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Beauveriolides I and III, which are naturally occurring cyclodepsipeptides, have been reported to bind to sterol O-acyltransferase (SOAT), inhibiting its ability to synthesize cholesteryl esters. To facilitate an analysis of the binding site(s) of these compounds, we designed beauveriolide analogues 1a-d wherein the Leu or D-allo-Ile residue was replaced by photoreactive amino acids possessing methyldiazirine or trifluoromethyldiazirine in the side chains. The methyldiazirine moiety was installed by reaction of methyl ketones with liquid ammonia to provide imine intermediates, followed by treatment with hydroxylamine-O-sulfonic acid to provide the diaziridines. Subsequent oxidation gave methyldiazirines. In contrast, trifluoromethyldiazirine derivatives were prepared from trifluoromethyl ketones via the oxime intermediates, which were transformed into diaziridines. Subsequent oxidation afforded trifluoromethyldiazirines. The synthesized photoreactive amino acids 3a-d were coupled with 3-hydroxy-4-methyloctanoic acid 4 and dipeptide 5, followed by macrolactamization to provide beauveriolide analogues 1a-d. The SOAT inhibitory activities of 1a-d were found to be as potent as those of beauveriolides I and III. Moreover, 1a-d inhibited SOAT1 selectively rather than SOAT2, which was also consistent with the behavior of beauveriolides I and III.

In protein evolution amino acid replacements occur more frequently between similar than dissimilar amino acids. Accordingly, the mutations obey a simple 'similar replaces similar' rule as disruption of the resulting protein structure is minimized by the modest alterations in the amino acid side chains. At laboratory level such non-destructive modifications have to be incorporated in a controlled manner by integrating the chemical diversity achieved in synthetic chemistry into proteins. For this purpose the most straightforward route is generation of the synthetic proteins by the cellular translational apparatus via insertion of isosteric synthetic amino acid analogs during gene expression. This leads to target proteins with chemical diversity not found in nature. Such genetic code engineering does not require any DNA mutagenesis step as it produces extensive sequence variations at the level of protein translation. It is generally achieved either by expansion of the existing amino acid repertoire or by introduction of novel coding units. Here we highlight the concept of using isosteric noncanonical amino acid analogs for in vivo protein synthesis as a useful tool to dissect, study and manipulate protein folding and conformational stability with two examples, the prion protein and green fluorescent protein. In the first example, we show how alternative translation of the single gene sequence by different synthetic amino acids enables the protein to fold into stable, but differing conformations. In particular, replacement of methionine by the isosteric analogs norleucine/metoxinine provide a chemical model to dissect the role of hydrophilicity/hydrophobicity in the alpha-->beta conversion of the prion protein structure. In the second example, proline residues in green fluorescent protein are replaced with (4S)-fluoroproline which improves folding and overall stability of the protein. Indeed, fluorination of the protein matrix creates a network of favorable local interactions absent in the parent (i.e. natural) protein. The generation of these novel properties is the first demonstration of a structural preogranisation principle in one complex globular protein molecule.

Synthesis of dihydroquinopymaric acid amides and their 2beta-succinyl and 2beta-phthalyl derivatives containing residues of amino acids was carried out for the first time. Antiviral properties of the compounds synthesized were investigated.

In order to study further the importance of the P'1 residue upon the activity of human PC1 and human furin, two important members of subtilisin/kexin family of enzymes, we have prepared by solid-phase Fmoc or recently introduced FastMoc chemistry a series of 10 peptidyl substrate analogs. The structures of these analogs are based upon the core sequence of pro-mPC1(83-93) namely, D-Tyr-Lys-Glu-Arg-Ser-Lys-Arg-Xaa-Val-Gln-Lys-Asp, where D-Tyr replaces the native L-Tyr residue and Xaa, representing the P'1 position, corresponds to L-Ser or to nonproteinacous amino acids such as Tle, Sarc, MLeu, Aib, D-Tic or L-Tic. Two more analogs with L-Tic at P'1 position but with one amino acid less, namely P5 Glu or P'3 Gln, and one with a Cit residue in place of Arg at P1 site of the dodecapeptide were also obtained. These peptides were all fully characterized by a combination of MS, 1H-NMR and amino acid analysis. In contrast to the Ser analog, which is an excellent substrate for both hPC1 and hfurin, these analogs displayed moderate to strong inhibition of both hPC1 and hfurin activity in a reversible competitive manner. They all exhibited higher potency for hfurin than for hPC1, with an inhibition constant (Ki) ranging from 0.8 to 10 microM and from 1.0 to 170 microM, respectively. Incorporation of L-Tic yielded an analog with a two to four-fold increased inhibition of either enzymes when compared to its D-Tic counterpart, the effect being more pronounced for hPC1 than for hfurin. Comparison of these data with those for the corresponding N-terminal Fmoc protected peptides revealed that the highly hydrophobic N-terminal Fmoc function occupying the P8 position can contribute positively or negatively towards proteinase inhibition depending on the nature of the unnatural amino acid at P'1 and the enzyme used. Finally, none of the analogs was significantly cleaved by either enzyme. FTIR data on these analogs revealed some important structural differences between the substrate and inhibitor analogs, as there appears to be a conformational shift from a more beta-sheet-like structure for the substrates to a more alpha-helical-like structure for the inhibitors.

Primary amino groups on the model peptide Xaa-Ala-Pro-Lys(ClZ)-Tyr(2BrZ), synthesized on a p-methylbenzhydryl amine resin with conventional Boc/benzyl protective group strategy, were reacted with 4,4'-dimethoxydityl chloride in dichloromethane, resulting in the introduction of the dimethoxydityl group, which is an acid-labile N-alkyl type of protective group. The secondary amino groups thereby formed can be methylated by treating the peptide-resin with formaldehyde and sodium cyanoborohydride in N,N-dimethylformamide. After the removal of the dimethoxydityl group with trifluoroacetic acid, the resulting N-methylated amino acid residues with a free secondary amino groups are accessible for acylation with the next activated Boc amino acid. With this method majority of the 20 common amino acids can be monomethylated directly on the resin and, in most cases, with very low levels of the side reactions. In the cases where the complete methylation is difficult to achieve, the remaining primary amino groups can be selectively acylated in the presence of secondary amino groups with trimethylacetic acid 1-hydroxybenzotriazole ester. The method provides a convenient general route to synthesize N-methylated derivatives of most of the occurring and synthetic amino acids.

Crystal structure data of 31 different globular proteins were analysed. Potentials for all 400 pairs of 20 amino acid residues in helix, extended structure, chain reversals and coil state were obtained. Analysis of these potentials showed that tripeptides of amino acid residues are not linear combinations of amino acids. The association effect of these tripeptides depends on the types of amino acids associated, the position of amino acids in the pair and the secondary structure in which the pair exists. This built-in information in tripeptides should be used in polypeptide and protein folding studies.

Despite astounding diversity in their structure and function, proteins are constructed from 22 protein or 'canonical' amino acids. Hundreds of amino acid analogues exist; many occur naturally in plants, some are synthetically produced or can be produced in vivo by oxidation of amino acid side-chains. Certain structural analogues of the protein amino acids can escape detection by the cellular machinery for protein synthesis and become misincorporated into the growing polypeptide chain of proteins to generate non-native proteins. In this review we seek to provide a comprehensive overview of the current knowledge on the biosynthetic incorporation of amino acid analogues into proteins by mammalian cells. We highlight factors influencing their incorporation and how the non-native proteins generated can alter cell function. We examine the ability of amino acid analogues, representing those commonly found in damaged proteins in pathological tissues, to be misincorporated into proteins by cells in vitro, providing us with a useful tool in the laboratory to generate modified proteins representing those present in a wide-range of pathologies. We also discuss the evidence for amino acid analogue incorporation in vivo and its association with autoimmune symptoms. We confine the review to studies in which the synthetic machinery of cell has not been modified to accept non-protein amino acids. © 2008 Elsevier Ltd. All rights reserved.

The L-type amino acid transporter 1 (LAT1, SLC7A5) is an Na+-independent neutral amino acid transporter the expression of which is located in retinal endothelial cells. Due to its broad substrate selectivity, LAT1 has been proposed to mediate the transport of amino acid-related drugs across the blood-tissue barriers. Here, we have investigated the transport screening of amino acid-mustards using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) which expresses LAT1. We synthesized 5 amino acid-mustards: tyrosine-mustard, phenylglycine-mustard, alanine-mustard, ornithine-mustard, and lysine-mustard. LAT1-mediated [3H]L-phenylalanine (Phe) uptake by TR-iBRB2 cells was inhibited in a competitive manner by tyrosine-mustard and phenylglycine-mustard as well as melphalan (phenylalanine-mustard). Phenylglycine-mustard was able to induce the efflux of [3H]Phe preloaded into the TR-iBRB2 cells expressing LAT1 through the obligatory exchange mechanism, although tyrosine-mustard, alanine-mustard, ornithine-mustard, lysine-mustard, and melphalan did not induce any significant efflux. These findings suggest that phenylglycine-mustard is a better substrate for LAT1 than melphalan and other amino acid-mustards.

Proteins are the central functional constituents in all living organisms ranging from viruses, bacteria, yeast, and plants to mammals. All of these biopolymers that are formed by natural biosynthetic pathways are composed of a genetically determined sequence of the 20 so-called natural amino acids. The physical and chemical properties of proteins are a reflection of the side chains of each of the component amino acids. However, for some purposes it would be very desireable to have amino acids with side chains of various selected physical chemical properties, such as a keto group, a crosslinker, or a NMR probe group, incorporated into the protein. Although chemical and biochemical methods for modifying amino acid moieties in proteins have been achieved, recent successes in incorporating unnatural amino acids in vivo open entirely new avenues for determining protein functions in vivo and for the creation of unnatural proteins with novel functionalities. Several examples by employing the novel activity of unnatural amino acids have shown significant roles in both basic research and biotechnology.

LAT1 (L-type amino acid transporter 1), one of the L-type amino acid transporters, transports the branched and aromatic amino acids. LAT1 requires the heavy chain of 4F2 antigen (4F2hc) for the functional expression as an amino acid transporter. The expression of this transporter is up-regulated in tumor cells and rapidly-growing cells to support their proliferation. Here, we studied the expression of LAT1 and 4F2hc in human cultured cells by real-time PCR and Western blot, and found that human brain astrocytomas, U343MGa, highly expressed LAT1 and 4F2hc mRNAs and proteins. The uptake of [14C]leucine by U343MGa cells is Na+-independent and inhibited by BCH (2-amino-2-norbornane carboxylic acid), and branched and aromatic amino acids, indicating that the LAT1 is expressed at the cell surface. Pulse chase labeling and surface labeling experiments of this cell line indicate that the protein synthesis of LAT1 and 4F2hc is slow, however, the heterodimeric complex assembled in the cells is very stable, and that the disulfide bond between the LAT1 and 4F2hc is not directly involved in the stability of the heterodimer.

The general amino acid permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring amino acids into yeast cells for use as a nitrogen source. Previous studies have shown that a nonubiquitinateable form of the permease, Gap1pK9R,K16R, is constitutively localized to the plasma membrane. Here, we report that amino acid transport activity of Gap1pK9R,K16Rcan be rapidly and reversibly inactivated at the plasma membrane by the presence of amino acid mixtures. Surprisingly, we also find that addition of most single amino acids is lethal to Gap1pK9R,K16R-expressing cells, whereas mixtures of amino acids are less toxic. This toxicity appears to be the consequence of uptake of unusually large quantities of a single amino acid. Exploiting this toxicity, we isolated gap1 alleles deficient in transport of a subset of amino acids. Using these mutations, we show that Gap1p inactivation at the plasma membrane does not depend on the presence of either extracellular or intracellular amino acids, but does require active amino acid transport by Gap1p. Together, our findings uncover a new mechanism for inhibition of permease activity in response to elevated amino acid levels and provide a physiological explanation for the stringent regulation of Gap1p activity in response to amino acids.

Drug design can benefit from the use of non-coded amino acids, such as alpha-amino isobutyric acids (Aib) or sarcosine (N-methyl-glycine). Non-coded amino acids can confer resistance to enzymatic degradation and increase the conformational stability of the peptides. We have simulated the conformational effects of combining N-methylation, bulky groups on the Calpha atom and/or thioamides using the class II CFF91 force field and our thioamide force field parameters. Although single amino acid substitutions (e.g. Aib) can restrict the available conformations, they do not necessarily lead to unique conformers, however, we predict that some of the amino acids described in this report will fold to a single phi, psi conformation (e.g. N-methylated and thioamide penicillamine). Several other amino acid/thiopeptide combinations were designed, which are predicted to prefer only two conformations. Novel amino acids of this type should prove useful for designing peptides with defined conformations.

In this paper, we describe the synthesis of novel endomorphin-2 analogs, containing N-methylated amino acids, consecutively in each position. The receptor-binding profile of the new analogs and their stability against enzymatic cleavage by commercially available peptidases, carboxypeptidase Y and aminopeptidase M, and a rat brain homogenate are reported. The best analog of this series, [Sar2]endomorphin-2, was almost equipotent with the parent peptide in the mu-receptor-binding assay and was also highly resistant to enzymatic degradation. This analog may be a suitable candidate for the in vivo antinociceptive studies.

We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.

reports. The likely requirements for intravenous amino acids were clearly spelt out and numerous commercial preparations (mostly for adults) became available in Europe. The composition of these preparations, however, was mainly based on oral requirement data in healthy man. The major nutrition efforts were directed to improve tolerance of the nitrogen load in the presence of illness rather than to provide specific nutrients for individual organs or tissues. At most instances, the efficacy of an amino acid therapy was monitored in blood (plasma). However, as shown over the past 20 years, changes in plasma-free amino acid concentrations may parallel those in intracellular compartment, but they frequently differ either quantitatively or qualitatively. The importance of this area was recognised early in the history of ESPEN when in the 1984 Arvid

Amino acids exert modulatory effects on proteins involved in control of mRNA translation in animal cells through the target of rapamycin (TOR) signaling pathway. Here we use oocytes of Xenopus laevis to investigate mechanisms by which amino acids are "sensed" in animal cells. Small ( approximately 48%) but physiologically relevant increases in intracellular but not extracellular total amino acid concentration (or Leu or Trp but not Ala, Glu, or Gln alone) resulted in increased phosphorylation of p70(S6K) and its substrate ribosomal protein S6. This response was inhibited by rapamycin, demonstrating that the effects require the TOR pathway. Alcohols of active amino acids substituted for amino acids with lower efficiency. Oocytes were refractory to changes in external amino acid concentration unless surface permeability of the cell to amino acids was increased by overexpression of the System L amino acid transporter. Amino acid-induced, rapamycin-sensitive activation of p70(S6K) was conferred when System L-expressing oocytes were incubated in extracellular amino acids, supporting intracellular localization of the putative amino acid sensor. In contrast to lower eukaryotes such as yeast, which possess an extracellular amino acid sensor, our findings provide the first direct evidence for an intracellular location for the putative amino acid sensor in animal cells that signals increased amino acid availability to TOR/p70(S6K).

Protein synthesis can be estimated by measuring the incorporation of a labeled amino acid into a proteolytic peptide. Although prelabeled amino acids are typically administered, recent studies have tested (2)H(2)O; the assumption is that there is rapid equilibration of (2)H (in body water) with the carbon-bound hydrogens of amino acids before those amino acids are incorporated into a protein(s). We have determined the temporal changes in (2)H labeling of body water and amino acids which should build confidence in (2)H(2)O-based studies of protein synthesis when one aims to measure the (2)H labeling of proteolytic peptides.

Amino acid type-selective experiments can help to remove ambiguities in automated assignment procedures for (15)N/(13)C-labeled proteins. Here we present five triple-resonance experiments that yield amino acid type-selective (1)H-(15)N correlations for aromatic amino acids. Four of the novel experiments are based on the MUSIC coherence transfer scheme that replaces the initial INEPT transfer and is selective for CH(2). The MUSIC sequence is combined with selective excitation pulses to create experiments for Trp (W-HSQC) as well as Phe, Tyr, and His (FYH-HSQC). In addition, an experiment selective for Trp H(epsilon1)-N(epsilon1) is presented. The new experiments are recorded as two-dimensional experiments and their performance is demonstrated with the application to a protein domain of 115 amino acids.

[see structure]. A fullerene derivative containing a free amino group has been condensed with N-Fmoc-L-glutamic acid alpha-tert-butyl ester to give a C60-functionalized amino acid. The carboxylic end of this amino acid has been deprotected in acidic conditions, and the resulting acid has been used for solid-phase peptide synthesis. The final peptide, cleaved from the resin, was very soluble in water solutions and showed antimicrobial activity against two representative bacteria.

Ssy1p and Ptr3p are known components of a yeast plasma membrane system that functions to sense the presence of amino acids in the extracellular environment. In response to amino acids, this sensing system initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and transport proteins, including multiple, genetically distinct amino acid permeases. We have found that SSY5 encodes a third component of this amino acid sensing system. Mutations in SSY5 manifest phenotypes that are indistinguishable from those resulting from either single ssy1 and ptr3 mutations or ssy5 ssy1 and ssy5 ptr3 double mutations. Although Ssy5p is predicted to be a soluble protein, it exhibits properties indicating that it is a peripherally associated plasma membrane protein. Each of the three sensor components, Ssy1p, Ptr3p, and Ssy5p, adopts conformations and modifications that are dependent upon the availability of amino acids and on the presence of the other two components. These results suggest that these components function as part of a sensor complex localized to the plasma membrane. Consistent with a sensor complex, the overexpression of SSY1 or the unique N-terminal extension of this amino acid permease homologue inactivates the amino acid sensor in a dominant-negative manner. Each of the components of the Ssy1p-Ptr3p-Ssy5p (SPS) signaling system undergoes rapid physical changes, reflected in altered electrophoretic mobility, when leucine is added to cells grown in media lacking amino acids. Furthermore, the levels of each SPS sensor component present in whole-cell extracts diminish upon leucine addition. The rapid physical alterations and reduced levels of sensor components are consistent with their being downregulated in response to amino acid availability. These results reveal the dynamic nature of the amino acid-initiated signals transduced by the SPS sensor.

D-amino acid-containing peptides with biological activities have been isolated from invertebrates and amphibians, and partial racemization of amino acid residues in mammalian peptides associated with aging and diseases have been discussed. Here, we review the amino acid configuration determination methods in these peptides and recent progress of simultaneous determination method for sequence and configuration of amino acid residues. The applicability of C-terminus sequence analysis and mass spectrometry to configuration determination of amino acids is also discussed.

A multifamily sequence alignment of the rabbit CYP4A members with the known structure of CYP102 indicates amino acid differences falling within the so-called substrate recognition site(s) (SRS). Chimeric proteins constructed between CYP4A4 and CYP4A7 indicate that laurate activity is affected by the residues within SRS1 and prostaglandin activity is influenced by SRS2-3. Site-directed mutant proteins of CYP4A7 found laurate and arachidonate activity markedly diminished in the R90W mutant (SRS1) and somewhat decreased in W93S. While PGE(1) activity was only slightly increased, the mutant proteins H206Y and S255F (SRS2-3), on the other hand, exhibited remarkable increases in laurate and arachidonate metabolism (3-fold) above wild-type substrate metabolism. Mutant proteins H206Y, S255F, and H206Y/S255F but not R90W/W93S, wild-type CYP4A4, or CYP4A7 metabolized arachidonic acid in the absence of cytochrome b(5). Stopped-flow kinetic experiments were performed in a CO-saturated environment performed to estimate interaction rates of the monooxygenase reaction components. The mutant protein H206Y, which exhibits 3-fold higher than wild-type substrate activity, interacts with CPR at a rate at least 10 times faster than that of wild-type CYP4A7. These experimental results provide insight regarding the residues responsible for modulation of substrate specificity, affinity, and kinetics, as well as possible localization within the enzyme structure based on comparisons with homologous, known cytochrome P450 structures.

A cDNA was isolated from rat C6 glioma cells by expression cloning which encodes a novel Na+-independent neutral amino acid transporter designated LAT1. For functional expression in Xenopus oocytes, LAT1 required the heavy chain of 4F2 cell surface antigen (CD98), a type II membrane glycoprotein. When co-expressed with 4F2 heavy chain, LAT1 transported neutral amino acids with branched or aromatic side chains and did not accept basic amino acids or acidic amino acids. The transport via LAT1 was Na+-independent and sensitive to a system L-specific inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. These functional properties correspond to those of the classically characterized amino acid transport system L, a major nutrient transporter. In in vitro translation, LAT1 was shown to be a nonglycosylated membrane protein consistent with the property of 4F2 light chain, suggesting LAT1 is at least one of the proteins formerly referred to as 4F2 light chain. LAT1 exhibits relatively low but significant amino acid sequence similarity to mammalian cationic amino acid transporters and amino acid permeases of bacteria and yeasts, indicating LAT1 is a new member of the APC superfamily. Because of highly regulated nature and high level of expression in tumor cell lines, LAT1 is thought to be up-regulated to support the high protein synthesis for cell growth and cell activation. The cloning of LAT1 is expected to facilitate the research on the protein-protein interaction in the transporter field and to provide a clue to the search for still unidentified transporters.

We have investigated the functional characteristics of the human amino acid transporter ATB degree using the Xenopus laevis oocyte expression system. When expressed in oocytes, ATB degree mediates the uptake of neutral amino acids in an Na(+)-dependent manner. In addition, this transporter is able to mediate the efflux of intracellular neutral amino acids in exchange with extracellular neutral amino acids. This homo- and hetero-exchange of amino acids is absolutely Na(+)-dependent and conforms strictly to the substrate specificity of ATB degree. Kinetic analysis indicates that the affinity of ATB degree for a given amino acid substrate is similar whether ATB degree catalyzes the influx of the amino acid or the amino acid-induced efflux of intracellular amino acids. These results demonstrate for the first time the ability of ATB degree to function as a homo- and hetero-exchanger for its substrates.

A novel and efficient method to prepare amino acid thiohydantoins, which are required as reference standards for development of C-terminal protein sequencing, is reported. Amino acid thiohydantoins were prepared using a straightforward method involving reaction of 20 free amino acids with acetyl chloride as activating reagent and trimethylsilyl isothiocyanate (TMS-ITC) as derivatizing reagent. The products were characterized by HPLC, uv spectra, amino acid analysis, MS, and NMR. Different reaction conditions were investigated and the chemical mechanism of the formation of amino acid thiohydantoins was illustrated.

A convenient and straightforward synthesis of an amino acid analog, [p-(N-alpha-Fmoc-L-aspartic acid-beta-amido)benzyl]-EDTA tetra-tert-butyl ester, compatible with Fmoc solid phase peptide synthesis strategy is described. This reagent was used to incorporate p-aminobenzyl-EDTA at an internal sequence position in an HIV-1 Tat protein fragment. After cleavage from the resin and standard deprotection, the peptide was purified by high-performance liquid chromatography and characterized by mass spectrometry. Through this methodology, flexible linkers of different lengths and containing various structures can be placed between the alpha-carbon backbone of peptides and metal chelates. These peptides will provide a new class of affinity cleaving reagents that can be directed against protein and nucleic acid targets.

The inhibition of D-amino acid oxidase by certain alpha-keto acids products of the reaction with D-amino acids, in particular alpha-keto acids that are analogs of the amino acids alanine, valine, leucine, phenylanaline, phenylglycine, tyrosine and tryptophan, is reported. All the alpha-keto acids assayed behaved as substrate competitive inhibitors of the enzyme. The relationship between the degree of inhibition and the structure of the inhibitor is discussed.

Reaction of 3,4-estrone o-quinone (3,4-EQ) with several amino acid side chain mimics, including 4-ethylphenol, 4-methylimidazole, acetic acid, and propanethiol, gave a mixture of several products including the catechol, Michael addition products, and dimeric products of the catechol. On the other hand, several other amino acid side chain mimics, including ethanol, acetamide, 1-ethylguanidine, and 3-methylindole, did not result in any addition products or catechol formation. Michael addition to 3,4-EQ with 4-methylimidazole, acetate, and 4-ethyl phenoxide resulted in 1,4-addition, leading to C-1 adducts while reaction with propanethiol gave the C-2 addition product.

As a prelude to engineering artificial energy conversion proteins emulating biology, we examine the inclusion of a synthetic naphthoquinone amino acid in a characterized host–guest protein and determine the effects of its quinone and hydroquinone forms on the helix-coil distribution.

Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein’s gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell. Because this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a “pulse” in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Noncanonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other useful ncAAs, including analogs that contain ketones or aryl halides. High-throughput screening strategies can identify aaRS variants that activate a wide range of ncAAs. Controlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective metabolic labeling of proteins. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. Proteins synthesized in cells not expressing the synthetase are neither labeled nor detected. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. In this Account, we summarize the tools and strategies that have been developed for interrogating cellular protein synthesis through residue-specific tagging with ncAAs. We describe the chemical and genetic components of ncAA-tagging strategies and discuss how these methods are being used in chemical biology.

Basic mechanisms that underlie the oxygen free radical-promoted oxidation of free amino acids and amino acid residues of proteins are derived from radiolysis studies. Results of these studies indicate that the most common pathway for the oxidation of simple aliphatic amino acids involves the hydroxyl radical-mediated abstraction of a hydrogen atom to form a carbon-centered radical at the alpha-position of the amino acid or amino acid residue in the polypeptide chain. Addition of O2 to the carbon-centered radicals leads to formation of peroxy radical derivatives, which upon decomposition lead to production of NH3 and alpha-ketoacids, or to production of NH3, CO2, and aldehydes or carboxylic acids containing one less carbon atom. As the number of carbon atoms in the amino acid is increased, hydrogen abstraction at other positions in the carbon chain becomes more important and leads either to the formation of hydroxy derivatives, or to amino acid cross-linked products as a consequence of carbon-centered radical recombination processes. alpha-Hydrogen abstraction plays a minor role in the oxidation of aromatic amino acids by radiolysis. Instead, the aromatic ring is the primary site of attack leading to hydroxy derivatives, to ring scission, and in the case of tyrosine to the formation of Tyr-Tyr cross-linked dimers. The basic pattern for the oxidation of amino acids by metal ion-catalyzed reactions (Fenton chemistry) is similar to the alpha-hydrogen abstraction pathway. But unlike the case of oxidation by radiolysis, this Fenton pathway is the major mechanism for the oxidation of all aliphatic amino acids, regardless of chain length, as well as for the oxidation of aromatic amino acids. Curiously, the Fe(III)-catalyzed oxidation of free amino acids is almost completely dependent upon the presence of bicarbonate ion, and is greatly stimulated by iron chelators at chelator/Fe(III) ratios less than 1.0, and is inhibited at chelator/Fe(III) ratios greater than 1.0. It is deduced that the most active catalytic complex is composed of two equivalents of HCO3-, an amino acid, and at least one equivalent of iron; however, two forms of iron, an iron-chelate and another form, must somehow be involved. In contrast to the situation with radiolysis, the aromatic rings of aromatic amino acids are only minor targets for metal-catalyzed reactions. All amino acid residues in proteins are subject to attack by hydroxyl radicals generated by ionizing radiation; however, the aromatic amino acids and sulfur-containing amino acids are most sensitive to oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)

OF several recent syntheses of dl-methionine, that commencing with y-butyrolactone1 is relatively long, while others2 are additionally inconvenient in that they employ methyl-B-chloroethylsulphide, a vesicant intermediate. It seems, therefore, desirable to state that the following series of reactions has been found to afford an overall yield of dl-methionine of 29 percent (that is, based on acrolein), and to be free from disadvantages such as those mentioned above

1. 1. An adreno-ferredoxin was purified from sheep adrenocortical mitochondria. 2. 2. Its amino acid composition and amino acid sequence were determined chemically. 3. 3. It was found to be composed of 127 amino acid residues, including two tyrosyl residues. 4. 4. The amino acid sequences of various ferredoxins of the two iron and two sulfur type were compared with respect to amino acid homology.

A number of unnatural amino acids and amino acid analogs with modified backbone structures were substituted for alanine-82 in T4 lysozyme. Replacements included alpha,alpha-disubstituted amino acids, N-alkyl amino acids, and lactic acid, an isoelectronic analog of alanine. The effects of these electronic and structural perturbations on the stability of T4 lysozyme were determined. The relatively broad substrate specificity of the Escherichia coli protein biosynthetic machinery suggests that a wide range of backbone and side-chain substitutions can be introduced, allowing a more precise definition of the factors affecting protein stability.

Amino acid analysis is used to determine the amino acid content of amino acid-, peptide- and protein-containing samples. With minor exceptions, proteins are long linear polymers of amino acids connected to each other via peptide bonds. The first step of amino acid analysis involves hydrolyzing these peptide bonds. The liberated amino acids are then separated, detected, and quantified. The method was first developed by Moore, Stein and coworkers in the 1950s using HCl acid hydrolysis, and, despite considerable effort by many workers, the basic methodology remains relatively unchanged. This unit provides an overview and strategic planning for amino acid analysis, discussing a range of methodologies and issues. In addition, several common methods used for analysis of L-amino acids are described in detail, including: HCl acid hydrolysis, performic acid oxidation for methionine and cysteine analysis, base hydrolysis for tryptophan analysis, analysis of free amino acids, and analysis of reactive lysine.

The effect of the addition of amino acids and amino acid derivatives on the crystallization of hemoglobin and ribonuclease A has been evaluated. The results showed that certain types of additives expand the concentration conditions in which crystals are formed., Determination of the appropriate conditions for protein crystallization remains a highly empirical process. Preventing protein aggregation is necessary for the formation of single crystals under aggregation-prone solution conditions. Because many amino acids and amino acid derivatives offer a unique combination of solubility and stabilizing properties, they open new avenues into the field of protein aggregation research. The use of amino acids and amino acid derivatives can potentially influence processes such as heat treatment and refolding reactions. The effect of the addition of several amino acids, such as lysine, and several amino acid derivatives, such as glycine ethyl ester and glycine amide, on the crystallization of equine hemoglobin and bovine pancreatic ribonuclease A has been examined. The addition of these amino acids and amino acid derivatives expanded the range of precipitant concentration in which crystals formed without aggregation. The addition of such additives appears to promote the crystallization of proteins.

Three groups of chloroenyne-modified amino acids were synthesized. Chloroenyne moiety was attached at the N- or C-terminal amino acid (Tyr, Phe, Val, Gly, Lys) position carrying different protecting groups. Prepared derivatives will be used as building blocks in the synthesis of enediyne-peptide conjugates. Furthermore, reactivity of modified amino acids in the peptide bond formation reaction was tested.

Recently, we have succeeded in incorporating various nonnatural amino acids into proteins by using four-base codon-anticodon pairs in Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system. Here, the reaction was conducted under various conditions in order to optimize the incorporation efficiency. The optimal concentration of aminoacyl-tRNA, reaction temperature, and reaction time were 2 nM, 25 degrees C, and 1.5 hr, respectively.

We have studied amino acid formation by UV (193 nm) irradiation to organic molecules (amines, alcohols and amides) in aqueous solution. Among several types of detected amino acids, small aliphatic amino acids (Gly and alpha-, beta-Ala and alpha-, beta-, gamma-ABA) were quantitatively identified. Among these small aliphatic amino acids, certain amino acids were formed in its free form, even before hydrolysis, contrary to the results of UV irradiation to a gas mixture of CO, NH3, and H2O, where amino acids were hardly detected before hydrolysis. The species distribution of identified amino acids showed a dependence on the starting organic molecules, and also on the presence of ammonia. The formation processes of the identified small aliphatic amino acids were investigated with the aid of electrospray ionization (ESI) MS and MS/MS measurements of photoproducts. Possible formation processes of these amino acid precursors from each starting molecules are proposed. By identifying the amino acid precursor, which has a chiral carbon atom, a new possibility is suggested for asymmetric photosynthesis of amino acid from achiral organic molecules.

Eight analogs 4a-7a and 4b-7b of philanthotoxin (PhTX) from wasp venom and nephilatoxin-8 (NPTX-8) from spider venom whose tyrosine or asparagine linker is replaced by squaryl (sq) amino acid or 4-amino squaryl (4-asq) amino acid have been synthesized in an efficient manner via coupling of N-acyl squaryl amino acid intermediate 19 or 26 with the corresponding polyamine part. Preliminary bioassay using crickets revealed that the analogs substituted by glutamate-type squaryl amino acid-containing NPTX 7a and 7b showed more potent paralytic activities than that of NPTX-8.

Mammalian cationic amino acid transporters (CAT) differ in their substrate affinity and sensitivity to trans-stimulation. The apparent Km values for cationic amino acids and the sensitivity to trans-stimulation of CAT-1, -2B, and -3 are characteristic of system y+. In contrast, CAT-2A exhibits a 10-fold lower substrate affinity and is largely independent of substrate at the trans-side of the membrane. CAT-2A and -2B demonstrate such divergent transport properties, even though their amino acid sequences differ only in a stretch of 42 amino acids. Here, we identify two amino acid residues within this 42-amino acid domain of the human CAT-2A protein that are responsible for the apparent low affinity of both the extracellular and intracellular substrate-binding sites. These residues are located in the fourth intracellular loop, suggesting that they are not part of the translocation pathway. Rather, they may be responsible for the low affinity conformation of the substrate-binding sites. The sensitivity to trans-stimulation is not determined by the same amino acid residues as the substrate affinity and must involve a more complex interaction between individual amino acid residues. In addition to the 42-amino acid domain, the adjacent transmembrane domain X seems to be involved in this function.

Hydrophobicity is a useful concept to rationalize the role played by amino acid residues in terms of buried or exposed conformation with regard to the aqueous environment in proteins. The relationship of this concept with distinct approaches to represent the molecular surface is analyzed by computing reliable surface areas for three definitions namely the van der Waals, solvent-accessible, and solvent-excluded molecular surfaces. The surface areas are obtained for all of the naturally occurring amino acids by first setting a proper reference standard state and then calculating their values for a database of proteins containing a total of 4297 residues. Despite the great differences in these molecular surfaces, proper indexes are here defined for handling the information of interest to study the hydrophobic behavior of amino acids provided by such surfaces.

A method for amino acid sequence and D/L configuration identification of peptides by using fluorogenic Edman reagent 7-[(N, N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+ to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their D/L configurations. Combination of the two HPLC systems, the amino acid sequence and D/L configuration of peptides could be determined. This method will be useful for searching D-amino-acid-containing peptides in animals.

The synthesis of several derivatives of a xenobiotic (phenoxyalkanecarboxylic acid) and an alpha-amino acid (L-lysine) is described. Various substituents were introduced in the aromatic ring of the phenoxyalkanecarboxylic acid and the side-chain was modified. They were tested for their effect on the transport of a neutral (L-threonine), an acidic (L-glutamic acid) and a basic (L-lysine) amino acid, and a sugar (sucrose). All compounds markedly inhibited threonine uptake by leaf tissues of Vicia faba L.--and more specifically phloem loading--with two exceptions, when the aromatic ring was substituted in the 4-position by a primary amino group or when D-lysine was used instead of L-lysine. By contrast, the addition of a chlorine atom in the 4-position of the aromatic ring enhanced the inhibitory activity. Similar results were obtained for inhibition of glutamate uptake and, to a lesser extent, for lysine uptake. pH dependence of the inhibitory activity as well as electrophysiological data indicate that permeases mediating active transport of amino acids are the target of these conjugates. These, in addition to other data obtained with other xenobiotics, show that the amino acid carrier system is capable of recognizing a wide range of conjugates of various sizes, structures and octanol/water partition coefficients.

Improved and efficient techniques have led to an explosive growth in the application of site-directed mutagenesis to the study of enzymes. However, the limited availability of only those 20 amino acids that are translated by the genetic code has prevented the systematic variation of an amino acid's properties in order to define more precisely its role in the catalytic mechanism of an enzyme. An approach is being examined that combines the high specificity of site-directed mutagenesis with the flexibility of chemical modification to overcome these limitations. A set of reagents has been synthesized and reacted with a cysteine model to produce a series of amino acid structural analogs at appreciable rates and in good overall yields. The selective incorporation of these analogs in place of important functional amino acids in a protein will allow a more detailed examination of the role of that amino acid.