Prothymosin alpha and parathymosin: amino acid sequences deduced from the cloned rat spleen cDNAs

A rat spleen cDNA library was screened for clones carrying the cDNAs for prothymosin alpha and parathymosin. Sequence analysis of a clone carrying the entire coding region for prothymosin alpha confirmed and completed the amino acid sequence for this polypeptide and established the number of amino acid residues as 111. Rat prothymosin alpha differs from human prothymosin alpha at six positions, including four substitutions and two insertions. The nucleotide sequences of the cDNAs for the rat and human polypeptides are more than 90% identical in the open reading frames, with significant homology extending into the 5' and 3' flanking regions. From the same library, we also isolated a clone carrying 80% of the coding region for rat parathymosin. The number of amino acid residues in rat parathymosin is 101, based on the sequence deduced from the cDNA insert and earlier information on the sequence in the amino-terminal portion of this polypeptide. Despite their similarity in size and amino acid composition, rat prothymosin alpha and rat parathymosin show only limited sequence homology, primarily in the segment including residues 14 through 25, where 10 of 12 positions are identical in the two polypeptides. this is also the region of significant sequence similarity to a 12-amino-acid segment in the p17 protein of the human immunodeficiency disease associated virus (HTLV-IIIB).     

Amino acid sequences of hemoglobins I and II from root nodules of the non-leguminous Parasponia rigida-rhizobium symbiosis, and a correction of the sequence of hemoglobin I from Parasponia andersonii

The amino acid sequence of hemoglobins I (pI 6.15 as oxyhemoglobin) and II (pI 5.64 as oxyhemoglobin) from the nitrogen-fixing root nodules of Parasponia rigida have been determined by protein sequencing. The sequence of hemoglobin I (pI 6.16, as oxyhemoglobin) from Parasponia andersonii was re-examined and the corrected primary structure, now in agreement with that predicted from the DNA sequence, is reported. The three Parasponia hemoglobins contain 161 amino acid residues (Mr approximately equal to 18,700 including the heme) with a single cysteine residue and five methionine residues. The N-terminal serine is blocked by an acetyl group. The primary structure of the Parasponia hemoglobins is highly conserved. Hemoglobins I from the two species of Parasponia are identical; both show microheterogeneity at position 30 (Asp/Glu substitution) and hemoglobin I from P. rigida shows microheterogeneity at position 150 (Ala/Val) while hemoglobin I from P. andersonii has only an Ala at 150. P. rigida hemoglobin II shows no microheterogeneity at these positions, having Asp and Val residues respectively, and it contains a single amino acid change of a Gln for an Arg at position 85, which accounts for the 0.5 unit difference in isoelectric point observed between hemoglobins I and II. The sequence data are consistent with allelic heterogeneity at a single locus rather than different genes.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Alpha-helix stability in proteins. I. Empirical correlations concerning substitution of side-chains at the N and C-caps and the replacement of alanine by glycine or serine at solvent-exposed surfaces.

The importance of amino acid side-chains in helix stability has been investigated by making a series of mutations at the N-caps, C-caps and internal positions of the solvent-exposed faces of the two alpha-helices of barnase. There is a strong positional and context dependence of the effect of a particular amino acid on stability. Correlations have been found that provide insight into the physical basis of helix stabilization. The relative effects of Ala and Gly (or Ser) may be rationalized on the basis of solvent-accessible surface areas: burial of hydrophobic surface stabilizes the protein as does exposure to solvent of unpaired hydrogen bond donors or acceptors in the protein. There is a good correlation between the relative stabilizing effects of Ala and Gly at internal positions with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Ala----Gly. The relationship may be extended to the N and C-caps by including an extra term in hydrophilic surface area for the solvent exposure of the non-intramolecularly hydrogen-bonded main-chain CO, NH or protein side-chain hydrogen bonding groups. The requirement for solvent exposure of the C-cap main-chain CO groups may account for the strong preference for residues having positive phi and psi angles at this position, since this alpha L-conformation results in the largest solvent exposure of the C-terminal CO groups. Glycine in an alpha L-conformation results in the greatest exposure of these CO groups. Further, the side-chains of His, Asn, Arg and Lys may, with positive phi and psi-angles, form a hydrogen bond with the backbone CO of residue in position C -3 (residues are numbered relative to the C-cap). The preferences at the C-cap are Gly much greater than His greater than Asn greater than Arg greater than Lys greater than Ala approximately Ser approximately greater than Asp. The preferences at the N-cap are determined by hydrogen bonding of side-chains or solvent to the exposed backbone NH groups and are: Thr approximately Asp approximately Ser greater than Gly approximately Asn greater than Gln approximately Glu approximately His greater than Ala greater than Val much greater than Pro. These general trends may be obscured when mutation allows another side-chain to become a surrogate cap.(ABSTRACT TRUNCATED AT 400 WORDS)     

The sequence of human parathymosin deduced from a cloned human kidney cDNA

The amino acid sequence of human parathymosin has been deduced from the cDNA sequence of a clone isolated from a human kidney cDNA library. Screening of the cDNA library with a probe containing a partial rat cDNA sequence yielded two clones containing inserts of 1200 and 1100 base pairs respectively, each including the complete open reading frame for human parathymosin. The open reading frame contains 306 nucleotides, including the codon for the initiator methionine. Analysis of the 5' flanking sequence excluded the presence of a hydrophobic signal peptide in the translated sequence. It may therefore be concluded that parathymosin, like prothymosin alpha, is synthesized without formation of a large precursor polypeptide. Comparison of the deduced amino acid sequence with the known primary structure of rat and bovine parathymosins shows that the primary structure of parathymosin is highly conserved among these species.     

Comparison of the three primary structures of deoxyribonuclease isolated from bovine, ovine, and porcine pancreas. Derivation of the amino acid sequence of ovine DNase and revision of the previously published amino acid sequence of bovine DNase

Based on the published bovine DNase sequence (Liao, T.-H., Salnikow, J., Moore, S., and Stein, W. H. (1973) J. Biol. Chem. 248, 1489-1495), the ovine DNase sequence is derived from the amino acid compositions of isolated short peptides covering all regions of the intact polypeptide. The sequence is substantiated by results of automated Edman degradation of the intact polypeptide and of the two middle CNBr fragments, and by elucidation of the complete sequence of the COOH-terminal CNBr peptide. The 12 changes from bovine to ovine DNase are at residues 22 (Ala to Ser), 29 (Val to Leu), 35 (Val to Ala), 54 (Tyr to Asp), 62 (Thr to Ser), 83 (Leu to Val), 121 (His to Pro), 127 (Glu to Ala), 132 (Ala to Pro), 159 (His to Asp), 163 (Val to Ile), and 231 (Ala to Val). A minor genetic variant form of ovine DNase has Val at residue 163. The data from automated Edman degradation of the largest CNBr peptide of bovine DNase show that the published bovine DNase sequence is in error and that an Ile-Val-Arg tripeptide must be inserted between Arg-27 and Arg-28. The corrected sequence is substantiated by two peptides covering this region each with three amino acids more than the published sequence. Comparison of the bovine, ovine, and porcine DNase sequences reveals the following: with the revised bovine sequence, all three DNase sequences can be aligned without a gap; all three DNases have a carbohydrate side chain at Asn-18, but only porcine DNase has carbohydrate at Asn-106; there are 12 changes between bovine and ovine DNases, 56 between bovine and porcine, and 50 between ovine and porcine; there are six highly variable regions and four invariable ones; bovine and ovine DNases have the same length while porcine DNase is longer by 2 amino acid residues at the COOH terminus; the residues around the nucleotide-binding site, the four pairs of salt bridges, and the essential His-134 groups are not changed.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: dissection of the phosphopeptide sequence specificity and coupling energetics

SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and Ile (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Gly), +2 (Gln, Asp, Ala, Gly), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (DeltaDeltaG degrees 相似文献   

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Molecular cloning and sequence variation of UDP-glucose pyrophosphorylase cDNAs from potatoes sensitive and resistant to cold sweetening

RT-PCR was used to isolate seven cDNAs encoding uridine diphosphate-glucose pyrophosphorylase (UGPase) from six potato cultivars that differed markedly in their ability to sweeten in cold storage (2-4 degrees C). These sequences were compared to two potato UGPase-cDNAs previously published. All cDNAs were highly conserved (97.6-99.9%) and coded for polypeptides with 477 amino acids. The cDNAs could be placed into two sequence classes depending on whether they contained a BamH1 site at nucleotide positions 1315-1320. The presence of the BamH1 site (substitution of a C for a T at bp position 1320) did not lead to a change of an amino acid in the mature protein. There were 27 nucleotide polymorphisms that co-segregated along with the BamH1 site, five of which led to an amino acid change (i.e., bp positions (5) Thr for Ala; (30) Glu for Asp; (82) Lys for Asn; (445) Lys for Glu; and (450) Val for Ile). All of the encoded polypeptides contained the five highly conserved lysine residues located at positions 263, 329, 367, 409 and 410 that have been demonstrated necessary for catalytic activity of UGPase. All polypeptides had putative glycosylation sites at amino acid positions 168 (NQS) and 307 (NLS). The Ser at position 420 provided a putative site for phosphorylation as well as a binding motif for 14-3-3 proteins.     

Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of alpha-chymases from different species indicated that certain rodent alpha-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the alpha-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His6 purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1'-(Xaa), and P2'-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat alpha-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human alpha-chymase.     

Primary structure of the casein macropeptide of caprine kappa casein]

The amino acid sequence of caprine CMP, the negatively charged C-terminal fragment released by chymosin (rennin EC 3.4.23.4) from goat K-casein at the initial stage of the milk-clotting process, has been investigated. The complete sequence has been determined by analysing chymotryptic and "thermolysin" fragments of the CMP. Caprine CMP contains 66 amino acid residues, 2 being phosphorylated. Asp2, Asn5, Thr11, Ser6, SerP2, Glu7, Gln2, Pro6, Ala9 Val5, Met1, Ile6, Lys3, His1, and the carbohydrate-free polypeptide chain has a molecular weight of 6,998 daltons. The occurrence in caprine CMP of an additional phosphate group, linked to serine 168 in the C-terminal region Thr-Ser168-Thr-Glu170-Val.OH of the polypeptide chain, has given support to the phosphorylation code for caseins that we postulated earlier [28, 27]. According to this hypothesis, a specific phosphoryl kinase may recognize an anionic phosphorylation site corresponding to the tripeptide sequence Thr/Ser-X-Glu, X being any amino acid residue. Since the C-terminal sequence of bovine and caprine CMPs differ by the substitution Ala/Glu170 (caprine), phosphorylation of caprine serine 168 could be explained by the occurrence of the new phosphorylation site Ser168-Thr-Glu170.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Effects of side-chain characteristics on stability and oligomerization state of a de novo-designed model coiled-coil: 20 amino acid substitutions in position "d"

We describe the de novo design and biophysical characterization of a model coiled-coil protein in which we have systematically substituted 20 different amino acid residues in the central "d" position. The model protein consists of two identical 38 residue polypeptide chains covalently linked at their N termini via a disulfide bridge. The hydrophobic core contained Val and Ile residues at positions "a" and Leu residues at positions "d". This core allowed for the formation of both two-stranded and three-stranded coiled-coils in benign buffer, depending on the substitution at position "d". The structure of each analog was analyzed by CD spectroscopy and their relative stability determined by chemical denaturation using GdnHCI (all analogs denatured from the two-stranded state). The oligomeric state(s) was determined by high-performance size-exclusion chromatography and sedimentation equilibrium analysis in benign medium. Our results showed a thermodynamic stability order (in order of decreasing stability) of: Leu, Met, Ile, Tyr, Phe, Val, Gln, Ala, Trp, Asn, His, Thr, Lys, Ser, Asp, Glu, Arg, Orn, and Gly. The Pro analog prevented coiled-coil formation. The overall stability range was 7.4 kcal/mol from the lowest to the highest analog, indicating the importance of the hydrophobic core and the dramatic effect a single substitution in the core can have upon the stability of the protein fold. In general, the side-chain contribution to the level of stability correlated with side-chain hydrophobicity. Molecular modelling studies, however, showed that packing effects could explain deviations from a direct correlation. In regards to oligomerization state, eight analogs demonstrated the ability to populate exclusively one oligomerization state in benign buffer (0.1 M KCl, 0.05 M K(2)PO(4)(pH 7)). Ile and Val (the beta-branched residues) induced the three-stranded oligomerization state, whereas Tyr, Lys, Arg, Orn, Glu and Asp induced the two-stranded state. Asn, Gln, Ser, Ala, Gly, Phe, Leu, Met and Trp analogs were indiscriminate and populated two-stranded and three-stranded states. Comparison of these results with similar substitutions in position "a" highlights the positional effects of individual residues in defining the stability and numbers of polypeptide chains occurring in a coiled-coil structure. Overall, these results in conjunction with other work now generate a relative thermodynamic stability scale for 19 naturally occurring amino acid residues in either an "a" or "d" position of a two-stranded coiled-coil. Thus, these results will aid in the de novo design of new coiled-coil structures, a better understanding of their structure/function relationships and the design of algorithms to predict the presence of coiled-coils within native protein sequences.     

Amino acid residue penultimate to the amino-terminal gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation

To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated model protein. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system or in transfected cells. It was found that the amino acid residue at position 3 in an N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the susceptibility of the protein to two different cotranslational protein modifications, N-myristoylation and N-acetylation; 10 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, and His) with a radius of gyration smaller than 1.80 A directed N-myristoylation, two negatively charged residues (Asp and Glu) directed N-acetylation, and two amino acids (Gly and Met) directed heterogeneous modification with both N-myristoylation and N-acetylation. The amino acid requirements at this position for the two modifications were dramatically changed when Ser at position 6 in the consensus motif was replaced with Ala. Thus, the amino acid residue penultimate to the N-terminal Gly residue strongly affected two cotranslational protein modifications, N-myristoylation and N-acetylation, and the amino acid requirements at this position for these two modifications were significantly affected by downstream residues.     

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).     

The complete amino acid sequence of rat submaxillary gland tonin does contain the aspartic acid at the active site: confirmation by protein sequence analysis

The revised amino acid sequence of rat submaxillary gland tonin, a serine protease, does contain the active site Asp residue. The active site of this kallikrein-related enzyme is thus made up of the same catalytic triad (Asp, Ser, and His) found in all known serine proteases. The important Asp residue has now been localized in a 16 amino acid peptide previously reported as missing in the tonin sequence. The complete amino acid sequence thus contains 235 residues corresponding to a molecular weight of 25,658, more in agreement with previously reported molecular weights. Moreover, the revised structure led (a) to the assignment of Arg, Asn, and Val residues instead of His, Asp, and Gly at positions 63, 165, and 169, respectively; (b) to the assignment of residues occupying an overlapping sequence at positions 165-171, and finally (c) to the localization of two N-glycosylation sites at positions 82 and 165. These results further document the close relationship of tonin to the ever expanding kallikrein family.