Water-soluble Polypeptides Research Articles

Using Enhanced Sampling Molecular Dynamics Techniques to Probe the Thermodynamics of pHLIP Peptide Insertion into a Model Lipid Bilayer

The molecular mechanism of spontaneous polypeptide folding and insertion into a membrane, as well as its exit and unfolding, is of interest from several perspectives, including the action of antimicrobial peptides, the folding and degradation of membrane proteins, and medical applications of pH-triggered insertion peptides. A notable example is pH (Low) Insertion Peptide (pHLIP), which is a water-soluble polypeptide derived from helix C of bacteriorhodopsin that has the ability to insert into a membrane at acidic pH to form a stable transmembrane α-helix. The insertion process takes place in three stages: pHLIP is unstructured and soluble in water at neutral pH(state I), unstructured and bound to the surface of a membrane at neutral pH(state II), and inserted into the membrane as an α-helix at low pH(state III). Our hypothesis is that, there is a connection between the free energy change of binding /insertion and pKa of insertion. To test this hypothesis, we used enhanced sampling molecular dynamics (MD) techniques (steered MD and umbrella sampling) to investigate the molecular interactions of a C-terminal-truncated pHLIP variant, pHLIP-1, with a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. The goal is to characterize the free energy of binding from state I to state II and the free energy of insertion from state II to state III (α-helical peptide insertion) and to identify key structural aspects that govern these processes. Our results agree well with the previous experiments, providing the first steps in establishing a direct relationship between the thermodynamics and pKa of pHLIP peptide insertion.

Ionic α-helical polypeptides toward nonviral gene delivery.

The advent of polymeric materials has significantly promoted the development and rapid growth of various technologies in biomedical applications, such as tissue engineering and controlled drug and gene delivery. Water-soluble polypeptides bearing functional side chains and adopting stable secondary structures are a new class of functional polymeric materials of potentially broad applications in medicine and biotechnology. In this article, we summarize our recent effort on the design and synthesis of the water-soluble α-helical ionic polypeptides originally developed in our laboratory and highlight their applications in cell membrane penetration and nonviral gene/small interfering RNA (siRNA) delivery.

Gold-decorated graphene nanosheets composed of a biocompatible non-charged water-soluble polypeptide

Nanocomposite materials derived from carbon/polymer hybrids and metallic nanoparticles are of great interest due to the wide spectrum of potential applications. In this work, the biocompatible, non-charged and independent of pH, water soluble polypeptide, poly(L-proline) (PLP), was covalently grafted on exfoliated graphene oxide (GO) by a ‘grafting to’ approach, through the ring opening reaction of the surface epoxide species, enabling thus the simultaneous reduction and stabilization of as-synthesized gold nanoparticles in water in one-step, without any toxic by-product formation. The PLP-mediated GO nanocomposites serve as a new scaffold for metallic particles supported on a carbon matrix through the polypeptide mediation, and could give rise to multiple applications, in catalysis and cancer therapies.

Processing of β-Glucosidase–Silk Fibroin Nanoparticle Bioconjugates and Their Characteristics

Silk fibroin derived from Bombyx mori is a biomacromolecular protein with excellent biocompatibility. The aim of this work was to develop silk fibroin nanoparticles (SFNs) derived from the fibrous protein, which is a novel vector for enzyme modification in food processing. Silk fibroin was dissolved in highly concentrated CaCl2 and subjected to lengthy desalting in water. The resulting liquid silk, which contained water-soluble polypeptides with molecular mass ranging from 10 to 200 kDa, and β-glucosidase were added rapidly into acetone. The β-glucosidase molecules were embedded into silk fibroin nanoparticles, forming β-glucosidase-silk fibroin nanoparticles (βG-SFNs) with a diameter of 50-150 nm. The enzyme activity of the βG-SFN bioconjugates was determined with p-nitrophenyl-β-D-glucoside as the substrate, and the optimum conditions for the preparation of βG-SFNs were investigated. The enzyme activity recovery of βG-SFNs was 59.2 % compared to the free enzyme (specific activity was 1 U mg(-1)). The kinetic parameters of the βG-SFNs and the free β-glucosidase were the same. The βG-SFNs had good operational stability and could be used repeatedly. These results confirmed that silk protein nanoparticles were good carriers as bioconjugates for the modification of enzymes with potential value for research and development. The method used in this study has potential applications in food processing and the production of flavour agents.

Water-Soluble Polypeptides with Elongated, Charged Side Chains Adopt Ultra-Stable Helical Conformations.

Water-soluble polypeptides adopting α-helical conformations with unprecedented high helicities were obtained by elongating the charge-containing side chains of the constituent amino acids to allow the terminal charges to be situated distally from the peptide backbone. Poly(γ-(4-aminoethylthiopropoxyl)-benzyl-(L)-glutamate) (PAOBLG-AET) with a charge-peptide backbone distance of 17 σ-bonds exhibited a remarkably high helical content (81%) at a degree of polymerization as low as 10. The helical conformations of these short polypeptides were very stable against various harsh, protein-denaturing conditions, such as extreme pH, high temperature, and high salt or urea concentrations.

Ionic polypeptides with unusual helical stability

Water-soluble peptides that adopt stable helical conformations are attractive motifs because of their importance in basic science and their broad utility in medicine and biotechnology. Incorporating charged amino-acid residues to improve peptide solubility, however, usually leads to reduced helical stability because of increased side-chain charge repulsion, reduced side-chain hydrophobicity and the disruption of intramolecular hydrogen bonding. Here, we show that water-soluble, ultra-stable α-helical polypeptides can be produced by elongating charge-containing amino-acid side chains to position the charges distally from the polypeptide backbone. The strategy has been successfully applied to the design and synthesis of water-soluble polypeptides bearing long, charged side chains and various functional moieties that possess unusual helical stability against changing environmental conditions, including changes in the pH and temperature and the presence of denaturing reagents.

Identification of Spoilage Marker Metabolites in Irish Chicken Breast Muscle Using HPLC, GC–MS Coupled with SPME and Traditional Chemical Techniques

The aim of this investigation was to determine the metabolites of spoilage present on the surface of Irish chicken breast muscle in order to identify biomarkers of microbial spoilage and to verify the results of a previous study which suggested that the increase of free amino acids is the main spectral influence factor leading to the near and middle infrared detection of microbial spoilage. Irish-reared chicken breast muscle samples were individually packed and stored at 4 °C for 8 days under aerobic conditions. Microbiological analysis revealed that Pseudomonas spp. and Brochothrix thermosphacta were the predominant organisms (total viable counts (TVC), 4.24, 6.37 and 8.6 colony forming unity (CFU) g−1 for days 0, 4 and 8, respectively, Pseudomonas 3.2, 5.1 and finally, on day 8 7.4 log CFU g−1). Glucose and L-lactate concentrations decreased but the concentration of water-soluble polypeptides and amino acids increased over storage time. HPLC analysis of free amino acids revealed an increase of the total concentration but the composition of the profiles did not change over time. Headspace analysis detected the following volatile compounds: ethanol, acetone, ethyl acetate, methyl benzoate, heptane, C15, C12, methyl ethyl ketone, carbon disulphide, dimethyl sulphide, hexanal, and toluene. Of interest is the fact that detection of sulphides and an increase of ethanol, acetone, and ethyl acetate concentrations occurred from day 4 to 8. The increase in free amino acids throughout storage and the production of volatile compounds after day 4 require further investigation but are selected as potential biomarkers of microbial spoilage as they could be analytically detected before the accepted levels of sensory spoilage detection.

Biosynthesis of β-Glucosidase-Silk Fibroin Nanoparticles Conjugates and Enzymatic Characteristics

Silk fibroin derived from Bombyx mori is a biomacromolecular protein with outstanding biocompatibility. When it was dissolved in highly concentrated CaCl2 solution and then the mixture of the protein and salt was subjected to desalting treatments for long time in flowing water, the resulting liquid silk was water-soluble polypeptides with different molecular masses, ranging from 10 to 200 kDa. When the liquid silk were introduced rapidly into acetone, silk protein nanoparticles (SFNs) with a range of 40~120 nm in diameter could be obtained. The crystalline silk nanoparticles could be conjugated with β-glucosidase with cross-linking of reagent glutaraldehyde. In this work, the activity of β-glucosidase-silk fibroin nanoparticles (βG-SFNs) bioconjugates was determined by p-nitrophenyl-β-D-glucoside (p-NPG) as a substrate and the optimal conditions for the biosynthesis of βG-SFNs bioconjugates were investigated. βG-SFN constructs obtained by 5h of covalent cross-linking time at the experimental conditions of 0.25% cross-linking reagent, 37 °C and the proportion of β-glucosidase and SFNs (75 : 100, U/mg) showed 46% of recoveries. Results showed that kinetic parameters of βG-SFNs were the same as for the free β-glucosidase. The optimal pH was 5.0 and the optimal temperature was 60°C. When β-glucosidase was coupled covalently with silk nanoparticles, the thermal stability of βG-SFNs was slightly enhanced as compared with free β-glucosidase. The apparent Km of βG-SFNs (1.41×10-3 mol•L-1) was near five-fold less than that of the free enzyme (7.26×10−3 mol•L-1), this fully shows that after the free enzyme conjugated with silk fibroin nanoparticles, the enzyme affinity with substrate increased. These results fully demonstrated the silk protein nanoparticles were good carriers as bioconjugation or modification of enzymes. Moreover, they had potential values for research and development in food processing and flavor processing agents.

Functionalization of magnetic nanoparticles with peptide dendrimers

Surface functionalization of magnetic nanoparticles (MNPs) has been an exciting area of interest for researchers in biomedicine. In this paper, we introduce a new family of peptide dendritic ligands for functionalizing MNPs of superior quality. L-Lysine- and L-glutamic acid-based dendritic ligands with dopamine located at the focal points were fully designed and synthesized before the functionalization. Then ligands of different dendritic generations (G1 to G3) were immobilized on the surface of oleic-acid-coated hydrophobic MNPsvia ligand-exchange method to realize phase transfer. The two series of modified MNPs were systematically studied viaFTIR, TGA, XRD, TEM, DLS, VSM and zeta potential measurements. The modified MNPs exhibited an adjustable number of terminal functional groups and superior stability in aqueous solutions in a broad pH range. The surface existence of water-soluble polypeptide ligands promoted monodispersity of the particles and led to an increased hydrodynamic diameter under 30 nm from G1 to G3. After the ligand exchange process, the superparamagnetic behavior was successfully retained. The two series of modified MNPs exhibited approximate magnetization in the same generation, while the saturation magnetization of the MNPs decreased with increasing surface dendritic generation. MNPs functionalized with G1L-glutamic acid dendritic ligands had the highest saturation magnetization (55 emu g−1), which was larger than for the initial MNPs. This novel functionalization strategy provides a potential platform for designing and preparing highly stable ultrafine MNPs with high magnetization for biomedicinal applications.

Minimalist design of water-soluble cross-β architecture

Demonstrated successes of protein design and engineering suggest significant potential to produce diverse protein architectures and assemblies beyond those found in nature. Here, we describe a new class of synthetic protein architecture through the successful design and atomic structures of water-soluble cross-beta proteins. The cross-beta motif is formed from the lamination of successive beta-sheet layers, and it is abundantly observed in the core of insoluble amyloid fibrils associated with protein-misfolding diseases. Despite its prominence, cross-beta has been designed only in the context of insoluble aggregates of peptides or proteins. Cross-beta's recalcitrance to protein engineering and conspicuous absence among the known atomic structures of natural proteins thus makes it a challenging target for design in a water-soluble form. Through comparative analysis of the cross-beta structures of fibril-forming peptides, we identified rows of hydrophobic residues ("ladders") running across beta-strands of each beta-sheet layer as a minimal component of the cross-beta motif. Grafting a single ladder of hydrophobic residues designed from the Alzheimer's amyloid-beta peptide onto a large beta-sheet protein formed a dimeric protein with a cross-beta architecture that remained water-soluble, as revealed by solution analysis and x-ray crystal structures. These results demonstrate that the cross-beta motif is a stable architecture in water-soluble polypeptides and can be readily designed. Our results provide a new route for accessing the cross-beta structure and expanding the scope of protein design.

Degradation of Poly(e-caprolactone) Fibers by Lipoprotein LipaseModified with a Water-soluble Polypeptide

Degradation of Poly(e-caprolactone) Fibers by Lipoprotein LipaseModified with a Water-soluble Polypeptide

Activity and Stability of Lipoprotein Lipase Modified with a Water-soluble Polypeptide

適切な水溶性高分子担体に酵素を共有結合で修飾して得られる修飾酵素が，高い酵素活性を維持し酵素安定性が改善される最適反応条件と技術の確立を目指すため，本研究では高分子担体としてコポリ(L-グルタミン酸:/L-アラニン)(EA)を選択し，酵素には脂質分解酵素であるリポ蛋白リパーゼ(LPL)を選んだ。種々の条件で調製した修飾LPLの酵素活性および安定性を実験的に評価した。本研究における修飾LPLは未修飾の場合に比べて相対活性を大きく低下させることなく，熱安定性，保存安定性を顕著に改善された。さらに，実用性の点からも，取扱いの簡便化，反復使用耐久性が得られることが示唆された。一方，水溶性（親水性）アミノ酸を主成分とし，それに適当な疎水性アミノ酸を共重合させることにより酵素-基質反応性を改善する可能性が認められた。

Biosynthesis of insulin-silk fibroin nanoparticles conjugates and in vitro evaluation of a drug delivery system

Silk fibroin derived from Bombyx mori is a biomacromolecular protein with outstanding biocompatibility. When it was dissolved in highly concentrated CaCl2 solution and then the mixture of the protein and salt was subjected to desalting treatments for long time in flowing water, the resulting liquid silk was water-soluble polypeptides with different molecular masses, ranging from 8 to 70 kDa. When the liquid silk was introduced rapidly into acetone, silk protein nanoparticles with a range of 40–120 nm in diameter could be obtained. The crystalline silk nanoparticles could be conjugated covalently with insulin alone with cross-linking reagent glutaraldehyde. In vitro properties of the insulin-silk fibroin nanoparticles (Ins-SFN) bioconjugates were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The optimal conditions for the biosynthesis of Ins-SFN bioconjugates were investigated. The Ins-SFN constructs obtained by 8 h of covalent cross-linking with 0.7% cross-linking reagent and the proportion of insulin and SFN being 30 IU: 15 mg showed much higher recoveries (90–115%). When insulin was coupled covalently with silk nanoparticles, the resistance of the modified insulin to trypsin digestion and in vitro stability in human serum were greatly enhanced as compared with insulin alone. The results in human serum indicated that the half-life in vitro of the biosynthesized Ins-SFN derivatives was about 2.5 times more than that of native insulin. Therefore, the silk protein nanoparticles have the potential values for being studied and developed as a new bioconjugate for enzyme/polypeptide drug delivery system.

Monoliths of Aligned Silica-Polypeptide Hexagonal Platelets

Water soluble alpha-helical polypeptides were used to prepare silica coated hexagonal single crystal platelets in concentrated solutions. To our knowledge, there is no other instance where polymer single crystals, typically formed under high dilution, can be grown in a bulk material. This unprecedented self-assembly process relies on complex cooperative interactions where silica condensation mediates the growth of polypeptide crystals, which in turn template silica overgrowth. The helices were also used to align samples giving monoliths composed of highly oriented layers of platelets. Overall, this procedure allows preparation of composites with good structural order and complexity via a simple biomimetic process.

Assembly of the Bi-component Leukocidin Pore Examined by Truncation Mutagenesis

Staphylococcal leukocidin (Luk) and alpha-hemolysin (alphaHL) are members of the same family of beta barrel pore-forming toxins (betaPFTs). Although the alphaHL pore is a homoheptamer, the Luk pore is formed by the co-assembly of four copies each of the two distantly related polypeptides, LukF and LukS, to form an octamer. Here, we examine N- and C-terminal truncation mutants of LukF and LukS. LukF subunits missing up to nineteen N-terminal amino acids are capable of producing stable, functional hetero-oligomers with WT LukS. LukS subunits missing up to fourteen N-terminal amino acids perform similarly in combination with WT LukF. Further, the simultaneous truncation of both LukF and LukS is tolerated. Both Luk subunits are vulnerable to short deletions at the C terminus. Interestingly, the N terminus of the LukS polypeptide becomes resistant to proteolytic digestion in the fully assembled Luk pore while the N terminus of LukF remains in an exposed conformation. The results from this work and related experiments on alphaHL suggest that, although the N termini of betaPFTs may undergo reorganization during assembly, they are dispensable for the formation of functional pores.

Subtle differences in structural transitions between poly-l- and poly-d-amino acids of equal length in water

Mirror-image asymmetric molecules, i.e., chiral isomers or enantiomers, are classically considered as chemically identical. Recent studies, however, have indicated that parity violation by the nuclear weak force induces a tiny energy difference between chiral isomers. Upon combination with a massive amplification process, expansion of this difference to a detectable macroscopic level may be achieved. Yet, experimental tests of this possibility, where one enantiomer is compared to the other in solution, are hampered by the possible presence of undetectable impurities. In this study we have overcome this problem by comparing structural and dynamic features of synthetic D- and L-polyglutamic acid and polylysine molecules each of 24 identical residues. In these water-soluble polypeptides helix formation is an intramolecular autocatalytic process amplified by each turn, which is actually unaffected by low level of putative impurities in the solvent. The helix and random coil configurations and their transition were determined in this study by circular dichroism (CD) and isothermal titration calorimetry (ITC) in water and deuterium oxide. Distinct differences in structure and transition energies between the enantiomeric polypeptides were detected by both CD and ITC when dissolved in water. Intriguingly, these differences were by and large abolished in deuterium oxide. Our findings suggest that deviation from physical invariance between the D- and L-polyamino acids is induced in part by different hydration in water which is eliminated in deuterium oxide. Based on the recent findings by Tikhonov and Volkov (V. I. Tikhonov and A. A. Volkov, Science 2002, 296, 2363) we suggest that ortho-H(2)O, which constitutes 75% of bulk H(2)O, has a preferential affinity to L-enantiomers. Differential hydration of enantiomers may have played a role in the selection of L-amino acids by early forms of life.

Effect of helix-coil transition on association behavior of both-ends hydrophobically-modified water-soluble polypeptide

Dodecyl and dodecanoyl groups (C12) are attached at the chain ends of poly[ N 5-(2-hydroxyethyl) l-glutamine] (PHEG), and association behavior of this both-ends hydrophobically modified water soluble polypeptide (C12–PHEG–C12) has been investigated by means of light scattering measurements. Water/ethylene glycol (EG) mixed solvents were used as selective solvent for PHEG block, and PHEG changed its structure from random-coil state to α-helix with increasing EG content in the mixed solvent ( W EG). When W EG is less than 0.5, flower-like micelle with C12 associated core and PHEG corona in loop conformation was suggested to be formed. Increase of W EG from 0.5 to 0.6 induced drastic increase of association number and size of the associate, in which many C12 associated cores may be connected by PHEG in bridge conformation. This structure change of associate is considered to be driven by the increase of helix content of PHEG with W EG, which enhances the possibility to form bridge conformation because of its rigidity. Solution preparation method, i.e. order of addition of solvent, was found to influence the structure of associate, although its effect on the helix content of PHEG was negligible.

Thymosin-loaded enteric microspheres for oral administration: Preparation and in vitro release studies

Thymosin, a water-soluble polypeptide compound, was encapsulated within enteric microspheres of acrylic acid resin II by modified oil in oil (o/o) emulsion solvent evaporation method. The mixture emulsifier composed of lecithin and Span 80 was critical to the formation of sphere-shaped thymosin microparticles. Optimizing process parameters, such as the volume ratio of organic solvent to water, initial drug feed and polymer concentration, resulted in high drug encapsulation efficiency of 89.7% (6% polymer concentration and 0.5% initial drug feed). In vitro release studies suggested that thymosin release from microspheres exhibited pH dependent profiles. For formulation with 6% polymer concentration and 0.5% initial drug feed, 68.7% thymosin was released within 4 h in pH 6.8 PBS buffer, while only 6.5% was observed in acid medium.

The 23-kDa polypeptide from Common Frog lens: isolation, characteristics and cellular localization

The major water-soluble polypeptide with molecular weight of approximately 23 kDa (the 23-kDa polypeptide) was identified in the lens of common frog Rana temporaria L. According to the gel filtration data, the peptide is a part of an oligomeric protein with molecular weight of more than 300 kDa (alpha-crystallin fraction). A highly pure fraction of the 23-kDa polypeptide was isolated by two-step ion-exchange chromatography and SDS electrophoresis and the specific antibodies were obtained. Immunohistochemistry showed the presence of the 23-kDa polypeptide in the cytoplasm of lens epithelial cells (including its central region) and in the zones neighbouring the plasma membranes in cortical fibers. The 23-kDa polypeptide was not found in the lens central zone (nucleus). It was also present in the retina (in the cells of inner nuclear layers), but not in the other tissues and organs of adult frog. Immunochemical analysis showed that the 23-kDa polypeptide was different from all known crystallins of frogs and other animals (bull, mouse, rat, and chicken). The nature of the 23-kDa polypeptide and the relation of its expression with lens cell differentiation are discussed.

Methylated Mono- and Diethyleneglycol Functionalized Polylysines: Nonionic, α-Helical, Water-Soluble Polypeptides

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTMethylated Mono- and Diethyleneglycol Functionalized Polylysines: Nonionic, α-Helical, Water-Soluble PolypeptidesMiaoer Yu, Andrew P. Nowak, Timothy J. Deming, and Darrin J. PochanView Author Information Departments of Materials and Chemistry University of California, Santa Barbara Santa Barbara, California 93106 Department of Materials Science and Engineering University of Delaware, Newark, Delaware 19716 Cite this: J. Am. Chem. Soc. 1999, 121, 51, 12210–12211Publication Date (Web):December 11, 1999Publication History Received11 October 1999Published online11 December 1999Published inissue 1 December 1999https://doi.org/10.1021/ja993637vCopyright © 1999 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views2161Altmetric-Citations116LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (91 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Monomers,Peptides and proteins,Polymers,Solubility,Solution chemistry Get e-Alerts