Reinvestigation of the N-terminal amino acid sequence of β-lipotropin from human pituitary glands

The N-terminal sequence of the first 60 amino acid residues of human β-LPH was reinvestigated by automated Edman degradation on the intact peptide. The result shows some differences with the recently proposed data. We have established the amino acid sequence of residues 9–26 to be Glu-Gly-Asp-Gly-Pro-Asp-Gly-Pro-Ala-Asp-Asp-Gly-Ala-Gly-Ala-Gln-Ala-Asp. From residues 1–8 and 27–60 the sequence is exactly identical with that of recently reported data.     

The amino acid sequence of ferredoxin from Sambucus nigra

The amino acid sequence of the ferredoxin from Sambucus nigra consists of a single polypeptide chain of 97 amino acid residues, 5 of which are cysteine. The positions of the 4 cysteine residues which bind the iron atoms of the active centre are identical to those of other ferredoxins. Due to difficulties of obtaining pure protein, residues 87–90 have only been identified from the amino acid analysis of peptide C 10 and by homology with other higher plant ferredoxins.     

Evaluation of the protein interfaces that form an intermolecular four-helix bundle as studied by computer simulation

Rational design of protein surface is important for creating higher order protein structures, but it is still challenging. In this study, we designed in silico the several binding interfaces on protein surfaces that allow a de novo protein–protein interaction to be formed. We used a computer simulation technique to find appropriate amino acid arrangements for the binding interface. The protein–protein interaction can be made by forming an intermolecular four-helix bundle structure, which is often found in naturally occurring protein subunit interfaces. As a model protein, we used a helical protein, YciF. Molecular dynamics simulation showed that a new protein–protein interaction is formed depending on the number of hydrophobic and charged amino acid residues present in the binding surfaces. However, too many hydrophobic amino acid residues present in the interface negatively affected on the binding. Finally, we found an appropriate arrangement of hydrophobic and charged amino acid residues that induces a protein–protein interaction through an intermolecular four-helix bundle formation.     

Primary structure of the amino-terminal (vitamin K-dependent) region of human prothrombin

The amino acid sequence of residues 1–51 of human prothrombin fragment 1 has been determined. This 51 residue peptide is 1 residue shorter than the comparable bovine region and 8 of the amino acid residues are different. The positions of 10 glutamic acid residues are identical to the γ-carboxylated ones in the bovine species. From this homology and additional evidence, these residues in human prothrombin are considered to be γ-carboxylated. The sequence was completed by automated Edman degradation of the reduced, alkylated fragment and its subfragmentation with trypsin, thermolysin and acid hydrolysis.     

Structure-wise discrimination of cytosine,thymine, and uracil by proteins in terms of their nonbonded interactions

The molecular recognition and discrimination of very similar ligand moieties by proteins are important subjects in protein–ligand interaction studies. Specificity in the recognition of molecules is determined by the arrangement of protein and ligand atoms in space. The three pyrimidine bases, viz. cytosine, thymine, and uracil, are structurally similar, but the proteins that bind to them are able to discriminate them and form interactions. Since nonbonded interactions are responsible for molecular recognition processes in biological systems, our work attempts to understand some of the underlying principles of such recognition of pyrimidine molecular structures by proteins. The preferences of the amino acid residues to contact the pyrimidine bases in terms of nonbonded interactions; amino acid residue–ligand atom preferences; main chain and side chain atom contributions of amino acid residues; and solvent-accessible surface area of ligand atoms when forming complexes are analyzed. Our analysis shows that the amino acid residues, tyrosine and phenyl alanine, are highly involved in the pyrimidine interactions. Arginine prefers contacts with the cytosine base. The similarities and differences that exist between the interactions of the amino acid residues with each of the three pyrimidine base atoms in our analysis provide insights that can be exploited in designing specific inhibitors competitive to the ligands.     

Molecular dynamics study of femtosecond events in photoactive yellow protein after photoexcitation of the chromophore

Molecular dynamics simulations were carried out to study what happens in a photoreceptor protein, photoactive yellow protein (PYP), immediately after the vertical transition of the chromophore from the ground to the excited state. A photon absorption simulation was performed to investigate the movement of amino acid residues upon photoexcitation. To calculate the excited state of the chromophore, SCF-CI calculation was carried out with INDO/S Hamiltonian. We observed that some amino acid residues have strong interactions with the chromophore. Most of these amino acid residues are conserved in PYPs from three different species of bacteria. This observation indicates the biological importance of these residues. Proteins 32:268–275, 1998. © 1998 Wiley-Liss, Inc.     

Amino Acid Sequence of Natural Tumor Necrosis Factor α Inhibitor Purified from Human Urine

Tumor necrosis factor α inhibitor (TNF–INH) was purified from human urine and it was composed of 161 amino acid residues. The complete amino acid sequence of TNF–INH found by sequence analysis agreed with that predicted from the cDNA structure for the extracellular domain (1–161 portion) of 55-kDa TNF receptor and its processing site at the C-terminal was Asn-161.     

Isolation and Amino Acid Sequences of Small Tryptic Peptides from the Oxidized Ala Chain of Ricin D

Twelve tryptic peptides as well as free arginine were isolated from the performic acid-oxidized Ala chain of ricin D by gel filtration on Sephadex G–25 and Dowex 1×2 column chromatography followed by paper chromatography. Total number of the amino acid residues in these peptides accounted for 90 out of 263 residues in the Ala chain of ricin D.

The amino acid sequences of nine peptides were determined by manual Edman degradation.     

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes

In most of homeodomain–DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1–DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein–DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein–DNA complexes. The order of stability of protein–DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein–DNA complexes. Among specific protein–DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.     

Epitope analysis using kinetic measurements of antibody binding to synthetic peptides presenting single amino acid substitutions

The fine modulation of peptide–antibody interactions was investigated with anti-peptide monoclonal antibodies recognizing peptide 125–136 of the coat protein of tobacco mosaic virus. Nine synthetic peptides presenting single amino acid substitutions were selected for detailed analysis on the basis of their reactivity in ELISA. Kinetic measurements of the binding of four antibodies to these peptides performed with a biosensor instrument (BIAcore^TM, Pharmacia) were used to quantify the contribution of individual residues to antibody binding. The results showed that even conservative exchanges of some residues in the epitope results in a small but significant decrease of the equilibrium affinity constant due mostly to a higher dissociation rate constant of the monoclonal antibodies. Two amino acid residues directly adjacent to the epitope, which appeared to play no role when tested by ELISA, were shown to influence the kinetics of binding. These data should be useful for computer modelling of the peptide–antibody interactions.     

Conversion of α-Terpineol to 8,9-Epoxy-p-menthan-1-ol

The complete amino acid sequence of pokeweed lectin-B (PL-B) has been analyzed by first sequencing seven lysylendopeptidase peptides derived from the reduced and S-pyridylethylated PL-B and then connecting them by analyzing the arginylendopeptidase peptides from the reduced and S-carboxymethylated PL-B. PL-B consists of 295 amino acid residues and two oligosaccharides linked to Asn96 and Asnl39, and has a molecular mass of 34,493 Da. PL-B is composed of seven repetitive chitin-binding domains having 48–79% sequence homology with each other. Twelve amino acid residues including eight cysteine residues in these domains are absolutely conserved in all other chitin-binding domains of plant lectins and class I chitinases. Also, it was strongly suggested that the extremely high hemagglutinating and mitogenic activities of PL-B may be ascribed to its seven-domain structure.     

Identification and analysis of novel amino acid sequence repeats and domains in Pyrobaculum aerophilum using computational tools

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.     

Site-directed Mutagenesis of Chitinase from Alteromonas sp. Strain O–7

Chitinase (Chi85) from Alteromonas sp. strain 0–7 contains the two conserved regions common to microbial and plant chitinases. We did site-directed mutagenesis of Chi85 to investigate the effects of the conserved amino acid residues on chitinase activity. We suggest that Asp-290 and Glu-292 of Chi85 may be the essential amino acid residues for the cleavage of β-glycosidic linkage of chitin.     

The independent distribution of amino acid near neighbor pairs into polypeptides

With the assumption that individual amino acids are independently distributed into naturally occurring polypeptide chains, it is shown that amino acid pairs with 0–2 arbitrary intervening residues are also independently distributed, with a few possible exceptions. This is not true of N- and C-terminal amino acids.     

Solid phase isotope exchange of deuterium and tritium for hydrogen in human recombinant insulin

Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B_1–13) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius, and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity.     

The amino-terminal hydrophilic region of the vacuolar transporter Avt3p is dispensable for the vacuolar amino acid compartmentalization of Schizosaccharomyces pombe

Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3^(?1–270) expressed in S. pombe avt3? cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3Δ cells in spore formation was rescued by the expression of avt3⁺ but was not completely rescued by the expression of avt3^(?1–270). The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.     

Plasmodium Falciparum: The Adherence of Erythrocytes Infected with Human Malaria Can Be Mimicked Using Pfalhesin-Coated Microspheres

Infection of human erythrocytes with the malaria parasite, Plasmodium falciparum, results in the exposure of amino acid residues 542–555 of the anion-exchange protein, band 3, in a conformation that enables the cell to adhere to C32 amelanotic melanoma cells. Attempts to isolate this adhesive form from infected cells by irnmunoaffinity were unsuccessful, and so other approaches were utilized. Chinese hamster ovary (CHO) cells tTansfected with cDNA encoding the first 578 amino acid residues of human band 3 protein transiently expressed the protein efficiently. A murine monoclonal antibody (MAb) that specifically recognizes the adhesin exposed on the surface of erythrocytes bearing mature stages of P. falciparum immunostained some transfected cells, confirming that the first 578 amino residues are sufficient for the adhesive conformation. As a more efficient alternative to transgenic expression of the adhesin, microspheres with covalently bound peptides fashioned on band 3 sequences previously found to be adherent (residues 546–553 and 820–829 and called pfalhesin) were produced. The pfalhesin-coated microspheres specifically bound to C32 amelanotic melanoma cells, whereas microspheres coupled with a scrambled version of residues 546–553 had little binding capacity for melanoma cells.

These results demonstrate that the previously identified band 3-related peptides that inhibit cytoadherence interact directly with target cells and suggest that microspheres with covalently coupled peptides might constitute novel ‘artificial’ P. falciparum-infected erythrocytes for use in in vitro and in vivo studies.     

The construction of an amino acid network for understanding protein structure and function

Amino acid networks (AANs) are undirected networks consisting of amino acid residues and their interactions in three-dimensional protein structures. The analysis of AANs provides novel insight into protein science, and several common amino acid network properties have revealed diverse classes of proteins. In this review, we first summarize methods for the construction and characterization of AANs. We then compare software tools for the construction and analysis of AANs. Finally, we review the application of AANs for understanding protein structure and function, including the identification of functional residues, the prediction of protein folding, analyzing protein stability and protein–protein interactions, and for understanding communication within and between proteins.     

Comparison of NMR and MD N–H bond order parameters: example of HIV-1 protease

It has been suggested that the cause of disagreements between molecular dynamics (MD) and NMR N–H bond order parameters is the fact that the NMR order parameter is determined for different amino acid residues at different time intervals, while the MD one is derived for all residues from the same MD trajectory of the same time interval. Therefore, it has been proposed for correct comparison with NMR data to calculate the MD order parameter for different amino acid residues separately for trajectory ranges close to NMR correlation time. The MD simulation of the human immunodeficiency virus type-1 protease (HIV-1 PR) with monoprotonated active centre was performed for verification of the proposition. It has been shown that the protease in aqueous solution adopts a set of conformations, which are intermediate between semiopen and closed ones. The calculated MD N–H bond order parameters are in agreement with literature NMR data in confidence interval limits.     

Truncated reactive center loop decrease the inhibitory activity of Antheraea pernyi serine protease inhibitor 6

Here, we assessed the effect of a systematic change in reactive center loop (RCL) length, N-terminal to the reactive center, on the inhibitory activity of the recombinant Apserpin-6. The domain prediction results indicated that the RCL is located between the amino acid numbered 359–379 at the C-terminal of Apserpin-6. The N-terminal variable region for amino acid positions P7–P1 of the RCL of Apserpin-6 was truncated or extended by residue deletion or insertion using site-directed mutagenesis. The recombinant Apserpin-6 with one or two residues insertion in RCL had no effect on prophenoloxidase (proPO) activity, whereas deletion of one or two residues in RCL lowered the efficiency of inhibition of Apserpin-6. The results of this study will facilitate the understanding of inhibition mechanism of RCL on proPO activity.