Effect Of Amino Acid Sequence Research Articles

Sequence-dependent dipeptide solubility in ethanol-water and DMSO-water solutions

Reliable solubility data is crucial for controlling and optimising crystallisation conditions, to provide a cost-effective purification method with the potential for scaling up. This study investigated the effect of amino acid sequence on determining peptides solubility behaviours. Four dipeptides with different sequences and combinations of alanine and glycine were selected to study their temperature dependent (283.15 K to 313.15 K) aqueous solubilities and solubility in the presence of ethanol and DMSO as antisolvent at 298.15 K. Aqueous solubility data of dipeptides presents the following order: glycyl-L-alanine (gly-ala) > L-alanyl-L-alanine (ala-ala) > glycyl glycine (gly-gly) > L-alanyl glycine (ala-gly). This solubility order has been substantiated by solvation free energy calculations using molecular dynamics (MD) simulations, providing insights into the underlying molecular interactions that govern these solubility patterns. The solubility data also demonstrated difference in the temperature dependency that: gly-gly > gly-ala > ala-gly > ala-ala, corresponding to their dissolution enthalpy order. In the presence of antisolvent range from 0 % to 80 % (by mass) concentration, peptide’s solubility was more susceptible to DMSO than ethanol. Accurate prediction of the solubilities validated the effectiveness of non-random two-liquid (NRTL) model for peptide studies.

Two-photon fluorescence depolarization dynamics of ionic strength biosensors

Compartmentalized, dynamic ionic strength within living cells influences numerous biochemical mechanisms such as catalytic function, protein folding, osmotic pressure, and energy production. Recent development of genetically encoded donor-linker-acceptor biosensors that undergo Förster resonance energy transfer (FRET) offers a promising methodology towards noninvasive, site-specific, quantitative, and sensitive mapping of in vivo ionic strength. Here, we investigate the effects of amino acid sequence in the flexible linker on the FRET efficiency and hence the sensitivity to ionic strength of varying KCl concentration.

Variants in ACE2; potential influences on virus infection and COVID-19 severity.

The third pandemic of coronavirus infection, called COVID-19 disease, was first detected in November 2019th. Various determinants of disease progression such as age, sex, virus mutations, comorbidity, lifestyle, host immune response, and genetic background variation have caused clinical variability of COVID-19. The causative agent of COVID-19 is an enveloped coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that invades host cells using an endocytic pathway. The SARS-CoV-2 spike protein is the main viral protein that contributes to the fusion of the virus particle to the host cell through angiotensin-converting enzyme 2 (ACE2). The highly conserved expression of ACE2 is found in various animals, which indicates its pivotal physiological function. The ACE2 has a crucial role in vascular, renal, and myocardial physiology. Genetic factors contributing to the outcome of SARS-CoV-2 infection are unknown; however, variants in the specific sites of ACE2 gene could be regarded as a main genetic risk factor for COVID-19. Given that ACE2 is the main site for virus landing on host cells, the effect of amino acid sequences of ACE2 on host susceptibility to COVID-19 seems reasonable. It would likely have a substantial role in the occurrence of a wide range of clinical symptoms. Several ACE2 variants can affect the protein stability, influencing the interaction between spike protein and ACE2 through imposing conformational changes while some other variants are known to cause a decrease or an increase in the ligand-receptor affinity. The other variations are located at the proteolytic cleavage site, which can influence virus infection; because soluble ACE2 can act as a decoy receptor for virus and decrease virus intake by cell surface ACE2. Notably, polymorphisms of regulatory and non-coding regions such as promoter in ACE2, can play crucial role in different expression levels of ACE2 among different individuals. Many studies should be performed to investigate the involvement of ACE2 polymorphism with susceptibility to COVID-19. Herein, we discuss some reported associations between variants of ACE2 and COVID-19 in details. In addition, the mode of action of ACE2 and its role in SARS-CoV-2 infection are highlighted which is followed by addressing the effects of several ACE2 variants on its protein stability, viral tropism or ligand-receptor affinity, secondary and tertiary structure or protein conformation, proteolytic cleavage site, and finally inter-individual clinical variability in COVID-19. The polymorphisms of regulatory regions of ACE2 and their effect on expression levels of ACE2 are also provided in this review. Such studies can improve the prediction of the affinity of mutant ACE2 variations with spike protein, and help the biopharmaceutical industry to design effective approaches for recombinant hACE2 therapy and vaccination of COVID-19 disease.

Self‐assembly of hairpin peptides mediated by Cu(II) ion: Effect of amino acid sequence

AbstractThe alteration of amino acid register in peptides could affect their folding properties and thus functions. The effect of sequence in metal ion induced folding of peptide/protein is important in the design of new functional protein structures, such as biomimetics. In this paper, a set of hairpin peptides with identical composition but different histidine locations have been designed to exploit the metal ion‐mediated peptide intramolecular folding transformation through the adjustment of histidine and lysine residue locations. The results demonstrated that the histidine positions in peptide sequence could dramatically affect the coordination modes between peptides and Cu(II) ion. The Cu(II) coordination could affect the secondary structure transformation and self‐assembly along with electrostatic interaction and hydrogen bonding synergistically. Three molecules with VHVH sequence at each side of the turn segment (VDPPT) were apt to coordinate with Cu(II) ion with three or four histidines. The closer the VHVH motifs were to the turn segment, the more easier the molecules were to form β sheet structure with the promotion of Cu(II) ions. These results demonstrated that the purposeful location of key amino acids in peptides could achieve the controllable conformation transformation of hairpin peptides, which provides useful strategies for biomimetic design, such as metalloenzymes.

The effects of amino acid sequence and solvent polarity on the self-assembling of cyclic peptide nanotubes and molecular channel formation inside the lipid bilayer

In this article, the effects of amino acid sequence and solvent polarity on the self-assembling process of cyclic peptides (CPs) were investigated by employing molecular dynamic (MD) simulations and quantum chemistry calculations. As a result, CP dimers are not stable in water, because of hydrogen bond (H-bond) lost between the CP units, while chloroform increases the stability of the CP dimers. MM-PBSA and MM-GBSA calculations confirmed that solvent polarity has an important effect on the stability of the CP dimers. Dynamical behavior of the cyclic peptide nanotubes (CPNTs) in chloroform indicates that CPNTs composed of leucine and phenylalanine are better molecular containers than that of isoleucine. At the next step, the ability of these CPNTs in molecular channel formation inside a fully hydrated DMPC (dimyristoylphosphatidylcholine) bilayer was investigated during 50 ns MD simulations. The obtained results show that only CPNT composed of isoleucine can form a molecular channel inside the DMPC membrane because isoleucine has a greater hydrophobicity than leucine and phenylalanine. This property increases the interactions between the CPNT and lipid residues, which elevates the stability of the CPNT inside the DMPC bilayer. Quantum chemistry calculations and non-covalent interactions analysis indicate that the solvent changes the stability and dynamical behavior of the CPNTs through the change in the H-bond strength. Finally, according to the different analyses, it can be concluded that the amino acid sequence in the CP units has an important role in designing specific nanostructures.

Rapid assembly and profiling of an anticoagulant sulfoprotein library

Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

Anisotropic Gold Nanostructures: Optimization via in Silico Modeling for Hyperthermia

Protein- and peptide-based manufacturing of self-assembled supramolecular functional materials has been a formidable challenge for biomedical applications, being complex in structure and immunogenic in nature. In this context, self-assembly of short amino acid sequences as simplified building blocks to design metal–biomolecule frameworks (MBioFs) is an emerging field of research. Here, we report a facile, bioinspired route of anisotropic nanostructure synthesis using gold binding peptides (10–15mers) secreted by cancer cells. The bioinformatics tool i-TASSER predicts the effect of amino acid sequences on metal binding sites and the secondary structures of the respective peptide sequence. Electron microscopy, X-ray, infrared, and Raman spectroscopy validated the versatile anisotropic gold nanostructures and the metal–bioorganic nature of this biomineralization. We studied the influence of precursor salt, pH, and peptide concentration on the evolution of nanoleaf, nanoflower, nanofiber, and dendrimer-like a...

Theory of Sequence Effects in Amyloid Aggregation.

We present a simple model for the effect of amino acid sequences on amyloid fibril formation. Using the HP model we find the binding lifetimes of four simple sequences by solving the first passage time for the intermolecular H-bond reaction coordinate. We find that sequences with identical binding energies have widely varying binding times depending on where the aggregation prone amino acids are located in the sequence. In general, longer binding times occur when the aggregation prone amino acids are clustered in a single "hot spot". Similarly, binding times are shortened by clustering weakly bound residues. Both of these effects are explained by an increase in the multiplicity of unbinding trajectories that comes from adding weak binding residues. Our model predicts a transition from ordered to disordered fibrils as the concentration of monomers increases. We apply our model to Aβ, IAPP, and apomyoglobin using binding energy estimates derived from bioinformatics. We find that these sequences are highly selective of the in-register state. This selectivity arises from the having strongly bound segments of varying length and separation.

Homing peptide-conjugated gold nanorods: the effect of amino acid sequence display on nanorod uptake and cellular proliferation.

Gold nanorods (GNRs) have attracted significant interest in the field of medicine as theranostic agents for both imaging and photothermal ablation of cancerous cells/tissues. Targeting theranostic GNRs specifically to cancer cells is necessary to enhance treatment efficacy and minimize undesired side effects. In this study, targeting functionalized GNR to EphA2 receptors that are overexpressed on prostate cancer cells was investigated as a strategy to achieve enhanced GNR uptake by cancer cells. In addition, the influence of targeting peptide orientation on functionalized GNR uptake by PC-3 cells was explored. GNRs of aspect ratio 4 were functionalized with an EphA2 homing peptide, YSA, using a layer-by-layer polypeptide wrapping approach. In parallel, an analogous population of YSA-modified GNRs, which display a reversed YSA peptide, with the N- and C- termini reversed, was also prepared. GC-MS analysis of the YSA-GNRs indicated that functionalized GNRs displayed approximately 3000 peptides/GNR. The functionalized GNRs remained well-dispersed in biological media for short times (<24 h). An increase in GNRs uptake of the YSA-GNRs by PC-3 cells, compared to the reversed YSA-GNRs, was observed under identical incubation conditions. Lastly, the effect of the YSA-GNRs binding to EphA2 receptors on prostate cancer cell proliferation was also studied. The YSA-functionalized GNRs inhibit PC-3 proliferation at a significantly lower effective dose than free YSA. Overall, the polypeptide LBL deposition technique provides a facile route to target nanoparticles to overexpressed cellular receptors, with the caveat that the specific orientation and display of the targeting moiety plays a critical role in the interaction between the nanoparticle and the cell.

Inhibition Strength of Short Peptides Derived from an ACE Inhibitory Peptide

A series of peptides, derived from an ACE inhibitory peptide (VTVNPYKWLP) found in our previous work, were synthesized. Their half maximal inhibition concentrations (IC(50)) for ACE inhibition have been determined. The effect of amino acid sequence on ACE inhibition was discussed on the basis of IC(50) of the synthetic peptides, and the following characteristics of the ACE inhibitory peptide have been clarified. First, the active portion of this peptide for ACE inhibition is KW. Second, the amino acid sequences near this dipeptide (KW) have a strong effect on the inhibitory activity. Especially, the proline residue in the C-terminal end strongly enhanced ACE inhibition. It should be noted that the IC(50) value of KWLP (5.5 μM) is the same as the ACE inhibitory peptide (VTVNPYKWLP) and that the IC(50) value of KW is 7.8 μM. The stability and absorption efficiency in vivo would be significantly improved by shortening the peptide length from 10 amino acids to four amino acids or two amino acids.

Molecular Simulations of Sequence-Specific Association of Transmembrane Proteins in Lipid Bilayers

Association of membrane proteins is central in material and information flow across the cellular membranes. Amino-acid sequence and the membrane environment are two critical factors controlling association, however, quantitative knowledge on such contributions is limited. In this work, we study the dimerization of helices in lipid bilayers using extensive parallel Monte Carlo simulations with recently developed algorithms [1]. The dimerization of Glycophorin A is examined employing a coarse-grain model that retains a level of amino-acid specificity, in three different phospholipid bilayers. Association is driven by a balance of protein-protein and lipid-induced interactions with the latter playing a major role at short separations. In all bilayers, sequence-specificity is evident by the formation of a clear interface between the helices that is modulated by the lipid environment. Extracted estimates of the dimerization affinity are in excellent agreement with experimental data [2]. Following a different approach, the effect of amino-acid sequence is studied using the four transmembrane domains of the epidermal growth factor receptor family in identical lipid environments. Detailed characterization of dimer formation and estimates of the free energy of association reveal that these helices present significant affinity to self-associate with certain dimers forming non-specific interfaces. We present results that support the role of lipid-mediated contributions to such effects with major implications on protein function. [1] Janosi L. and Doxastakis M., “Accelerating flat-histogram methods for potential of mean force calculations”, J. Chem. Phys., 131, 054105 (2009) [2] Janosi L., Prakash A. and Doxastakis M., “Lipid-Modulated sequence-specific association of Glycophorin A in membranes”, Biophys. J., 99, 284-292 (2010)

Conformational study on dipeptides containing phenylalanine: A DFT approach

DFT calculations has been done applying 6-31G ∗ basis set on a series of dipeptides where the N-terminus position is fixed with phenylalanine and the C-terminus is varied with eight different amino acids. Different geometrical parameters (bond angle, bond length, geometry around α-carbon atom) are thoroughly investigated to study the effect of amino acid sequence on dipeptide. Dihedral angle data analysis shows the deviation of amide plane from planarity, which is due to the combined effect of the steric hindrance of –R group and hydrogen bonding. The λ max value for phenylalanine has been calculated, which shows good agreement with the experimental value. A rigid potential energy scan is performed on phenylalanine by rotating –CH 2Ph, –COOH and –NH 2 groups separately to get some idea about the conformational stability.

Investigation on the structure of dipeptides: A DFT study

We present here the hybrid DFT level study of a series of dipeptide at 6-31g ∗ basis. A rigorous computation has been performed on a series of dipeptide (keeping valine as a fixed component in the N-terminus and the component in the C-terminus are varied with eight different combinations) to investigate various geometrical parameters to understand the effect of amino acid sequence in the dipeptides. Bond lengths, bond angles have been reported. Geometry around α-carbon atom is discussed. The analysis of dihedral angles shows deviations from planarity. This has been explained in terms of the cumulative effect of steric hindrance of –R group and H-bonding. To get an idea about its conformational stability, a rigid potential energy scan has also been performed on valine by rotating –COOH and –CH(CH 3) 2 groups separately.

Structure–permeation relationships for the non-invasive transdermal delivery of cationic peptides by iontophoresis

Transdermal iontophoresis enables the controlled, non-invasive administration of peptide therapeutics. The aims of this study were (i) to evaluate the effect of amino acid sequence and the spatial distribution of peptide physicochemical properties on electrotransport, and (ii) to develop a quantitative model to predict peptide transport rates. Experimental results showed that the distribution of molecular properties over the peptide surface significantly affected iontophoretic delivery: different arrangements of the same residues resulted in different transport behavior. Computational studies generated three-dimensional quantitative structure–permeation relationships (3D-QSPR) based on 3D descriptors. The model predicted that iontophoresis was favored by peptide hydrophilicity but hindered by voluminous, localized hydrophobicity. Molecular characteristics that favor electrotransport are the converse of those required for passive diffusion across biological membranes. The data represent the first analysis of peptide electrotransport in terms of the spatial distribution of molecular properties and provide insight into the ab initio prediction of transdermal iontophoretic peptide delivery.

Effect of amino acid sequence on transdermal iontophoretic peptide delivery

The objective of this study was to investigate the effect of amino acid sequence on the transdermal delivery of peptides by iontophoresis. Structurally related, cationic tripeptides based on the residues at positions (i) 6–8 in LHRH (Ac-X-Leu-Arg-NH 2) and (ii) 3–5 in octreotide (Ac-X- dTrp-Lys-NH 2) were studied. Iontophoretic transport experiments were conducted using porcine skin in vitro to investigate the dependence of flux on peptide concentration. Co-iontophoresis of acetaminophen enabled deconvolution of the contributions of electromigration (EM) and electroosmosis (EO) and the calculation of an electroosmotic inhibition factor (IF). A two-fold increase in donor peptide concentration increased iontophoretic flux for most peptides, and electroosmotic inhibition for dNal-containing tripeptides. The improvement in transport and the impact on the EM and EO components were peptide-specific. A reduction in the number of competing ions in the formulation significantly increased transport and, specifically, the EM contribution; it also increased IF of compounds with a propensity to interact with the membrane. No monotonic dependence of flux on either molecular weight or lipophilicity was observed. Iontophoretic peptide transport could not be rationalized in terms of either peptide molecular weight or computational 2D estimates of lipophilicity. Data suggest that a more complex three-dimensional approach is required to develop structure permeation relationships governing iontophoretic peptide delivery.

Effect of amino acid sequence on the hydrophobicity of small peptides

Hydrophobic adsorbents, which contain alkyl ligands of various chain lengths, were used to study the hydrophobic interactions between alkyl chains and small peptides. We investigated the adsorption of four similar peptides: GWWG, GWGW, WGWG, WGGW. All of them contained two glycines (G) and two tryptophans (W) but the amino acids were arranged in different orders. The capacity factors of peptides between 10 and 35 °C were measured and then the thermodynamic parameters, such as enthalpy and entropy changes (Δ H° and Δ S°) of adsorption, were estimated. It implied that enthalpy was the major driving force in all the adsorption processes. Furthermore, Δ H° and Δ S° became more negative as the alkyl chain length was increased. It revealed that the van der Waal's interaction had greater influence on the adsorption as the chain length increased. It was also found that, the contribution of each amino acid to peptide's hydrophobicity was affected by the position of the amino acid. When a hydrophobic amino acid was positioned in the middle of a peptide chain, it exhibited the highest hydrophobicity. Interestingly, tryptophan at the carboxyl end was found more hydrophobic than it at the amino end of the peptide.

Linear analysis of carbon-13 chemical shift differences and its application to the detection and correction of errors in referencing and spin system identifications

Statistical analysis reveals that the set of differences between the secondary shifts of the alpha- and beta-carbons for residues i of a protein (Deltadelta13C(alpha)i - Deltadelta13C(beta)i) provides the means to detect and correct referencing errors for 1H and 13C nuclei within a given dataset. In a correctly referenced protein dataset, linear regression plots of Deltadelta13C(alpha)i, Deltadelta13C(beta)i, or Deltadelta1H(alpha)i vs. (Deltadelta13C(alpha)i - Deltadelta13C(beta)i) pass through the origin from two directions, the helix-to-coil and strand-to-coil directions. Thus, linear analysis of chemical shifts (LACS) can be used to detect referencing errors and to recalibrate the 1H and 13C chemical shift scales if needed. The analysis requires only that the signals be identified with distinct residue types (intra-residue spin systems). LACS allows errors in calibration to be detected and corrected in advance of sequence-specific assignments and secondary structure determinations. Signals that do not fit the linear model (outliers) deserve scrutiny since they could represent errors in identifying signals with a particular residue, or interesting features such as a cis-peptide bond. LACS provides the basis for the automated detection of such features and for testing reassignment hypotheses. Early detection and correction of errors in referencing and spin system identifications can improve the speed and accuracy of chemical shift assignments and secondary structure determinations. We have used LACS to create a database of offset-corrected chemical shifts corresponding to nearly 1800 BMRB entries: 300 with and 1500 without corresponding three-dimensional (3D) structures. This database can serve as a resource for future analysis of the effects of amino acid sequence and protein secondary and tertiary structure on NMR chemical shifts.

Effect of amino acid sequence and pH on nanofiber formation of self-assembling peptides EAK16-II and EAK16-IV.

Atomic force microscopy (AFM) and axisymmetric drop shape analysis-profile (ASDA-P) were used to investigate the mechanism of self-assembly of peptides. The peptides chosen consisted of 16 alternating hydrophobic and hydrophilic amino acids, where the hydrophilic residues possess alternating negative and positive charges. Two types of peptides, AEAEAKAKAEAEAKAK (EAK16-II) and AEAEAEAEAKAKAKAK (EAK16-IV), were investigated in terms of nanostructure formation through self-assembly. The experimental results, which focused on the effects of the amino acid sequence and pH, show that the nanostructures formed by the peptides are dependent on the amino acid sequence and the pH of the solution. For pH conditions around neutrality, one of the peptides used in this study, EAK16-IV, forms globular assemblies and has lower surface tension at air-water interfaces than another peptide, EAK16-II, which forms fibrillar assemblies at the same pH. When the pH is lowered below 6.5 or raised above 7.5, there is a transition from globular to fibrillar structures for EAK16-IV, but EAK16-II does not show any structural transition. Surface tension measurements using ADSA-P showed different surface activities of peptides at air-water interfaces. EAK16-II does not show a significant difference in surface tension for the pH range between 4 and 9. However, EAK16-IV shows a noticeable decrease in surface tension at pH around neutrality, indicating that the formation of globular assemblies is related to the molecular hydrophobicity.

Monte Carlo simulation of proteins through a random walk in energy space

A Monte Carlo algorithm that performs a random walk in energy space has been used to study random coil–helix and random coil–beta sheet transitions in model proteins. This method permits estimation of the density of states of a protein via a random walk on the energy surface, thereby allowing the system to escape from local free-energy minima with relative ease. A cubic lattice model and a knowledge based force field are employed for these simulations. It is shown that, for a given amino acid sequence, the method is able to fold long polypeptides reproducibly. Its results compare favorably with those of annealing and parallel tempering simulations, which have been used before in the same context. This method is used to examine the effect of amino acid sequence and chain length on the folding of several designer polypeptides.

Oligopeptides as an Oral Delivery System II. Effect of Amino Acid Sequences on Aggregation Behavior

The effect of amino acid sequences on the aggregation and the subsequent sphere formation behavior of tetrapeptides, pEE(a)X(y) involving glutamic acid (E), phenylalanine (F), tyrosine (Y), alanine (A), and leucine (L) were investigated by light scattering and light microscopy. At 0.1 M pEE(a)Y(y)Y and pEE(a)F(y)F showed similar pH and concentration-dependent aggregation behaviors. These tetrapeptides also showed similar aggregation profiles in the presence of macromolecular drugs. Only pEE(a)F(y)F produced discrete spheres in the presence of protein drugs. Neither pEE(a)A(y)A nor pEE(a)L(y)L gave aggregates even at 0.1 M at pH 2. Consequently, the amino acids terminal positions are important in the aggregation characteristics of the tetrapeptides.