Peptoid residues and beta-turn formation.

A set of terminally protected tripeptoids containing a residue of either N-methylglycine or N-isobutylglycine in position i + 1/i + 2 were synthesized and tested for intramolecularly H-bonded beta-turn formation. By exploiting FT-IR absorption and 1H NMR techniques, their folding tendencies were compared with those of a variety of reference peptides. The amount of beta-turn induction and the relative extent of the various types of intramolecularly H-bonded beta-turn conformers were determined in chloroform solution.     

The status of half-cystine residues and locations of N-glycosylated asparagine residues in human eosinophil peroxidase

The determination by protein chemistry methods of the half-cystine status in human eosinophil peroxidase (EPO) is reported. EPO is two-chained and has a total of 14 half-cystine residues. Cys141 and Cys152 form an intrachain bridge in the light chain of EPO. Disulfide bridges connect Cys253 and Cys263, Cys257 and Cys287, Cys359 and Cys370, Cys570 and Cys635, and Cys676 and Cys701, forming five intrachain disulfide bridges in the heavy chain of EPO. Cys291 and Cys455 are found to be unpaired, containing free sulfhydryl groups. The pattern of disulfide bridges is in agreement with that predicted from the X-ray crystallographic structure of canine myeloperoxidase (MPO) (Zeng, J., and Fenna, R. E. (1992) J. Mol. Biol. 226, 185-207) to be general for the class of mammalian peroxidases, including EPO, MPO, lactoperoxidase (LPO), and thyroid peroxidase (TPO). Of four candidate sites in EPO for attachment of glucosamine-based carbohydrate, Asn327 and Asn363 are occupied, whereas Asn700 and Asn708 are unsubstituted. Furthermore, a discrepancy in the literature regarding the sequence of residues 645-659 is resolved.     

The conversion of phosphoserine residues to selenocysteine residues on an opal suppressor tRNA and casein

This study has been undertaken in order to elucidate the mechanisms of incorporation of Se into glutathione peroxidase (GSHPx), in which selenocysteine corresponds to the opal termination codon UGA on the mRNA. We studied the above mechanisms using an opal suppressor tRNA, prepared from bovine liver, and casein as a model protein for the GSHPx apo-enzyme which might contain phosphoserine. The results showed that opal suppressor tRNA did not accept selenocysteine (lower than 0.1 mmol/mol) under the standard conditions. A trace amount of phosphoseryl-tRNA was converted to selenocysteyl-tRNA by incubation with H2Se and some enzymes. Meanwhile, a number of phosphoserine residues in casein were converted to selenocysteine residues by incubation with H2Se and enzymes. These results suggest that opal suppressor tRNA plays a role in synthesizing GSHPx via co- and/or post-translational mechanisms.     

Novel feature for catalytic protein residues reflecting interactions with other residues

Owing to their potential for systematic analysis, complex networks have been widely used in proteomics. Representing a protein structure as a topology network provides novel insight into understanding protein folding mechanisms, stability and function. Here, we develop a new feature to reveal correlations between residues using a protein structure network. In an original attempt to quantify the effects of several key residues on catalytic residues, a power function was used to model interactions between residues. The results indicate that focusing on a few residues is a feasible approach to identifying catalytic residues. The spatial environment surrounding a catalytic residue was analyzed in a layered manner. We present evidence that correlation between residues is related to their distance apart most environmental parameters of the outer layer make a smaller contribution to prediction and ii catalytic residues tend to be located near key positions in enzyme folds. Feature analysis revealed satisfactory performance for our features, which were combined with several conventional features in a prediction model for catalytic residues using a comprehensive data set from the Catalytic Site Atlas. Values of 88.6 for sensitivity and 88.4 for specificity were obtained by 10-fold cross-validation. These results suggest that these features reveal the mutual dependence of residues and are promising for further study of structure-function relationship.     

Ionization states of residues in OmpF and mutants: effects of dielectric constant and interactions between residues

To understand ion permeation, one must assign correct ionization states to titratable amino acid residues in protein channels. We report on the effects of physical and methodological assumptions in calculating the protonation states at neutral bulk pH of titratable residues lining the lumen of the native Escherichia coli OmpF channel, and five mutants. We systematically considered a wide range of assumed protein dielectric constants and all plausible combinations of protonation states for electrostatically interacting side chains, and three different levels of accounting for solute shielding: 1), full nonlinear Poisson-Boltzmann; 2), linearized Poisson-Boltzmann; and 3), neglect of solute shielding. For this system we found it acceptable to neglect solute shielding, a result we postulate to be generalizable to narrow lumens of other protein channels. For the large majority of residues, the protonation state at neutral bulk pH was found to be independent of the assumed dielectric constant of the protein, and unambiguously determined by the calculation; for native OmpF only Asp-127 has a protonation state that is sensitive to the assumed protein dielectric constant. Our results are significant for understanding two published experimental observations: the structure of the narrow part of the channel, and the ionic selectivity of OmpF mutants.     

Improving prediction of burial state of residues by exploiting correlation among residues

Background

Residues in a protein might be buried inside or exposed to the solvent surrounding the protein. The buried residues usually form hydrophobic cores to maintain the structural integrity of proteins while the exposed residues are tightly related to protein functions. Thus, the accurate prediction of solvent accessibility of residues will greatly facilitate our understanding of both structure and functionalities of proteins. Most of the state-of-the-art prediction approaches consider the burial state of each residue independently, thus neglecting the correlations among residues.

Results

In this study, we present a high-order conditional random field model that considers burial states of all residues in a protein simultaneously. Our approach exploits not only the correlation among adjacent residues but also the correlation among long-range residues. Experimental results showed that by exploiting the correlation among residues, our approach outperformed the state-of-the-art approaches in prediction accuracy. In-depth case studies also showed that by using the high-order statistical model, the errors committed by the bidirectional recurrent neural network and chain conditional random field models were successfully corrected.

Conclusions

Our methods enable the accurate prediction of residue burial states, which should greatly facilitate protein structure prediction and evaluation.

     

Partial retro-inverso analogues of somatostatin: pairwise modifications at residues 7 and 8 and at residues 8 and 9

Peptide bonds between residues 7 and 8 residues 8 and 9, postulated internal cleavage sites of the peptide hormone somatostatin, were subjected to "pairwise" retro-inverso modification, where atoms of these peptide bonds were interchanged to give the analogues [gPhe7-m-(RS)-Trp8]somatostatin (I) and [gTrp8-m-(RS)-Lys9]somatostatin (II). Key fragments containing the modifications were synthesized by using [bis(trifluoroacetoxy)iodo]benzene for the generation of gem-diaminoalkyl-containing precursors from peptide amides. The versatility of solution synthetic methods was utilized to allow the incorporation of the modified segments. Protecting groups, removable selectively and under mild conditions, included tert-butyl-based groups for the side chains and the tert-butylmercapto group for the cysteine thiols. The excellent results obtained in the syntheses of analogues I and II, and previously of somatostatin on a larger scale [Moroder, L., Gemeiner, M., Goehring, W., Faeger, E., Thamm, P., & Wunsch, E. (1981) Biopolymers 20, 17-31], suggest the general feasibility of this route for the synthesis of centrally modified analogues. The purification of the products by Sephadex LH-20 chromatography afforded the separation of diastereomers of both analogues. The two isomers of I showed significant but different activities while those of analogue II were marginally active.     

Arginyl residues and thermal stability in proteins

Guanidination and amidination of bovine serum albumin, yeast enolase and yeast alcohol dehydrogenase were accompanied by increases in thermal stability at lower extents of modification. Decreases in thermal stability result from greater modification. These results support suggestions that surface guanidino groups (arginyl groups) are an important factor in thermal stability of proteins.     

Carboxyl-terminal residues of mammalian fibrinogen and fibrin

The carboxyl-terminal residues of mammalian fibrinogens of six different species and the chain peptides, alpha(A), beta(B) and gamma, isolated from these fibrinogens were determined by hydrazinolysis, digestion with carboxypeptidases and selective tritium labelling. The C-terminal ends of bovine fibrinogen and fibrin were identified as proline and valine, in the molar ratio of approximately 1:2. Proline was identified as the C-terminus of the alpha(A)-chain, and C-terminal valine was found on both the beta(B)- and gamma-chains. On hydrazinolysis after selective tritium labelling of fibrinogen, radioactive C-terminal valine was also identified. The same C-terminal ends as those of bovine fibrinogen were found on the corresponding chain peptides isolated from sheep fibrinogen. The C-terminal residues of all the chain peptides of human and horse fibrinogens, however, were valine. In hog and dog fibrinogens, proline was identified at the C-termini of the alpha(A)-chains, and C-terminal valine and isoleucine were found on the beta(B)- and gamma-chains, respectively. Thus, the C-terminal amino acid residues of the fibrinogens of all mammalian species tested were very similar. It should be noted that hydrophobic amino acids, like isoleucine, valine and proline, are mainly located in the C-terminal ends of all three chain peptides in the fibrinogen molecule.     

Sugar residues on proteins

Glycoproteins have become increasingly important in the structure and function of many different mammalian systems; for example, membrane glycoproteins and glycoprotein hormones. It is, therefore, important to understand their chemistry, which would include an understanding of both the carbohydrate and protein parts of the molecule. Since the chemical characterization of the protein moiety has been extensively examined and the techniques for its characterization are well worked out, only the carbohydrate portion of glycoproteins will be reviewed in this article. The chemical nature of the carbohydrate moiety of glycoproteins will be examined. First, the types of monosaccharides present in animal systems, especially those in the mammalian systems, will be described. Next, various types of simple and complex carbohydrate chains will be discussed to establish the diversity, size, and number of chains present in the carbohydrate units in different glycoproteins. Then, the type of linkages of the carbohydrate to the protein will be examined to determine if the primary sequence of protein is important in determining the size and type of carbohydrate chains present in glycoproteins. Finally, the current methods of structural elucidation such as monosaccharide sequence, intersugar bonds, and anomeric linkages in the carbohydrate moiety of glycoproteins will be reviewed. These methods include the techniques of periodate oxidation, methylation, partial acid hydrolysis, and specific glycosidase digestion of glycoproteins, as well as the latest techniques using micromethods of carbohydrate quantitation and characterization involving gas chromatography and mass spectrometry. The function of the carbohydrate in glycoproteins will also be considered. First, hormone glycoproteins will be discussed in their relationship to the immunological and biological function of the glycoprotein when the carbohydrate is sequentially removed. Next, the function of the carbohydrate in the turnover of glycoproteins will be discussed. These topics will be considered in order to develop an understanding of a specific function(s) of the carbohydrate in glycoproteins.     

Conservation of metal-coordinating residues

As a result of rapid advances in genome sequencing, the pace of discovery of new protein sequences has surpassed that of structure and function determination by orders of magnitude. This is also true for metal-binding proteins, that is, proteins that bind one or more metal atoms necessary for their biological function. While metal binding site geometry and composition have been extensively studied, no large scale investigation of metal-coordinating residue conservation has been pursued so far. In pursuing this analysis, we were able to corroborate anecdotal evidence that certain residues are preferred to others for binding to certain metals. The conservation of most metal-coordinating residues is correlated with residue preference in a statistically significant manner. Additionally, we also established a statistically significant difference in conservation between metal-coordinating and noncoordinating residues. These results could be useful for providing better insight to functional importance of metal-coordinating residues, possibly aiding metal binding site prediction and design, metal-protein complex structure prediction, drug discovery, as well as model fitting to electron-density maps produced by X-ray crystallography.     

Phytotoxicity from plant residues

Summary Proximity of new wheat straw residues to sown wheat seed has an effect on germination, plant growth and ultimate yield. Decomposition of wheat straw may produce toxins or it may cause immobilization of nitrogen in, or applied to the soil. In pot experiments, it has been shown that germination of wheat was depressed when large amounts of straw were decomposed on the surface for up to 18 days; after 54 days it had no effect on germination. Immobilization of nitrogen occurred mainly when the straw was mixed with the soil, or when surface-rotted straw was ploughed into the soil just before seeding. The latter effect could not be overcome by the addition of mineral nitrogen. Part II, Plant and Soil 38, 347–361 (1973).