Human leukocyte interferon: Isolation and characterization of several molecular forms

Purification of human leukocyte interferon by high-performance liquid chromatography yielded eight major interferon species. These were characterized by their molecular weight, amino acid composition, and antiviral activity on human and bovine cells. The molecular weights of the purified species ranged from 16,000 to 21,000. The tryptic peptide profiles of all the pure species were compared. Both amino acid compositions and tryptic peptide profiles revealed a structural similarity among the various species.     

Isolation and chemical characterization of the phosphoproteins of chicken bone matrix: heterogeneity in molecular weight and composition

Ethylenediaminetetraacetic acid and HCl extracts of calcified chicken bone were fractionated by a variety of techniques, including molecular sieving in guanidinium chloride, ion-exchange chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC), reverse-phase HPLC, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using several different experimental schemas, we isolated 14 apparently homogeneous components varying in molecular weight from approximately 150K to approximately 4K-5K. The compositions of all of the phosphoproteins were characterized by high concentrations of Asp, Glu, Ser, Gly, and Ala. Seven of the components which were analyzed contained concentrations of carbohydrate varying from approximately 4% to approximately 17%. Three of the components containing O-phosphoserine which behaved as single bands on SDS-PAGE with molecular weights of approximately 150K, approximately 90K, and approximately 70K contained Hyp and Hyl or Hyl alone and may represent covalently bonded or strongly associated collagen-phosphoprotein complexes or hydroxylated Pro and/or Lys residues of the phosphoproteins. The findings that the amino acid compositions of several of the components were very similar and that N-terminal partial amino acid sequences of the approximately 90- and approximately 60-kilodalton (kDa) and of the approximately 150- and approximately 32-kDa components, respectively, were identical make it clear that some of the lower molecular weight components are derived by proteolysis from higher molecular weight species. In addition to proteolysis, we speculate that it is possible, from the N-terminal amino acid sequence data and preliminary cross-reaction studies of antibodies to four of the phosphoproteins, that the heterogeneity observed in the phosphoprotein components may also be due in part to there being more than one independent gene product for chicken bone phosphoproteins.     

Complete amino acid sequence of a subunit from rapeseed high molecular weight protein

A subunit (Mr 15,600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 x 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.     

Validation of molecular force field parameters for peptides including isomerized amino acids

Recently, stereoinversions and isomerizations of amino acid residues in the proteins of living beings have been observed. Because isomerized amino acids cause structural changes and denaturation of proteins, isomerizations of amino acid residues are suspected to cause age‐related diseases. In this study, AMBER molecular force field parameters were tested by using computationally generated nonapeptides and tripeptides including stereoinverted and/or isomerized amino acid residues. Energy calculations by using density functional theory were also performed for comparison. Although the force field parameters were developed by parameter fitting for l ‐α‐amino acids, the accuracy of the computational results for d ‐amino acids and β‐amino acids was comparable to those for l ‐α‐amino acids. The conformational energies for tripeptides calculated by using density functional theory were reproduced more accurately than those for nonapeptides calculated by using the molecular mechanical force field. The evaluations were performed for the ff99SB, ff03, ff12SB, and the latest ff14SB force field parameters.     

Compositional changes in RNA,DNA and proteins for bacterial adaptation to higher and lower temperatures

It is known that in thermophiles the G+C content of ribosomal RNA linearly correlates with growth temperature, while that of genomic DNA does not. Although the G+C contents (singlet) of the genomic DNAs of thermophiles and methophiles do not differ significantly, the dinucleotide (doublet) compositions of the two bacterial groups clearly do. The average amino acid compositions of proteins of the two groups are also distinct. Based on these facts, we here analyzed the DNA and protein compositions of various bacteria in terms of the optimal growth temperature (OGT). Regression analyses of the sequence data for thermophilic, mesophilic and psychrophilic bacteria revealed good linear relationships between OGT and the dinucleotide compositions of DNA, and between OGT and the amino acid compositions of proteins. Together with the above-mentioned linear relationship between ribosomal RNA and OGT, the DNA and protein compositions can be regarded as thermostability measures for RNA, DNA and proteins, covering a wide range of temperatures. Both the DNA and proteins of psychrophiles apparently exhibit characteristics diametrically opposite to those of thermophiles. The physicochemical parameters of dinucleotides suggested that supercoiling of DNA is relevant to its thermostability. Protein stability in thermophiles is realized primarily through global changes that increase charged residues (i.e., Glu, Arg, and Lys) on the molecular surface of all proteins. This kind of global change is attainable through a change in the amino acid composition coupled with alterations in the DNA base composition. The general strategies of thermophiles and psychrophiles for adaptation to higher and lower temperatures, respectively, that are suggested by the present study are discussed.     

Impact of potassium,sodium, and salinity on the protein-and free amino acid content of wheat grain

A lysimeter study was conducted on Cajeme wheat (Triticum aestivum L.) to investigate the impact of salinity on protein and free amino acid content of the grain. Cross correlations were obtained between 16 different soil-plant-water based parameters and the concentration and total accumulation of amino acids. The results indicated that after 3 years of irrigation, the majority of protein bound and free amino acids increased in concentration in the grain. However, both free tryptophan and free proline revealed decreasing concentrations with increasing salinity. Free tryptophan showed a synergism between total accumulation, yield and concentration. Free proline concentrations decreased in association with increasing protein concentrations. Cross correlations of the 16 soil-plant-water based parameters with free and protein bound amino acids revealed significant correlations for free aspartic acid and glycine with total accumulation but not with concentrations. Only methionine plus cystine was lower than suggested FAO levels for essential amino acids and was lower in the third year than in the first year.     

Two human trypsinogens. Purification, molecular properties, and N-terminal sequences

The two human trypsinogens have been isolated from human pancreatic juice in a sufficient amount to study molecular and structural properties. The purification procedure included filtration on Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The two trypsinogens represent 19% of total proteins of pancreatic juice. Trypsinogen 1, the major form, is present in a quantity twice that of trypsinogen 2, which is the most anionic protein in human pancreatic juice. The two proteins have partial immunological identity, close molecular weights (23 438 and 25 006 for trypsinogens 1 and 2, respectively) and similar amino acid compositions. The N-terminal sequences are the same for the first 9 residues: Ala-Pro-Phe-Asp4-Lys-Ile. The two proteins differ in the activation peptides released during the transformation to trypsins. Trypsinogen 2 liberates one octapeptide Ala-Pro-Phe-Asp4-Lys while trypsinogen 1 liberates two peptides, the same octapeptide and the pentapeptide (Asp)4-Lys.     

Fluorescence and amino acid characteristics of molecular size fractions of DOM in the waters of Lake Biwa

Dissolved organic matter (DOM) in the waters from Lake Biwa, Japan was fractionated using tangential flow ultrafiltration, and subsequently characterized by fluorescence properties and amino acids. While major dissolved organic carbon (DOC), UV absorbance (Abs), humic-like fluorescence (Flu) and total hydrolyzed amino acids (THAA) occurred in the less than 5 kDa molecular size fraction, they were not evenly distributed among various molecular size fractions. Flu/Abs ratios increased, and THAA/DOC ratios decreased with decreasing molecular size. Humic-like fluorescence occurred in all molecular size fractions, but protein-like fluorescence only occurred in the 0.1 m-GF/F fraction. Subtle differences in amino acid compositions (both individuals and functional groups) were observed between various molecular size fractions, this may indicate the occurrence of DOM degradation from higher to lower molecular weight. The results reported here have significance for further understanding the sources and nature of DOM in aquatic environments.     

Yolk Phosphoprotein Metabolism during Early Development of the Fish, Oryzias latipes

Changes in yolk phosphoproteins of Oryzias latipes embryos during early development were examined by electrophoresis. Four phosphoproteins were identified in the yolk of blastulae by polyacrylamide gel electrophoresis. Two of them were high molecular weight phosphoproteins containing 0.7% (w/w) phosphorus and with similar amino acid compositions to that of vitellogenin. The other two were low molecular weight phosphoproteins, characterized by high contents of phosphorus [12.2% (w/w)] and serine (44.8 mole%) and low contents of aromatic amino acid residues. From these characteristics, together with their behaviors on DEAE-cellulose chromatography and electrophoresis and low stainability with dyes, the latter two were concluded to be phosvitins. These phosvitins were isolated and partially characterized.
The yolk phosphoproteins, especially the phosvitins, were degraded, their amounts in embryos decreased throughout early development. Studies on the mechanism of endogenous phosphoprotein degradation strongly suggested the participation of some protease(s) that was precipitated on centrifugation of the egg homogenate at 14,000 × g for 10 min.     

Unexpected amino acid composition of modern Reptilia and its implications in molecular mechanisms of dinosaur extinction

Dinosaur extinction is a great challenge to evolutionary biology. Although accumulating evidence suggests that an abrupt change of environment, such as a long period of low temperature induced by asteroid hit or other disasters, may be responsible for dinosaur extinction, little is known about the underlying molecular mechanisms. By analyzing the amino acid compositions of 13 biological classes, we found that the charged amino acid content of modern Reptilia, the sibling of dinosaur, is strikingly different from those of other classes, which inspires us to propose a possible molecular mechanism for dinosaur extinction.     

Polypeptide molecular weights of the (Na+,K+)-ATPase from porcine kidney medulla

The molecular weights of the polypeptide chains from (Na+,K+)-ATPase of porcine kidney medulla have been determined by analytical sedimentation equilibrium. The alpha-subunit molecular weight is 93 900, and the beta subunit is a glycoprotein with a polypeptide molecular weight of 32 300 (41 400 including protein and carbohydrate). Amino acid and carbohydrate compositions are presented together with related properties (i.e., partial specific volumes, extinction coefficients, and hydrophobic/hydrophilic amino acid content).     

Heme-linked spectral changes of the protein moiety of hemoproteins in the near ultraviolet region

Spectral changes of hemoproteins in the near ultraviolet region on binding to a ligand and on oxidation-reduction of the heme-iron were studied by computer-controlled spectrophotometry. Near ultraviolet difference spectra between the low spin and high spin forms of ferric hemoproteins were classified into three groups: Those showing two absorption peaks having maxima at around 285 and 295 nm, those showing a peak at around 275 nm, and those showing a peak at around 300 nm. No corresponding absorption peak was observed with model heme complexes of low molecular weight. The intensity of the peak in cyanide difference spectra of catalase and horseradish peroxidase in the near ultraviolet region was dependent on the concentration of added cyanide and paralleled the intensity of the spectral changes in the Soret region. The spectral changes in both the near ultraviolet and Soret regions developed within 6 ms after the addition of cyanide. Difference spectra between the reduced and oxidized forms of cytochrome c, cytochrome oxidase-cyanide complex, hemoglobin, and lactoperoxidase-cyanide complex showed a characteristic peak at around 285-290 nm. Various difference spectra of hemoglobin in the near ultraviolet region were also measured. The observed positions, shapes, combinations, and relative intensities of the peaks were compared with those of solvent perturbation difference spectra and pH difference spectra of proteins and aromatic amino acids and also with the diacetylchitobiose-induced difference spectrum of lysozyme. The kinds of aromatic amino acid residues possibly responsible for the observed difference peaks were discussed on the basis of the results of the comparison. Based on the results obtained, the common occurrence of a heme-linked functional response of the hemoprotein conformation was suggested.     

Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat-brain synaptosomes

Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 X 10(7) mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits. Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody. The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.     

Computing and visually analyzing mutual information in molecular co-evolution

Background  

Selective pressure in molecular evolution leads to uneven distributions of amino acids and nucleotides. In fact one observes correlations among such constituents due to a large number of biophysical mechanisms (folding properties, electrostatics, ...). To quantify these correlations the mutual information -after proper normalization - has proven most effective. The challenge is to navigate the large amount of data, which in a study for a typical protein cannot simply be plotted.     

Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation

The lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data.     

The amino acid composition of secreted mouse prolactin,growth hormone,and hamster prolactin: The presence of one tryptophan in mouse prolactin

The amino acid compositions and the electrophoretic properties of secreted mouse prolactin (mPRL), mouse growth hormone (mGH), and hamster prolactin (haPRL) were determined. The amino acid compositions of secreted mPRL and haPRL were similar to the compositions of other rodent prolactins, except that secreted mPRL contained only one tryptophan residue rather than the usual two. The composition of secreted mGH was similar to that of rat growth hormone. On 10% alkaline polyacrylamide gels, mPRL, mGH, and haPRL migrated with R_f's of 0.54, 0.21, and 0.69, respectively. The molecular weights of mPRL, mGH, and haPRL, determined by SDS-gel electrophoresis, were 23,000, 21,000, and 22,000, respectively.     

On the trends in protein molecular evolution: Amino acid composition

Data on the amino acid composition of proteins having various functions from organisms representing different evolutionary levels (83 superfamilies) are used in order to elucidate the trends in protein molecular evolution. The interconnections evolutionary rate (rate of mutation acceptance) — amino acid composition, and evolutionary level of the organism — amino acid composition (in case of proteins of the same or very similar function) are studied. The amino acid compositions of proteins performing jointly an evolutionarily old functions are also juxtaposed. The mean contemporary protein composition is used as a basis for comparison. The obtained results are evidence in favour of the existence of a trend for an increase of the special amino acids (Met, Ile, Gln, His, Lys, Asn, Phe, Tyr, Trp, Cys) at the expense of the usual ones (Thr, Pro, Ala, Ser, Arg, Gly, Leu, Val, Glu, Asp). The tests of statistical significance of the obtained results (comparison of the mean compositions of proteins from low evolutionary level organisms with that of all sequenced proteins; comparison of the mean contemporary protein composition with that obtained after simulation of the evolutionary process) confirm and universalize the observed trend. The above results direct the attention to the concept of a smaller number of amino acids in the ancient proteins and respectively simpler genetic code. A fluctuation around the initial primitive level is suggested to explain the conservatism of proteins of the same function in evolutionarily low level organisms. The observed trend could be applied for designing new proteins.     

Shape-based virtual screening,docking, and molecular dynamics simulations to identify Mtb-ASADH inhibitors

Aspartate β-semialdehyde dehydrogenase (ASADH) is a key enzyme for the biosynthesis of essential amino acids and several important metabolites in microbes. Inhibition of ASADH enzyme is a promising drug target strategy against Mycobacterium tuberculosis (Mtb). In this work, in silico approach was used to identify potent inhibitors of Mtb-ASADH. Aspartyl β-difluorophosphonate (β-AFP), a known lead compound, was used to understand the molecular recognition interactions (using molecular docking and molecular dynamics analysis). This analysis helped in validating the computational protocol and established the participation of Arg99, Glu224, Cys130, Arg249, and His256 amino acids as the key amino acids in stabilizing ligand–enzyme interactions for effective binding, an essential feature is H-bonding interactions with the two arginyl residues at the two ends of the ligand. Best binding conformation of β-AFP was selected as a template for shape-based virtual screening (ZINC and NCI databases) to identify compounds that competitively inhibit the Mtb-ASADH. The top rank hits were further subjected to ADME and toxicity filters. Final filter was based on molecular docking analysis. Each screened molecule carries the characteristics of the highly electronegative groups on both sides separated by an average distance of 6?Å. Finally, the best predicted 20 compounds exhibited minimum three H-bonding interactions with Arg99 and Arg249. These identified hits can be further used for designing the more potent inhibitors against ASADH family. MD simulations were also performed on two selected compounds (NSC4862 and ZINC02534243) for further validation. During the MD simulations, both compounds showed same H-bonding interactions and remained bound to key active residues of Mtb-ASADH.     

Probing origins of molecular interactions stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin

Single-molecule atomic force microscopy and spectroscopy were applied to detect molecular interactions stabilizing the structure of halorhodopsin (HR), a light-driven chloride pump from Halobacterium salinarum. Because of the high structural and sequence similarities between HR and bacteriorhodopsin, we compared their unfolding pathways and polypeptide regions that established structurally stable segments against unfolding. Unfolding pathways and structural segments stabilizing the proteins both exhibited a remarkably high similarity. This suggests that different amino acid compositions can establish structurally indistinguishable energetic barriers. These stabilizing domains rather result from comprehensive interactions of all amino acids within a structural region than from specific interactions. However, one additional unfolding barrier located within a short segment of helix E was detected for HR. This barrier correlated with a Pi-bulk interaction, which locally disrupts helix E and divides a structural stabilizing segment.     

The Protein Amino Acid Compositions of Amoeba proteus,A. discoides,A. dubia and Pelomyxa carolinensis*

SYNOPSIS. A comparison has been made of the protein amino acid compositions of Amoeba proteus, A. discoides, A. dubia and Pelomyxa carolinensis. The protein amino acid compositions of each of these species differed in 1–3 of the following amino acids: arginine, aspartic acid-asparagine, threonine and glycine. The possibility of using these characteristics as acceptable genetic markers is discussed.