Quantitation of the nearest-neighbour effects of amino acid side-chains that restrict conformational freedom of the polypeptide chain using reversed-phase liquid chromatography of synthetic model peptides with L- and D-amino acid substitutions

Side-chain backbone interactions (or "effects") between nearest neighbours may severely restrict the conformations accessible to a polypeptide chain and thus represent the first step in protein folding. We have quantified nearest-neighbour effects (i to i+1) in peptides through reversed-phase liquid chromatography (RP-HPLC) of model synthetic peptides, where L- and D-amino acids were substituted at the N-terminal end of the peptide sequence, adjacent to a L-Leu residue. These nearest-neighbour effects (expressed as the difference in retention times of L- and D-peptide diastereomers at pHs 2 and 7) were frequently dramatic, depending on the type of side-chain adjacent to the L-Leu residue, albeit such effects were independent of mobile phase conditions. No nearest-neighbour effects were observed when residue i is adjacent to a Gly residue. Calculation of minimum energy conformations of selected peptides supported the view that, whether a L- or D-amino acid is substituted adjacent to L-Leu, its orientation relative to this bulky Leu side-chain represents the most energetically favourable configuration. We believe that such energetically favourable, and different, configurations of L- and D-peptide diastereomers affect their respective interactions with a hydrophobic stationary phase, which are thus quantified by different RP-HPLC retention times. Side-chain hydrophilicity/hydrophobicity coefficients were generated in the presence of these nearest-neighbour effects and, despite the relative difference in such coefficients generated from peptides substituted with L- or D-amino acids, the relative difference in hydrophilicity/hydrophobicity between different amino acids in the L- or D-series is maintained. Overall, our results demonstrate that such nearest-neighbour effects can clearly restrict conformational space of an amino acid side-chain in a polypeptide chain.     

Design,synthesis and structural analysis of mixed α/β-peptides that adopt stable cyclic hairpin-like conformations

The strategic replacement of four α-amino acid residues of a cyclo-(ααααα)₂ peptide by β-, β²- or β³-amino acids residues provided a series of novel 2:1 α/β-mixed peptides that were designed to adopt cyclic hairpin-like structures. It was shown that conformationally stable cyclo-(αβαβα)₂ isomers can be obtained using both enantiomers of the central two basic α-amino acid residues, a known α-amino acid turn sequence and several combinations of facing β-amino acid residues with no side chain or a hydrophobic side chain having specific regio- and stereochemistry. The X-ray analysis of two derivatives provides molecular details of the intra-molecular hydrogen bonding interaction, dihedral angles of the backbone and side chain positioning of the novel cyclic hairpin-like structures. One of these isomers forms an unprecedented hexagon-shaped nano-channel assembly in the crystal structure. Well-defined cyclic hairpin-like structures as described here and derivatives that can be readily designed based on this research can be used as scaffolds onto which functional groups can be grafted in a spatially controlled manner and as β-hairpin mimics with specific biological properties.     

Preparation and characterization of multipoint yeast D-amino acid oxidase mutants with improved stability and activity

D-amino acid oxidase (DAAO) is an FAD-containing oxidoreductase that stereospecifically oxidases D-amino acids to produce α-keto-acids, an ammonium ion, and hydrogen peroxide. The most important biotechnological process involving DAAO is the production of 7-amino cephalospranic acid (7-ACA) from cephalosporin C. The reaction product, 7-ACA, is then used as a precursor for the synthesis of cephalosporin antibiotics of different generations. We previously obtained mutant DAAOs from the yeast Trigonopsis variabilis (TvDAAO). The mutants with point amino acid substitutions were characterized by either an increased thermal stability or improved catalytic properties in the oxidation of cephalosporin C. In the present study, we obtained two new mutant TvDAAOs with two and four amino acid substitutions, respectively. The catalytic constants of these mutant TvDAAOs for the oxidation of cephalosporin C were 1.8 and 4 times higher than the respective parameter of the wild-type enzyme (wt-TvDAAO). The combination of substitutions increased the thermal stabilities of both mutant TvDAAOs by a factor of 2–3 as compared with the wt- TvDAAO.     

Molecular Mechanisms of Sweet Taste 1: Sweet and Non-Sweet Tasting Amino Acids

ABSTRACT

A novel mechanism is proposed for the AH/B interaction of sweet molecules, such as D-α-amino acids with the L-asparagine unit at the N-terminus of a receptor protein which has a right-handed α-helical conformation. The lipophilic, dispersive bonding occurs between the side chain of the fifth amino acid residue, from the N-terminus, and either the side chain of the α-amino acid or its methine group at the carbon 2. The sequence of AH, B and X groups on the sweet amino acids occurs in a clockwise orientation, when viewed from the receptor.     

The tunability of the electronic structures for poly(carbosilylsilanes): a theoretical study

To tune purposefully the electronic structures of poly(carbosilylsilanes), a theory study has been investigated using the density functional theory combined with AM1 method. Attentions were paid to the dependence of molecular geometries and absorption spectra on the backbone conformation and the various substituting groups. The strong electronegative substituents can more effectively tune the geometries and spectra of the polysilanes than the alkyl ones. Their main-chain substitutions can induce the great red-shift of the absorption spectra, and the side-chain substitutions can induce the blue-shift. The length of methylene chain in the carbosilyl groups exerts the small effect on the absorption spectra, but with the lengthening of side chain, poly(carbosilylsilanes) have a preference for the all-trans conformation with the loose helix backbone. Different from the alkyl side chain in poly(alkylsilanes), the lengthening of carbosilyl chain leads to the decrease of the positive charges of silicon backbone.     

Automated NMR assignment of protein side chain resonances using automated projection spectroscopy (APSY)

This paper describes an automated method for sequence-specific NMR assignment of the aliphatic resonances of protein side chains in small- and medium-sized globular proteins in aqueous solution. The method requires the recording of a five-dimensional (5D) automated projection spectroscopy (APSY-) NMR experiment and the subsequent analysis of the APSY peak list with the algorithm ALASCA (Algorithm for local and linear assignment of side chains from APSY data). The 5D APSY-HC(CC-TOCSY)CONH experiment yields 5D chemical shift correlations of aliphatic side chain C-H moieties with the backbone atoms H(N), N, and C'. A simultaneous variation of the TOCSY mixing times and the projection angles in this APSY-type TOCSY experiment gives access to all aliphatic C-H moieties in the 20 proteinogenic amino acids. The correlation peak list resulting from the 5D APSY-HC(CC-TOCSY)CONH experiment together with the backbone assignment of the protein under study is the sole input for the algorithm ALASCA that assigns carbon and proton resonances of protein side chains. The algorithm is described, and it is shown that the aliphatic parts of 17 of the 20 common amino acid side chains are assigned unambiguously, whereas the remaining three amino acids are assigned with a certainty of above 95%. The overall feasibility of the approach is demonstrated with the globular 116-residue protein TM1290, for which reference assignments are known. For this protein, 97% of the expected side chain carbon atoms and 87% of the expected side chain protons were detected with the 5D APSY-HC(CC-TOCSY)CONH experiment in 24 h of spectrometer time, and all these resonances were correctly assigned by ALASCA. Based on the experience with TM1290, we expect that the approach presented in this work is routinely applicable to globular proteins with sizes up to at least 120 amino acids.     

Engineering the properties of D-amino acid oxidases by a rational and a directed evolution approach

D-amino acid oxidase (DAAO) is a FAD-containing flavoprotein that dehydrogenates the D-isomer of amino acids to the corresponding imino acids, coupled with the reduction of FAD. The cofactor then reoxidizes on molecular oxygen and the imino acid hydrolyzes spontaneously to the alpha-keto acid and ammonia. In vitro DAAO displays broad substrate specificity, acting on several neutral and basic D-amino acids: the most efficient substrates are amino acids with hydrophobic side chains. D-aspartic acid and D-glutamic acid are not substrates for DAAO. Through the years, it has been the subject of a number of structural, functional and kinetic investigations. The most recent advances are represented by site-directed mutagenesis studies and resolution of the 3D-structure of the enzymes from pig, human and yeast. The two approaches have given us a deeper understanding of the structure-function relationships and promoted a number of investigations aimed at the modulating the protein properties. By a rational and/or a directed evolution approach, DAAO variants with altered substrate specificity (e.g., active on acidic or on all D-amino acids), increased stability (e.g., stable up to 60 degrees C), modified interaction with the flavin cofactor, and altered oligomeric state were produced. The aim of this paper is to provide an overview of the most recent research on the engineering of DAAOs to illustrate their new intriguing properties, which also have enabled us to pursue new biotechnological applications.     

Radical Stability Directs Electron Capture and Transfer Dissociation of β‐Amino Acids in Peptides

We report on the characteristics of the radical‐ion‐driven dissociation of a diverse array of β‐amino acids incorporated into α‐peptides, as probed by tandem electron‐capture and electron‐transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β‐amino acids than for their α‐form counterparts. In particular, only aromatic (e.g., β‐Phe), and to a substantially lower extent, carbonyl‐containing (e.g., β‐Glu and β‐Gln) amino acid side chains, lead to N? C_β bond cleavage in the corresponding β‐amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical‐driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α‐amino acids, ECD of peptides composed mainly of β‐amino acids reveals a shift in cleavage priority from the N? C_β to the C_α? C bond. The incorporation of CH₂ groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical‐driven β‐amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.     

Unnatural multidentate metal ligating α-amino acids

A family of penta- and hexadentate metal ligating α-amino acids, suitably protected for Fmoc solid-phase chemistry, has been prepared. These residues incorporate the mono-amides of ethanolaminetriacetic acid, ethylenediaminetriacetic acid, and ethylenediaminetetraacetic acid as side chains. Side chains are tethered varying distances (n) from the Cα-carbon to allow metal binding events to occur at distinct distances from the peptide backbone. These residues are designed to allow the facile installation of metal chelates along a peptide backbone.     

Homochirality and life

Before the emergence of life, left-handed amino acids (L-enantiomers) were selected and right-handed amino acids (D-enantiomers) were eliminated on the primal earth. Nevertheless, with the progress of analytical methods, D-amino acids have recently been found in higher order living organisms in the form of free amino acids, peptides, and proteins. Free D-amino acids have numerous physiological functions. D-amino acids containing animal peptides are well known as opioid peptides. D-amino acids in protein are related to aging. In this review, we describe the D-amino acids that are present and function as D-amino acid biosystems in our bodies.     

The design, synthesis and application of stereochemical and directional peptide isomers: a critical review

Physiological processes are regulated to a large extent by physical and chemical interactions between polypeptides. Although many small molecules have been discovered that can modulate such interactions and may be useful as drugs, the design of these agents purely from the knowledge of the details of a given protein-protein interaction, or through screening, remains difficult. Therefore, the peptidomimetic process, which aims at using peptides derived from either polypeptide binding partner directly, or after modification to improve affinity and physicochemical properties, continues to be attractive. The vast majority of naturally occurring polypeptides are composed of L-amino acids. Because natural proteins need to be metabolised, L-amino acid polypeptides are very prone to proteolytic degradation, a property that severely limits their therapeutic application. The proteolytic machinery is not well equipped to deal with D-amino acid polypeptides, however, and it is this finding above all else that has spurned research into stereochemical and directional manipulation of peptide chains. The expectation has been that systematic inversion of the stereochemistry at the peptide backbone alpha-carbon atoms, if accompanied by chain reversal, should yield proteolytically stable retro-inverso peptide isomers, whose side chain topology, in the extended conformation, corresponds closely to that of a native sequence, and whose biological activity emulates that of a parent polypeptide. The actual structural implications of modifying amino acid stereochemistry and peptide bond direction are reviewed critically here and the reasons for the lack of general success with this strategy are discussed. The application of polypeptides is particularly pertinent to synthetic vaccine design. Interestingly, the retro-inverso strategy has been more successful for immunological applications than elsewhere; recent finding are collated in this review. Partial rather than global retro-inversion holds much promise since the loss of crucial backbone hydrogen-bonding through peptide bond reversal can be avoided, while still permitting stabilisation of selected hydrolysis-prone peptide bonds. Generically applicable synthetic methods for such partially modified retro-inverso peptides are not as yet available; progress towards this goal is also summarised.     

Hydroxamic acid polymers. Effect of structure on the chelation of iron in water: II

The effect of structure on the ability of hydroxamic acid polymers to chelate iron(III) was examined. The polymers were derived from acryloyl or methacryloyl backbones that bore side chains terminated in hydroxamic acids. The side chain length, which establishes the atomic chain distances between hydroxamic acid groups, had the most pronounced effect on the stability constant of the iron chelate. It was this atomic chain distance that determined how easily the three neighboring hydroxamic acids could fit the octahedral sphere of the iron. Other structural changes such as the presence or absence of methyl groups on the backbone or on the hydroxamic acid nitrogen had little measurable effect. The stability of the iron complexes appeared to be optimum at an 11-atom spacing between hydroxamic acids and decreased with shorter or longer spacing distances.     

Harnessing Aromatic-Histidine Interactions through Synergistic Backbone Extension and Side Chain Modification

Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high-resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor-1, which is targeted by osteoporosis drugs. These results should motivate other structure-based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins.     

Differential ordering of the protein backbone and side chains during protein folding revealed by site-specific recombinant infrared probes

The time scale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and ordering of the side chains is particularly important for the formation of β-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed α/β structure. We have developed a general approach for introducing site-specific IR probes for the side chains (azide) and backbone ((13)C═(18)O) using recombinant protein expression. Temperature-jump time-resolved IR spectroscopy combined with site-specific labeling enables independent measurement of the respective backbone and side-chain dynamics with single residue resolution. We have found that side-chain ordering in a key region of the β-sheet structure occurs on a slower time scale than ordering of the backbone during the folding of NTL9, likely as a result of the transient formation of non-native side-chain interactions.     

The dehydroalanine effect in the fragmentation of ions derived from polypeptides

The fragmentation of peptides and proteins upon collision‐induced dissociation (CID) is highly dependent on sequence and ion type (e.g. protonated, deprotonated, sodiated, odd electron, etc.). Some amino acids, for example aspartic acid and proline, have been found to enhance certain cleavages along the backbone. Here, we show that peptides and proteins containing dehydroalanine, a non‐proteinogenic amino acid with an unsaturated side‐chain, undergo enhanced cleavage of the N—Cα bond of the dehydroalanine residue to generate c‐ and z‐ions. Because these fragment ion types are not commonly observed upon activation of positively charged even‐electron species, they can be used to identify dehydroalanine residues and localize them within the peptide or protein chain. While dehydroalanine can be generated in solution, it can also be generated in the gas phase upon CID of various species. Oxidized S‐alkyl cysteine residues generate dehydroalanine upon activation via highly efficient loss of the alkyl sulfenic acid. Asymmetric cleavage of disulfide bonds upon collisional activation of systems with limited proton mobility also generates dehydroalanine. Furthermore, we show that gas‐phase ion/ion reactions can be used to facilitate the generation of dehydroalanine residues via, for example, oxidation of S‐alkyl cysteine residues and conversion of multiply‐protonated peptides to radical cations. In the latter case, loss of radical side‐chains to generate dehydroalanine from some amino acids gives rise to the possibility for residue‐specific backbone cleavage of polypeptide ions. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantitative determination of short branches in high-pressure polyethylene by gamma radiolysis

A careful study of the radiolysis products of a series of ethylene α-olefin copolymers and ethylene homopolymers has shown that if a correction is applied, to take into account the fragments arising from scission at chain ends, the remaining products can be quantitatively accounted for as entirely due to scission of side branches introduced onto the backbone chain by the α-olefin comonomer. The cleavage of branches takes place, for all practical purposes, exclusively at the branch points at which the branches are attached to the backbone chain. The same data together with similar radiolysis data of poly(3-methyl pentene-1) and poly(4-methyl pentene-1) have further shown that all branches cleave with equal efficiency, regardless of their length. Radiolysis does, therefore, provide a reliable and convenient tool for the quantitative characterization of high-pressure polyethylene with regard to the unique short-chain branching distribution that is characteristic of each.     

Clathration-induced asymmetric transformation of cefadroxil

[structure: see text] The cephalosporin antibiotic Cefadroxil can be epimerized at the alpha-carbon of its amino acid side chain using pyridoxal as the mediator. By clathration with 2,7-dihydroxynaphthalene, the desired diastereomer can be selectively withdrawn from the equilibrating mixture of epimers. In this way, an asymmetric transformation of Cefadroxil can be accomplished. This opens the possibility of the production of Cefadroxil starting from racemic p-hydroxyphenylglycine, in contrast to the current industrial synthesis that employs the D-amino acid in enantiopure form.     

Occurrence and functions of free D-aspartate and its metabolizing enzymes

D-Aspartate is one of a few D-amino acids that attracted attention at an early date, since it was detected in various tissues of mammals as a protein component. The occurrence of free D-aspartate in nature was recognized a little later, and raised questions about its physiological functions and metabolism. This amino acid has been gradually accepted, based on various experimental observations, to be a physiological substrate of D-aspartate oxidase, whose role had been considered enigmatic since its early discovery in the 1940s. Mammalian enzymes that serve to liberate D-aspartyl residue in proteins have been identified. One enzyme hydrolyzes peptide bond at the amino side of D-aspartyl residue in a dipeptide and another enzyme hydrolyzes that at the carbonyl side of the residue in proteins. The first pyridoxal 5'-phosphate-dependent aspartate racemase has been purified and cloned from a bivalve species. The enzyme supports the high contents of D-aspartate comparable to those of L-aspartate in the bivalve, and the enantiomers are consumed when hypoxia is imposed on the bivalve. In some yeast species, assimilation of D-aspartate has been found to depend on inducible D-aspartate oxidase, which also serves to detoxify acidic D-amino acids.     

Solvation free energy of amino acids and side-chain analogues

The solvation free energies of amino acids and their side-chain analogues in water and cyclohexane are calculated by using Monte Carlo simulation. The molecular interactions are described by the OPLS-AA force field for the amino acids and the TIP4P model for water, and the free energies are determined by using the Bennett acceptance method. Results for the side-chain analogues in cyclohexane and in water are used to evaluate the performance of the force field for the van der Waals and the electrostatic interactions, respectively. Comparison of the calculated hydration free energies for the amino acid analogues and the full amino acids allows assessment of the additivity of the side chain contributions on the number of hydrating water molecules. The hydration free energies of neutral amino acids can be reasonably approximated by adding the contributions of their side chains to that of the hydration of glycine. However, significant nonadditivity in the free energy is found for the zwitterionic form of amino acids with polar side chains. In serine and threonine, intramolecular hydrogen bonds are formed between the polar side chains and backbone groups, leading to weaker solvation than for glycine. In contrast, such nonadditivity is not observed in tyrosine, in which the hydroxyl group is farther separated from, and therefore cannot form an intramolecular hydrogen bond with, the backbone. For histidine we find that a water molecule can form a bridge when the intramolecular hydrogen bond between the polar group and the backbone is broken.     

On the consequences of side chain flexibility and backbone conformation on hydration and proton dissociation in perfluorosulfonic acid membranes

The flexibility of the side chain and effects of conformational changes in the backbone on hydration and proton transfer in the short-side-chain (SSC) perfluorosulfonic acid fuel cell membrane have been investigated through first principles based molecular modelling studies. Potential energy profiles determined at the B3LYP/6-31G(d,p) level in the two pendant side chain fragments: CF(3)CF(-O(CF(2))(2)SO(3)H)-(CF(2))(7)-CF(-O(CF(2))(2)SO(3)H)CF(3) indicate that the largest CF(2)-CF(2) rotational barrier along the backbone is nearly 28.9 kJ mol(-1) higher than the minimum energy staggered trans conformation. Furthermore, the calculations reveal that the stiffest portion of the side chain is near to its attachment site on the backbone, with CF-O and O-CF(2) barriers of 38.1 and 28.0 kJ mol(-1), respectively. The most flexible portion of the side chain is the carbon-sulfur bond, with a barrier of only 8.8 kJ mol(-1). Extensive searches for minimum energy structures (at the B3LYP/6-311G(d,p) level) of the same polymeric fragment with 4-7 explicit water molecules reveal that the perfluorocarbon backbone may adopt either an elongated geometry, with all carbons in a trans configuration, or a folded conformation as a result of the hydrogen bonding of the terminal sulfonic acids with the water. These electronic structure calculations show that the fragments displaying the latter 'kinked' backbone possessed stronger binding of the water to the sulfonic acid groups, and also undergo proton dissociation with fewer water molecules. The calculations point to the importance of the flexibility in both the backbone and side chains of PFSA membranes to effectively transport protons under low humidity conditions.