes, was found to bind quite strongly. These findings demonstrate that peptide binding to class I proteins is due to a combination of all the anchor residues, which may be occupied also by unnatural amino acids–a necessary step towards the development of peptidic or non-peptidic antagonists for immunomodulation.     

Interaction of nucleolar phosphoprotein B23 with nucleic acids

The interaction of eukaryotic nucleolar phosphoprotein B23 with nucleic acids was examined by gel retardation and filter binding assays, by fluorescence techniques, and by circular dichroism. All studies utilized protein prepared under native conditions by a newly developed purification procedure. Electrophoretic gel mobility shift assays with phage M13 DNA suggested that protein B23 is a single-stranded nucleic acid binding protein. This was confirmed in competition binding assays with native or heat-denatured linearized plasmid pUC18 DNA where the protein showed a marked preference for the denatured form. In other competition assays, there was no apparent preference for single-stranded synthetic ribo- versus deoxyribonucleotides. Equilibrium binding with poly(riboethenoadenylic acid) indicated cooperative ligand binding with a protein binding site size of 11 nucleotides and an apparent binding constant (K omega) of 5 x 10(7) M-1 which includes an intrinsic binding constant (K) of 6.3 x 10(4) M-1 and a cooperativity factor (omega) of 800. In circular dichroism (CD) studies, protein B23, when combined with the single-stranded synthetic nucleic acids poly(rA) and poly(rC), effected a decrease in ellipticity and a shift of the positive peak at 260-270 nm toward higher wavelengths, indicating helix destabilizing activity. No CD changes were seen with double-stranded poly(dA.dT). The change in ellipticity of poly(rA) was sigmoidal upon addition of protein, confirming the cooperative behavior seen with fluorescence methods. These studies indicate that protein B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity. These features may be related to its role in ribosome assembly.     

mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence.

We identified and produced antibodies to the major proteins that interact with poly(A)+ RNAs in the yeast Saccharomyces cerevisiae. The major proteins which were cross-linked by UV light to poly(A)+ RNA in intact yeast cells had apparent molecular weights of 72,000, 60,000, and 50,000. The poly(A) segment of the RNA was selectively cross-linked to the 72,000-molecular-weight protein (72K protein). Mice immunized with purified UV-cross-linked RNA-protein (RNP) complexes produced antibodies to the three major RNP proteins. A yeast genomic DNA library constructed in the lambda gt11 expression vector was screened with the anti-RNP serum, and recombinant bacteriophage clones were isolated. One recombinant phage, lambda YPA72.1, bearing a 2.5-kilobase insert, produced a large beta-galactosidase-RNP fusion protein. Affinity-selected antibodies from the anti-RNP serum on this fusion protein recognized a single 72K protein which was cross-linked to the poly(A) segment of RNA in the intact cell. Furthermore, the fusion protein of lambda YPA72.1 had specific poly(A)-binding activity. Therefore, lambda YPA72.1 encodes the 72K poly(A)-binding protein. Immunofluorescence microscopy showed that this protein was localized in the cytoplasm. Hybrid-selected mRNA translated in vitro produced the 72K poly(A)-binding protein, and mRNA blot analysis detected a single 2.1-kilobase mRNA. DNA blot analysis suggested a single gene for the poly(A)-binding protein. DNA sequence analysis of genomic clones spanning the entire gene revealed a long open reading frame encoding a 64,272-molecular-weight protein with several distinct domains and repeating structural elements. A sequence of 11 to 13 amino acids is repeated three times in this protein. Strikingly, this repeated sequence (RNP consensus sequence) is highly homologous to a sequence that is repeated twice in a major mammalian heterogeneous nuclear RNP protein, A1. The conservation of the repetitive RNP consensus sequence suggests an important function and a common evolutionary origin for messenger RNP and heterogeneous nuclear RNP proteins.     

An alpha-helical peptide model for electrostatic interactions of proteins with DNA. The N terminus of RecA

A series of synthetic peptides have been studied as models for non-specific protein-DNA interactions. In an alpha-helical conformation, the charged amino acid residues of the N-terminal 24 residues of RecA protein are asymmetrically distributed; at neutral pH there is a +4 charge on one face of the helix and a -3 charge on the other face. Modeling suggests that the positive face of the helix can bind five DNA phosphate groups by electrostatic interactions. Circular dichroism (c.d.) spectra indicate that the analogous peptide, Rec24 (AIDENKQKALAAALGQIEKQFGKG-amide), is largely unstructured in water but becomes highly helical in the presence of DNA. Peptide titrations of fluorescent etheno-DNA confirm that the changes in the c.d. spectrum of the peptide are associated with binding, although a dependence of the c.d. signal on the degree of DNA saturation is observed, indicating that peptide can be bound in more than one conformation. At saturation the peptide binds to 5.0(+/- 0.5) DNA phosphate groups as predicted and the electrostatic nature of the binding is confirmed by a strong dependence on salt concentration. A "mutant" peptide where an acidic glutamate residue replaces an alanine on the basic face of the Rec24 helix exhibits weaker binding to single-stranded DNA, also consistent with the electrostatic nature of the proposed peptide-DNA interaction. Extending Rec24 by ten amino acid residues, where the additional residues do not participate in the helical motif, does not noticeably affect binding. Thus, we show experimentally that an asymmetric charge distribution on an alpha-helix can represent an important element for binding nucleic acids.     

Hybridization and partial cDNA sequence analyses of bovine lung angiotensin I-converting enzyme

The mRNA encoding angiotensin I-converting enzyme, a zinc-metallo dipeptidyl carboxyhydrolase, has been identified in extracts prepared from bovine lung tissue. Bovine lung poly(A) + mRNAs were subjected to electrophoresis and northern blot hybridization analysis using a radiolabeled synthetic 24-deoxyoligonucleotide probe complementary to eight codons for amino acids at the active-site of the enzyme (Harris, R.B. & Wilson, I.B., J. Biol. Chem. 260, 2208-2211, 1985). This amino acid sequence contains the catalytic glutamic acid residue. A single RNA species (approximately equal to 4 kb) was detected which is 1 kb larger than predicted from the molecular weight of the enzyme. The excess nucleic acid composition may be due to leader and/or trailer sequences or the RNA may encode a high molecular weight precursor form of the enzyme. We have cloned an EcoR1-HindIII digest fragment (1400 bp) of the duplex cDNA derived from the bovine lung converting enzyme poly(A) + mRNA and also Bal31 deletion fragments generated from the 1400 bp clone. Several of the Bal31 clones contain the active-site sequence codons of the enzyme and the complete cDNA sequence of one of these (72 bp) has been determined. We found the amino acid sequence at the active site to be -Phe-Thr-Glu-Leu-Ala-Asn-Ser-, containing the catalytic Glu residue. This sequence is identical with the sequence that we previously determined by manual Edman degradation analysis of the appropriate active-site peptide except that we now find Asn instead of Asp. We have sequenced 670 bp of the 1400 bp clone but have not yet overlapped the active-site sequence.(ABSTRACT TRUNCATED AT 250 WORDS)