Rigid Amino Acid Research Articles

Prevalence and mechanism of synergistic carboxylate-cation-water interactions in halophilic proteins

The cytoplasmic proteins of some halophilic organisms remain stable and functional at multimolar concentrations of KCl, i.e., under conditions that most mesophilic proteins cannot withstand. Their stability arises from their unusual amino acid composition. The most dramatic difference between halophilic and mesophilic proteins is that the former are rich in acidic amino acids. It has been proposed that one of the evolutionary driving forces for this difference is the occurrence of synergistic interactions between multiple acidic amino acids at the surface of the protein, the potassium cations in solution, and water. We investigate this possibility with molecular dynamics simulations, using high-quality force fields for the protein-water, protein-ion, and ion-ion interactions. We create a rigorous thermodynamic definition of interactions between acidic amino acids on proteins that can be used to distinguish between synergistic, noninteracting and interfering interactions. Our results demonstrate that synergistic interactions between neighboring acidic amino acids in halophilic proteins are frequent at multimolar KCl concentration. Synergistic interactions have an electrostatic origin, and are associated with stronger water-to-carboxylate hydrogen bonds than for acidic amino acids without synergistic interactions. Synergistic interactions are not observed in minimal systems of carboxylates, indicating that the protein environment is critical for their emergence. Our results demonstrate that synergistic interactions are neither associated with rigid amino acid orientations nor with highly structured and slow moving water networks, as had been originally proposed. Moreover, synergistic interactions can also be found in unfolded protein conformations. However, because these conformations are only a small subset of the unfolded state ensemble, synergistic interactions should contribute to the net stabilization of the folded state.

Investigation of sequence features of hinge-bending regions in proteins with domain movements using kernel logistic regression

BackgroundHinge-bending movements in proteins comprising two or more domains form a large class of functional movements. Hinge-bending regions demarcate protein domains and collectively control the domain movement. Consequently, the ability to recognise sequence features of hinge-bending regions and to be able to predict them from sequence alone would benefit various areas of protein research. For example, an understanding of how the sequence features of these regions relate to dynamic properties in multi-domain proteins would aid in the rational design of linkers in therapeutic fusion proteins.ResultsThe DynDom database of protein domain movements comprises sequences annotated to indicate whether the amino acid residue is located within a hinge-bending region or within an intradomain region. Using statistical methods and Kernel Logistic Regression (KLR) models, this data was used to determine sequence features that favour or disfavour hinge-bending regions. This is a difficult classification problem as the number of negative cases (intradomain residues) is much larger than the number of positive cases (hinge residues). The statistical methods and the KLR models both show that cysteine has the lowest propensity for hinge-bending regions and proline has the highest, even though it is the most rigid amino acid. As hinge-bending regions have been previously shown to occur frequently at the terminal regions of the secondary structures, the propensity for proline at these regions is likely due to its tendency to break secondary structures. The KLR models also indicate that isoleucine may act as a domain-capping residue. We have found that a quadratic KLR model outperforms a linear KLR model and that improvement in performance occurs up to very long window lengths (eighty residues) indicating long-range correlations.ConclusionIn contrast to the only other approach that focused solely on interdomain hinge-bending regions, the method provides a modest and statistically significant improvement over a random classifier. An explanation of the KLR results is that in the prediction of hinge-bending regions a long-range correlation is at play between a small number amino acids that either favour or disfavour hinge-bending regions. The resulting sequence-based prediction tool, HingeSeek, is available to run through a webserver at hingeseek.cmp.uea.ac.uk.

Novel synthetic approach to non-natural conformationally rigid spiro-linked amino acids

1-Aminospiro[2.3]hexane-1,5-dicarboxylic and 1-ammo-5-(ammomethyl)spiro[2.3]hexane-1-carboxylic acids, which are the conformationally rigid analogues of glutamic acid and lysine, respectively, were synthesized from 3-methylidenecyclobutanecarbonitrile. The synthetic route includes a catalytic [1+2]-cycloaddition of ethyl 2-diazo-2-nitroacetate to a double bond of the unsaturated nitrile, a subsequent chemoselective reduction of the nitro and cyano groups, and a hydrolysis of the ester moiety.

Tiny Particles with Big Impacts on Clean Future Energy

Tiny Particles with Big Impacts on Clean Future Energy

Ab initio study of electronic interaction energies and desolvation energies for dopaminergic ligands in the catechol-O-methyltransferase active site

Catechol-O-methyltransferase (COMT) deactivates dopamine and other catecholic substrates via the methylation of a hydroxyl group. The targeted inhibition of COMT can prevent premature metabolism of both endogenous and exogenous ligands; for example, it can prolong the effectiveness of l-DOPA treatments for Parkinson’s disease. The inhibitory ability of a suite of dopaminergic derivatives against COMT has been studied using electronic interaction energies between each ligand and the enzymatic active site as well as desolvation energies for each ligand. A crystal structure of the COMT active site in complex with a known COMT inhibitor, BIA 8-176, was isolated from the Protein Data Bank (PDB ID: 2CL5). The positions of a suite of dopaminergic derivatives in this same active site were optimized using M062X/6-31G with three different model chemistries: in vacuo with rigid amino acid side chains, with implicit solvation and rigid amino acid side chains, and with implicit solvation and flexible amino acid side chains. Electronic interaction energies between the ligands and the active site residues were calculated for each model using M06L and MP2 with the 6-311+G∗ basis set. Ligands with a nitrile substituent were favored over other substituents in vacuo, but this preference was not retained when the same ligands were optimized with implicit solvation; the preference was regained when using implicit solvation and a relaxed active site. Desolvation energies of the ligands were calculated using a series of hydration shells (n=7, 9, 11 and 13) with M062X and the aug-cc-pvdz, cc-pvdz, and cc-pvtz basis sets. Ligands with carboxyl and nitro substituents exhibited the least favorable desolvation energies, whereas ligands with the nitrile substituents exhibited the most favorable desolvation energies.

Marked difference in self-assembly, morphology, and cell viability of positional isomeric dipeptides generated by reversal of sequence.

In this study two positional isomeric dipeptides Boc-m-ABA-Aib-OMe () and Boc-Aib-m-ABA-OMe () synthesized by reversal of the positions of two rigid amino acids (m-ABA: m-aminobenzoic acid, Aib: α-aminoisobutyric acid) showed marked difference in morphology under the same environmental conditions. Investigation of single crystal structures reveals the difference in crystal packing and higher order self-assembly pattern for both the isomeric peptides, which might be the responsible factor for their different morphological patterns. Moreover, these isomeric dipeptides have produced different cellular viability effects towards normal bone cells. These two peptides would have utilities in the model study of isomeric peptides/proteins, where morphological difference under identical conditions brings changes in their individual bio-activities and where the reversal of sequence causes different cellular viability and generates health hazard.

Effects of implicit solvent and relaxed amino acid side chains on the MP2 and DFT calculations of ligand–protein structure and electronic interaction energies of dopaminergic ligands in the SULT1A3 enzyme active site

The electronic interaction energies between eight dopaminergic ligands and resveratrol and the SULT1A3 active site have been calculated using MP2 and M062X with the 6-311+G∗ basis set. The SULT1A3 active site was isolated from the crystal structure with dopamine bound (PBD ID 2A3R). Structures for the nine ligands bound in the active site were obtained by three different optimization approaches with M062X/6-31G: by optimization in vacuo with rigid amino acid residue side chains (i.e. using the side chain structures from the crystal structure; the ligand and all protons were optimized), with implicit solvation by water with rigid amino acid residue side chains, and with implicit solvation by water and relaxed amino acid residue side chains (i.e. the ligand and all amino acid residue side chains were optimized). The calculations show that the use of implicit solvent and a relaxed active site offer considerable improvement in ligand interaction energies (more strongly binding), and that the solvated-relaxed model shows less variability in ligand interaction than the other two models. Finally, 6-carboxydopamine was shown to be the most strongly binding of the nine ligands, with an interaction energy over 100kcal/mol stronger than its nearest neighbor. Four of the eight non-dopamine ligands studied interact with SULT1A3 more strongly than the endogenous ligand, dopamine, in the solvated-relaxed model.

Inhibitors of peptidyl proline isomerases as antivirals in hepatitis C and other viruses.

Viruses have small genomes with limited coding capacity. A common strategy by which viral genomes maximize their coding capacity is to express multifunctional proteins that promiscuously interact with various cellular partners to perform an array of essential functions. These interactions often involve flexible, and in some cases intrinsically disordered, viral domains or entire proteins that assume distinct conformations only upon binding cellular partners (see review, [1]). Viral coding capacity is further enhanced by relying on host factors and protein folding machinery to access different conformations and functions. These disordered peptide regions can be computationally recognized by features such as glycine, serine, and proline residues in contexts that are not conducive to β–strands or α–helices (reviewed by [2]). Bioinformatic analysis was used to predict the rigidity of proteins encoded by nearly 3,500 genomes from archaea, bacteria, eukaryotes, and viruses. This analysis suggests that almost all genomes with greater than 50% of their encoded residues in a predicted disordered state are viral genomes [3]. Thus, disordered proteins are enriched in the viral proteome and are common features to a large number of viruses. Flexible viral proteins and/or domains interact with the cellular folding machinery, including proline isomerases. While proline is traditionally thought of as being a rigid amino acid that can “kink” the polypeptide chain, prolines can slowly rotate between two energetically similar configurations, cis or trans. This rotation is only fast enough to be physiologically relevant when facilitated by proline isomerases (rotamases) such as mammalian cyclophilins [4]. At least four structurally distinct classes of cellular proline isomerases exist in bacteria and eukaryotes, and some viruses encode their own proline isomerase [4], [5]. Identification of the host isomerases exploited by viruses and the viral proteins that require them to perform essential viral functions for replication in culture, or more importantly, in animals, presents an obvious antiviral strategy. Whether or not inhibition of host proline isomerases could be an antiviral strategy for hepatitis C virus (HCV) was a subject of debate for several years.

Synthetic adhesive oligopeptides with rigid polyhydroxylated amino acids

Synthetic oligopeptides containing polyhydroxylated bicyclic dipeptide (Glc=Tap) are investigated for their adhesion properties. The non-natural amino acid building block composed of Glc=Tap is derived from glucuronic acid and mimics the hydroxyl-amino acids of the natural proteins. Peptide oligomers of Glc=Tap flanked by the amino acids Tyr and Lys were synthesized and characterized. Solution structural studies performed by circular dichromism spectroscopy suggests that poly(Lys-Glc=Tap-Tyr) and poly(Glc=Tap-Tyr) adopts extended helical structures. Adhesion of these oligomers to the mica surface is shown by atomic force microscopy spectroscopy. Studies indicate that extended polyproline II polyhydroxylated peptide chains, which bear additional phenolic as well as cationic side chains, can mimic some of the adhesion properties of the natural protein models. Furthermore, obtained data suggest that poly(Glc=Tap-Tyr) and poly(Lys-Glc=Tap-Tyr) as outstanding adhesive compounds, which combine efficient synthetic accessibility with promising adhesive properties.

Investigation of the role of the amino acid residue at position 230 for catalysis in monomeric carbonyl reductase 3

Monomeric carbonyl reductase 3 (CBR3) is a member of the short-chain dehydrogenase/reductase family. CBR3 exhibits much lower activity than monomeric carbonyl reductase 1 (CBR1) in humans and Chinese hamsters although they are highly homologous to each other in amino acid sequence levels. In the present study, we first cloned the CBR3 gene of rat origin (rCBR3), and characterized its enzymatic activity. rCBR3 also exhibited a limited catalytic efficiency similarly to the other CBR3 orthologues of humans and Chinese hamsters. Among the CBR3 orthologues, the human enzyme showed considerably lower activity. Compared with the amino acid sequences of CBR1 and CBR3 among humans, rats, Chinese hamsters, and mice, the tryptophan residue at position 230 is highly conserved while human CBR3 possesses rigid amino acid, proline, at that position instead. Thus, the Trp-230 was expected to be one of the important residues for catalysis since it locates in the hinge region at the substrate-binding loop. The substitution of tryptophan for proline in hCBR3 failed to affect the enzymatic characteristics. Similarly, the substitution of proline for tryptophan in either Chinese hamster CBR3 (CHCR3) or rCBR3 showed no significant change in the catalytic properties. These results suggest that limited catalytic efficiency of carbonyl reductase activity of CBR3 is a common property among animal species, and the substitution of the amino acid residue at position 230 alone has no apparent impact on their enzymatic activities.

Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR

Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-008-9248-0) contains supplementary material, which is available to authorized users.

Amino acid oxidase‐catalysed resolution and Pictet–Spengler reaction towards chiral and rigid unnatural amino acids

AbstractBACKGROUND: The biosynthesis of structurally complex isoquinoline alkaloids and other natural products occurs via aromatic amino acids such as tyrosine, and chiral and rigid amino acids. These structures are also key building blocks of many active pharmaceutical ingredients. The aim of this work was the exploration of a rapid and straightforward route to chiral 6‐hydroxy‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid.RESULTS: The preparation of (S)‐meta‐tyrosine from racemic meta‐tyro‐ sine with aminoacidoxidase has been developed with ee > 99% and 88% yield. The combination of this resolution with a subsequent Pictet–Spengler reaction enables straightforward and versatile access to chiral (S)‐6‐hydroxy‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid in 30% yield.CONCLUSIONS: This new short chemoenzymatic route to (S)‐6‐hydroxy‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid from commercially available DL‐m‐tyrosine is more convenient than other chemical procedures and establishes a new link between the pool of easily accessible racemic aromatic amino acids and the corresponding chiral rigidified amino acids, which are of interest as structural elements of many active pharmaceutical ingredients. These results facilitate synthetic access to a range of active pharmaceutical ingredients and metabolites in chiral form from the oxidation of amino acids. This advances the opportunities to study the molecular interactions with enzymes, receptors and effectors more precisely than with the racemic forms. Copyright © 2007 Society of Chemical Industry

Viscoelastic Properties of Self-Assembled Type I Collagen Fibers: Molecular Basis of Elastic and Viscous Behaviors

We have studied the strain rate dependence of incremental stress-strain curves of self-assembled type I collagen fibers in an effort to understand the molecular phenomena that contribute to the macroscopic mechanical behavior of tendons. Results of viscoelastic tests at strain rates between 10% and 1000% per min suggest that the slope of the elastic stress-strain curve is to a first approximation independent of strain rate while the slope of the viscous stress-strain curve increases with increased strain rate. After correction of the slope of the viscous stress-strain curve for the changes in strain rate, it is observed that the apparent viscosity decreases with increased strain rate. It is concluded that the approximate strain rate independence of the elastic spring constant of collagen is consistent with the spring-like behavior of the 12 flexible regions that make up the collagen D-period. These regions are poor in the rigid amino acid residues proline and hydroxyproline. In contrast, the thixotropy of collagen is consistent with the slippage of subfibrillar subunits during tensile deformation. It is hypothesized that at high strain rates subfibrillar subunits appear to "hydroplane" by each other on a layer of loosely bound water.

Viscoelastic Properties of Self-Assembled Type I Collagen Fibers: Molecular Basis of Elastic and Viscous Behaviors

We have studied the strain rate dependence of incremental stress-strain curves of self-assembled type I collagen fibers in an effort to understand the molecular phenomena that contribute to the macroscopic mechanical behavior of tendons. Results of viscoelastic tests at strain rates between 10% and 1000% per min suggest that the slope of the elastic stress-strain curve is to a first approximation independent of strain rate while the slope of the viscous stress-strain curve increases with increased strain rate. After correction of the slope of the viscous stress-strain curve for the changes in strain rate, it is observed that the apparent viscosity decreases with increased strain rate. It is concluded that the approximate strain rate independence of the elastic spring constant of collagen is consistent with the spring-like behavior of the 12 flexible regions that make up the collagen D-period. These regions are poor in the rigid amino acid residues proline and hydroxyproline. In contrast, the thixotropy of collagen is consistent with the slippage of subfibrillar subunits during tensile deformation. It is hypothesized that at high strain rates subfibrillar subunits appear to "hydroplane" by each other on a layer of loosely bound water.

Flexibility of amino acid residues at position four of nonapeptides enhances their binding to human leucocyte antigen (HLA) molecules.

The binding affinity of synthetic nonapeptides to human leucocyte antigens (HLA) molecules of the A0201 allotype, the most common in Caucasian, is enhanced or reduced by suitable amino acid substitutions at position 4, as a result of increased or decreased chain flexibility. A higher flexibility of the bond at this position correlates with an easier accommodation of the fragment into the HLA groove, while rigidity of the peptide chain appears to interfere. These data are based on two lines of evidence: a) most natural high affinity ligands for HLA-A0201 possess, at position 4, flexible residues b) substitutions of such residues by rigid amino acids results in a decrease of binding affinity.

New energy transfer dyes for DNA sequencing.

We have synthesized a set of four energy transfer dyes and demonstrated their use in automated DNA sequencing. The donor dyes are the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes are a novel set of four 4,7-dichloro-substituted rhodamine dyes which have narrower emission spectra than the standard, unsubstituted rhodamines. A rigid amino acid linker, 4-aminomethylbenzoic acid, was used to separate the dyes. The brightness of each dye in an automated sequencing instrument equipped with a dual line argon ion laser (488 and 514 nm excitation) was 2-2.5 times greater than the standard dye-primers with a 2 times reduction in multicomponent noise. The overall improvement in signal-to-noise was 4- to 5-fold. The utility of the new dye set was demonstrated by sequencing of a BAC DNA with an 80 kb insert. Measurement of the extinction coefficients and the relative quantum yields of the dichlororhodamine components of the energy transfer dyes showed their values were reduced by 20-25% compared with the dichlororhodamine dyes alone.

A versatile synthesis of a β-turn peptidomimetic scaffold: An approach towards a designed model antagonist of the tachykinin NK-2 receptor

A general and stereocontrolled synthesis of an azabicyclo[4.3.0]nonane amino acid template with an appended substitutent at C-5 was developed. The approach allows for stereochemical and functional variations that extend the utility of these rigid amino acid motifs as peptidomimetics. The synthesis of a weakly active but specific NK-2 receptor antagonist is described.

Qβ replicase: Mapping the functional domains of an RNA-dependent RNA polymerase

We have localized a functional region of the RNA bacteriophage Qβ replicase following an extensive mutational analysis. Using the method of oligonucleotide linker-insertion mutagenesis, we specifically introduced mutations into a cloned DNA copy of the Qβ replicase gene so that the resulting replicase products would putatively contain small amino acid insertions. In a selective phenotypic assay, we screened mutant replicases for RNA-directed replication activity in vivo. Analysis of 37 different mutant clones indicated that Qβ replicase can accept amino acid substitutions and insertions at several sites at the amino and carboxy termini without abolishing functional activity in vivo or in vitro. However, disruption within the internal amino acid sequence resulted almost exclusively in nonfunctional enzyme. The results suggest that the central region of the replicase protein contains a rigid amino acid composition that is required for replicase function, whereas the amino and carboxy termini are much more receptive to small amino acid insertions and substitutions. These experiments should further enable us to analyze the coding function of the Qβ replicase gene independently of other phage RNA functions contained within this nucleotide region.

Amino acid derivatives that stabilized secondary structures of polypeptides—III. β-Enaminonitriles as analogs of secondary amides. The MCC group—1-acylamino-2-aminoalkyl-3-cyano-2-cyclopentenes as amino acid analogs.

The properties of β-cyanoenamines as analogs of amides are studied with derivatives of a rigid amino acid equivalent—Mcc = 1,2-diamino-3-cyanocyclopentene, prepared in two steps from methyl 2-(N-Boc-amino)-5-cyanopentanoate. Racemation of Mcc and incorporation into a cyclic pentapeptide are described.

Rigid amino acids related to alpha-methyldopa.

Rigid amino acids related to alpha-methyldopa.