Dha Residues Research Articles

Post-translational mutagenesis for installation of natural and unnatural amino acid side chains into recombinant proteins.

Methods for installing natural and unnatural amino acids and their modifications into proteins in a benign and precise manner are highly sought-after in protein science. Here we describe a protocol for 'post-translational mutagenesis' that enables the programmed installation of protein side chains through the use of rapid, mild and operationally simple free-radical chemistry performed on recombinantly expressed and purified proteins. By introduction of protein dehydroalanine (Dha) residues (in this instance, from a unique cysteine residue introduced by site-directed mutagenesis) as free-radical trapping 'tags' for downstream modification, exquisite control over the site of subsequent modification is achieved. Using readily available alkyl halide precursors and simple borohydride salts, alkyl radicals can be generated in aqueous solution. These alkyl radicals react rapidly with protein-bound Dha residues to yield functionalized protein products with new carbon-carbon bonds. Once the Dha is installed, the introduction of the desired functionality is limited only by the requirement for polarity matching of the generated radical with the Dha 'acceptor', the solubility of the alkyl halide precursors in aqueous solution and the kinetics of the reaction itself. For example, methylated derivatives of lysine, arginine and glutamine are readily accessible. Furthermore, as the side chains are constructed chemically, many unnatural modifications can also be directly introduced as part of the side chain, including isotope reporters (19F, 13C) that can be used in biophysical experiments such as protein NMR. From a suitable cysteine mutant of the target protein, the entire procedure for this chemical post-translational mutation takes 2 d and is readily performed by nonchemists.

Theoretical Study of the Phosphoryl Transfer Reaction from ATP to Dha Catalyzed by DhaK from Escherichia coli.

Protein kinases, representing one of the largest protein families involved in almost all aspects of cell life, have become one of the most important targets for the development of new drugs to be used in, for instance, cancer treatments. In this article an exhaustive theoretical study of the phosphoryl transfer reaction from adenosine triphosphate (ATP) to dihydroxyacetone (Dha) catalyzed by DhaK from Escherichia coli (E. coli) is reported. Two different mechanisms, previously proposed for the phosphoryl transfer from ATP to the hydroxyl side chain of specific serine, threonine, or tyrosine residues, have been explored based on the generation of free energy surfaces (FES) computed with hybrid QM/MM potentials. The results suggest that the substrate-assisted phosphoryl and proton-transfer mechanism is kinetically more favorable than the mechanism where an aspartate would be activating the Dha. Although the details of the mechanisms appear to be dramatically dependent on the level of theory employed in the calculations (PM3/MM, B3LYP:PM3/MM, or B3LYP/MM), the transition states (TSs) for the phosphoryl transfer step appear to be described as a concerted step with different degrees of synchronicity in the breaking and forming bonds process in both explored mechanisms. Residues of the active site belonging to different subunits of the protein, such as Gly78B, Thr79A, Ser80A, Arg178B, and one Mg2+ cation, would be stabilizing the transferred phosphate in the TS. Asp109A would have a structural role by posing the Dha and other residues of the active site in the proper orientation. The information derived from our calculations not only reveals the role of the enzyme and the particular residues of its active site, but it can assist in the rational design of new more specific inhibitors.

Solid-phase peptide synthesis of analogues of the N-terminus A-ring fragment of the lantibiotic nisin: Replacements for the dehydroalanine (Dha) residue at position 5 and the first incorporation of a thioamide residue

A number of A-ring analogues of the lantibiotic nisin, containing replacements for the Dha residue at position 5, have been successfully prepared by solid-phase peptide synthesis. The Dha replacements include glycine, alanine, phenylalanine, serine and 1-aminocyclopropyl carboxylic acid (ACCa). The incorporation of a thioamide-isoleucine residue at position 4 is also described and represents the first reported preparation of a lantibiotic ring fragment containing a thioamide link.

The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages.

We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. Graphical Abstract ᅟ.

Chemical Modification of Proteins

The invention relates to methods for selectively converting a cysteine residue in a peptide or protein to the dehydroalanine (Dha) residue. The method also works on selenocysteine and substituted cysteine and selenocysteine residues, resulting in the Dha residue which may be converted to any natural or unnatural amino acid residue desired without the alteration of the remainder of the peptide or protein. The invention also allows ligation of a desired peptide at any point rather than at a point where there should be a naturally occurring cysteine, thereby allowing native chemical ligation to be used in the synthesis of peptides that do not contain cysteine. The methodology allows for the synthesis of very large peptides.

Rhizobium rubiT: A Gram‐Negative Phytopathogenic Bacterium Expressing the Lewis B Epitope on the Outer Core of its Lipooligosaccharide Fraction

The structure of the core oligosaccharide from the phytopathogenic bacterium Rhizobium rubi was deduced by combining information from complementary chemical approaches (alkaline and acid hydrolysis), similar to the "overlap peptide" strategy. This structure is new and it contains two main oligosaccharide backbones that differ in the substitution degree of the external Kdo unit. The relevant feature shared by both oligosaccharides is the presence of a tetrasaccharide motif that is similar to the blood group Lewis B antigen (Le(B)). This epitope differs from Le(B) in the glycosidic configuration of the glucosamine unit (alpha and not beta) and in the occurrence of acetyls substituents at O3 and/or O4 of the galactose moiety. Other notable structural features are the location of the Dha residue, the presence of a alpha-glucose unit that is linked to the inner Kdo unit, the high number of acid sugars and the highly branched core structure.

Mechanistic Investigations of the Dehydration Reaction of Lacticin 481 Synthetase Using Site-Directed Mutagenesis

Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.

Dehydroalanine-containing peptides: preparation from phenylselenocysteine and utility in convergent ligation strategies

This protocol describes the methodology for the synthesis of dehydroalanine (Dha)-containing peptides and illustrates their use in convergent ligation strategies for the preparation of peptide conjugates. A nonproteinogenic amino acid, Fmoc-Se-phenylselenocysteine (SecPh), can be prepared in high yield over four synthetic steps and be conveniently incorporated into peptides by standard solid-phase peptide synthesis techniques. Globally deprotected peptides containing phenylselenocysteine can be converted to dehydrated peptides following a chemoselective, mild oxidation with hydrogen peroxide or sodium periodate (i.e., the phenylselenocysteine side chain is converted to that of Dha). Dha residues are electrophilic handles for the preparation of glycopeptides, lipopeptides or other peptide conjugates; one such transformation will be outlined here. The preparation of Dha-containing peptides, including the synthesis of SecPh, peptide elongation and oxidative treatment of phenylselenocysteine-containing peptides can be completed by one person in approximately 3-5 weeks. However, once SecPh is in hand, the time required for the preparation of peptides is significantly shorter and comparable to that for any peptide synthesis.

Method for analysis of TAG formed by reaction of fish oil with hydrogenated soybean oil

AbstractAn HPLC method was developed for analysis of the TAG formed during interesterification of a fish oil rich in DHA residues and of a fully hydrogenated soybean oil. TAG species were separated using a three‐phase (acetone/acetonitrile/chloroform) solvent system. Peak identities were assigned on the basis of a multiple linear regression analysis by using factors such as carbon number, number of double bonds, and number of PUFA in the molecule as predictors for TAG retention time. Good agreement between experimental and predicted retention times was observed when the effect of the PUFA was separated in the regression model from that of the monounsaturated FA. In addition, the new method permits one to determine tristearin at concentrations up to 3 mg/mL without encountering the problem of partial retention of this TAG in the column that was observed when chloroform is not incorporated in the mobile phase.

Role of the leader and structural regions of prelantibiotic peptides as assessed by expressing nisin-subtilin chimeras in Bacillus subtilis 168, and characterization of their physical, chemical, and antimicrobial properties.

Biosynthesis of lantibiotics such as nisin and subtilin involves post-translational modifications, including dehydration of serines and threonines, formation of thioether cross-linkages, translocation, cleavage of a leader sequence, and release into the medium. We have studied the cellular machinery that performs the modifications by constructing and expressing nisin-subtilin chimeric prepeptides in a strain of Bacillus subtilis 168 that possesses all of the cellular machinery for making subtilin except for the presubtilin gene. The chimeras consisted of a normal subtilin leader region (SL), fused to nisin-subtilin chimeric structural regions, one of which was SL-Nis1-11-Sub12-32, in which the N-terminal portion of the structural region was derived from nisin, and the C-terminal portion derived from subtilin. This chimera was accurately and efficiently converted to the corresponding mature lantibiotic, as established by reverse phase high performance liquid chromatography profiles, proton NMR spectroscopy, mass spectral analysis, and biological activity. A succinylated form of the chimera was also produced. Another chimera was in the reverse sense, with subtilin sequence at the N terminus and nisin sequence at the C terminus of the structural region (SL-Sub1-11-Nis12-34). It was processed into a heterogeneous mixture of products, none of which had the characteristics of a correctly processed polypeptide, but did contain a minor component that was active, with a specific activity that considerably exceeded nisin itself. These results, together with results published earlier, establish that processing requires specific recognition between the prelantibiotic peptide and the processing machinery, and in order for the processing to occur correctly, there must be an appropriate combination of the N-terminal part of the leader region and the C-terminal part of the structural region of the prepeptide.

A cassette vector for protein engineering the lantibiotic nisin

Expression vectors are described that make use of a plasmid-encoded nisA cassette encoding the peptide component of nisin. Specific mutations can readily be incorporated throughout the coding region of nisA. Using this vector in a nisA mutant host, three variant nisins, with dehydroalanine (Dha) residues changed to Ala residues, were generated.

The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms

Subtilin is a ribosomally synthesized antimicrobial peptide that contains several unusual amino acids as a result of posttranslational modifications. Site-directed mutagenesis was employed to construct a structural variant of subtilin in which the unusual dehydroalanine (Dha) residue at position 5 was changed to alanine. Proton nuclear magnetic resonance spectroscopy, amino acid composition, and N-terminal sequence analysis established that the mutation did not disrupt posttranslational processing of the precursor peptide. This mutant subtilin was devoid of antimicrobial activity as assessed by its lack of inhibitory effects on outgrowth of Bacillus cereus T spores. However, this same mutant subtilin was fully active with respect to its ability to induce lysis of vegetative B. cereus T cells. Because an intact Dha-5 residue is required in the one instance but not in the other, it was concluded that the molecular mechanism by which subtilin inhibits (without lysis) spore outgrowth is not the same as the mechanism by which it inhibits (with lysis) vegetative cells.