Dynamics and orientation of transmembrane peptide from bacteriorhodopsin incorporated into lipid bilayer as revealed by solid state (31)P and (13)C NMR spectroscopy.

13C and (31)P NMR spectra of a transmembrane peptide, [1-(13)C]Ala(14)-labeled A(6-34), of bacteriorhodopsin incorporated into dimyristoylphosphatidylcholine (DMPC) bilayer were recorded to clarify its dynamics and orientation in the lipid bilayer. This peptide is shown to take an alpha-helical form both in liquid crystalline and gel phases, as viewed from the conformation dependent (13)C chemical shifts. In addition, this peptide undergoes rapid rigid-body rotation about the helical axis at ambient temperature as viewed from the axially symmetric (13)C chemical shift anisotropy, whereas this symmetric anisotropy is changed to an asymmetric pattern at temperatures below 10 degrees C. We further incorporated the peptide into the spontaneously aligned DMPC bilayer to applied magnetic field, induced by dynorphin (dynorphin:DMPC =1:10), a heptadeca-opioid peptide with very high affinity to opioid receptor, in order to gain insight into its orientation in the bilayer. This magnetically aligned system turned out to be persistent even at 0 degrees C as viewed from (31)P NMR spectra of the lipid bilayer, after this peptide was incorporated into this system [A(6-34): dynorphin: DMPC = 4:10:100]. It was found from the (13)C NMR spectra of [1-(13)C]Ala(14) A(6-34) that the helical axis of A(6-34) is oriented parallel to the bilayer normal irrespective of the presence or absence of reorientation motion about the helical axis at a temperature above the lowered gel to liquid crystalline phase transition.     

Cosolvent,ions, and temperature effects on the structural properties of cecropin A‐Magainin 2 hybrid peptide in solutions

Antimicrobial peptides are promising alternative to traditional antibiotics and antitumor drugs for the battle against new antibiotic resistant bacteria strains and cancer maladies. The study of their structural and dynamics properties at physiological conditions can help to understand their stability, delivery mechanisms, and activity in the human body. In this article, we have used molecular dynamics simulations to study the effects of solvent environment, temperature, ions concentration, and peptide concentration on the structural properties of the antimicrobial hybrid peptide Cecropin A–Magainin 2. In TFE/water mixtures, the structure of the peptide retained α‐helix contents and an average hinge angle in close agreement with the experimental NMR and CD measurements reported in literature. Compared to the TFE/water mixture, the peptide simulated at the same ionic concentration lost most of its α‐helix structure. The increase of peptide concentration at both 300 and 310 K resulted in the peptide aggregation. The peptides in the complex retained the initial N‐ter α‐helix segment during all the simulation. The α‐helix stabilization is further enhanced in the high salt concentration simulations. The peptide aggregation was not observed in TFE/water mixture simulations and, the peptide aggregate, obtained from the water simulation, simulated in the same conditions did dissolve within few tens of nanoseconds. The results of this study provide insights at molecular level on the structural and dynamics properties of the CA‐MA peptide at physiological and membrane mimic conditions that can help to better understand its delivery and interaction with biological interfaces. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 1–14, 2015.     

The structure-antiaggregative activity relationship in a series of Arg-Gly-Asp analogues

A series of analogues of Arg-Gly-Asp tripeptide, the common structural element of most of the integrin receptor ligands, possessing different conformational features for the interaction with platelet receptors, were synthesized by the methods of conventional peptide chemistry for use in the search for antithrombotic agents. The distance between the guanidine group of Arg and the β-carboxyl group of Asp was shown to affect the antiaggregative activity. A potent inhibitor of platelet aggregation, tripeptide Arg-βAla-Asp, with IC₅₀ 10.6 = 10.6 μM (ADP, 1.5 μM) was revealed.     

Hydrophobic tail length plays a pivotal role in amyloid beta (25–35) fibril–surfactant interactions

The amyloid β‐peptide fragment comprising residues 25–35 (Aβ_25‐35) is known to be the most toxic fragment of the full length Aβ peptide which undergoes fibrillation very rapidly. In the present work, we have investigated the effects of the micellar environment (cationic, anionic, and nonionic) on preformed Aβ_25‐35 fibrils. The amyloid fibrils have been prepared and characterized by several biophysical and microscopic techniques. Effects of cationic dodecyl trimethyl ammonium bromide (DTAB), cetyl trimethylammonium bromide (CTAB), anionic sodium dodecyl sulfate (SDS), and nonionic polyoxyethyleneoctyl phenyl ether (Triton X‐100 or TX) on fibrils have been studied by Thioflavin T fluorescence, UV–vis spectroscopy based turbidity assay and microscopic analyses. Interestingly, DTAB and SDS micelles were observed to disintegrate prepared fibrils to some extent irrespective of their charges. CTAB micelles were found to break down the fibrillar assembly to a greater extent. On the other hand, the nonionic surfactant TX was found to trigger the fibrillation process. The presence of a longer hydrophobic tail in case of CTAB is assumed to be a reason for its higher fibril disaggregating efficacy, the premise of their formation being largely attributed to hydrophobic interactions. Proteins 2016; 84:1213–1223. © 2016 Wiley Periodicals, Inc.     

Spectroscopic investigation on gel-forming beta-sheet assemblage of peptide derivatives

The conformational studies of peptide derivatives A and B in a gel state were studied by using circular dichroism (CD), Fourier transformed infrared (FTIR), and fluorescence spectroscopic techniques. Birefringence and electron microscopic studies were carried out to characterize the morphological aspects of the fibrils in the gel. The FTIR spectra of the peptides show the absence of free NH in the gel state, implying that the intermolecular hydrogen-bond formation is the driving force for the aggregation. The CD spectrum of the peptide gels shows the presence of antiparallel and parallel beta-sheet conformation for peptide derivatives A and B, respectively. Electron microscopic studies (EM) of the peptide derivatives A and B reveal that peptide A formed rigid, rod-like structures without cross-linking and peptide B formed loose fibrils organized into highly noncovalently cross-linked mesh-like structural aggregates. Peptide A was much more soluble in alcoholic solvents than peptide B, and no birefringence was observed with Congo red (CR) staining in the temperature range of 0-80 degrees C. The spectroscopic studies indicate that peptide B consists of domains having a significant amount of beta-sheet structure and exhibiting golden yellow birefringence between 53 and 56 degrees C when stained with Congo red. On the other hand, peptide A gives no evidence of birefringence under polarized light. Fluorescence probe binding studies with pyrene in gel state with peptides A and B indicates the polarity in the interior of the aggregates. The data presented in the present work indicate that peptide B forms fibrils, which is similar to amyloid aggregates that are present in biological systems.     

Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's Aβ peptide

The oligomerization of Aβ peptide into amyloid fibrils is a hallmark of Alzheimer’s disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aβ and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aβ peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aβ fibrillation. The three histidine residues at positions 6, 13 and 14 of Aβ(1–40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.     

抗凝活性肽RGD226基因构建、表达、产物纯化及活性分析

通过PCR法 ,将来源于蛇毒蛋白质eristostatin中的一段含有RGD(Arg Gly Asp)序列的十三肽(SRVARGDWNDDYS)的基因 ,以一个无胰岛素活性但保留天然免疫原性的胰岛素原突变体(PJG4 0 1)基因为模板 ,置换其B2 8和A1位之间的连接肽基因 ,构建成了能展示RGD功能序列的人源化分子 (RGD2 2 6 )的基因 .通过该基因在大肠杆菌中的表达、产物分离纯化 ,得到高纯度RGD2 2 6 .该RGD肽对由ADP诱导的体外人血小板聚集的半抑制浓度IC50 为 2 2 3μmol L .其胰岛素免疫活性是PJG4 0 1的 15 1% ,提示其在一定程度上保留了胰岛素原的免疫原性 .其受体结合活性不到PJG4 0 1的 0 1% ,说明其胰岛素的生物活性基本丧失 .动物实验证实 ,RGD2 2 6在延长小鼠出血时间上具有较明显的作用     

一种用于穿透多肽筛选的随机文库的构建及筛选

以增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP)为示踪物,在pET-14b载体上构建编码12个氨基酸的随机多肽表达文库.建立一种简便、经济、有效的文库筛选方法,从所构建的文库中筛选出细胞穿透多肽（cell-penetrating peptide, CPP）. 采用点突变技术,首先在pET-14b载体多克隆位点NdeⅠ和XhoⅠ之间加入4个限制性内切酶位点,随后在BamH Ⅰ位点后加入三联终止密码子,接着再利用亚克隆的方法在Kpn Ⅰ 和XhoⅠ之间插入EGFP,形成一个新的用于原核表达示踪蛋白的载体pET-14bMCStop/EGFP.最后再利用点突变技术在上述构建的示踪载体的多克隆位点XhoⅠ和BamH Ⅰ之间插入36个随机碱基序列.以His-Tat-EGFP作为工具建立有效的筛选方法,利用这种方法对文库进行筛选. 酶切和测序表明,示踪载体的构建是正确的,且在大肠杆菌中可有效地表达出His标记的EGFP.在示踪载体的基础上构建的随机多肽文库至少包含了10⁵个独立克隆,其中90%以上的克隆插入的随机片段都是36个碱基.建立的筛选方法是可行的,并用此方法进行了初步的筛选.     

Effects of interfaces on aggregates of peptides derived from pancreatic islet amyloid polypeptide

Recent experimental studies indicate that oligomeric complexes of misfolded proteins and peptides are the primary agents of cytotoxicity in amyloid-related diseases. Given the prevalence of mixed-polarity interfaces in physiological environments, an understanding of the mechanisms of interactions between amorphous (pre-fibrillar) aggregates and surfaces is important for completing our knowledge of the behaviour of peptide aggregation phenomena. We have employed fully solvated molecular dynamics simulations to study the morphology, interactions and peptide conformations of disordered aggregates of the amyloidogenic NFGAIL (derived from human islet amyloid polypeptide) and non-amyloidogenic AGAIL peptides upon adsorption to vapour–water, decane–water, bilayer and solid–water interfaces. All of the interfaces studied promote elongation and surface-spreading of both peptide aggregates, with the liquid–liquid interface being particularly efficient at altering the gross morphology of disordered aggregates. NFGAIL aggregates are more effective at disrupting lipid bilayers compared to AGAIL. Additionally, the interfaces studied cause greater changes in peptide conformations within the NFGAIL aggregates compared to AGAIL. We propose that simulations may elucidate the capability of interfaces to effect changes in the behaviour of disordered peptide aggregates, which may also serve to provide measures of the intrinsic fibrillogenicity of a given peptide sequence.     

Charged surfactants induce a non‐fibrillar aggregation pathway of amyloid‐beta peptide

The amyloid β‐peptide with a sequence of 42 amino acids is the major constituent of extracellular amyloid deposits in Alzheimer's disease plaques. The control of the peptide self‐assembly is difficult to achieve because the process is fast and is affected by many variables. In this paper, we describe the effect of different charged and non‐charged surfactants on Aβ_(1‐42) fibrillation to define common alternate aggregation pathways. The characterization of the peptide‐surfactant interactions by ultra‐structural analysis, thioflavin T assay and secondary structure analysis, suggested that charged surfactants interact with Aβ_(1‐42) through electrostatic interactions. Charged micelles slow down the aggregation process and stabilize the peptide in the oligomeric state, whereas non‐charged surfactants promote the Aβ_(1‐42) fibril formation. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Gel-phase synthesis of a difficult sequence peptide on 1,4-butanediol dimethacrylate-crosslinked polystyrene support

Summary The synthetic usefulness of the protocol using NMP/DMSO and DIEA for the synthesis of difficult sequence peptides on amphiphilic and flexible 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) support was demonstrated by synthesizing [DAla¹⁷] analogue of gonadotropin releasing hormone precursor protein fragment (14–36) [hGnRH (14–36)] using Boc chemistry. The swelling capacity of the peptidyl resin was followed as a measure of the aggregation of pendant peptide chains on the support. The progress of chain assembly was monitored by quantitative ninhydrin test and amino acid analysis. The purity of the peptide was checked by reverse phase HPLC and characterized by amino acid analysis and electrospray ionisation mass spectrometry (ESI-MS).     

利钠肽家族基因与心血管疾病研究新进展

利钠肽家族是一组由心肌细胞分泌的激素,主要包括A型、B型和C型利钠肽,具有相似的基因结构和生理学效应,可对心血管系统产生血压调节、抗心肌肥厚、抗心肌纤维化和抗心肌弛缓等保护作用。利钠肽受体A、B和C亦介导多种生理活性,调节心血管稳态。利钠肽受体A选择性结合A型、B型利钠肽。利钠肽受体B结合C型利钠肽。利钠肽受体C结合各型利钠肽,通过受体介导的内化和退化作用清除血液循环中利钠肽。对利钠肽家族及其受体基因单核甘酸多态性及功能研究显示,其与多种心血管疾病(房颤、高血压、心力衰竭等)的易感性相关。利钠肽家族及其受体基因缺失的转基因小鼠表现为心肌肥厚、心肌纤维化,与高血压、心肌病及心力衰竭的发生发展相关。各种导致心肌肥厚和缺血性损伤的刺激均参与利钠肽及其受体基因的表达调控。临床将脑钠肽作为左室功能障碍和心力衰竭失代偿的一个预测指标。静脉注射重组脑钠肽已经成为治疗急性心力衰竭的有效手段。深入了解利钠肽家族基因变异及其信号调控有助于探索心血管疾病的病理生理机制,为临床诊疗开辟新思路。     

Probing the efficacy of peptide-based inhibitors against acid- and zinc-promoted oligomerization of amyloid-β peptide via single-oligomer spectroscopy

One avenue for prevention and treatment of Alzheimer's disease involves inhibiting the aggregation of amyloid-β peptide (Aβ). Given the deleterious effects reported for Aβ dimers and trimers, it is important to investigate inhibition of the earliest association steps. We have employed quantized photobleaching of dye-labeled Aβ peptides to characterize four peptide-based inhibitors of fibrillogenesis and/or cytotoxicity, assessing their ability to inhibit association in the smallest oligomers (n = 2-5). Inhibitors were tested at acidic pH and in the presence of zinc, conditions that may promote oligomerization in vivo. Distributions of peptide species were constructed by examining dozens of surface-tethered monomers and oligomers, one at a time. Results show that all four inhibitors shift the distribution of Aβ species toward monomers; however, efficacies vary for each compound and sample environment. Collectively, these studies highlight promising design strategies for future oligomerization inhibitors, affording insight into oligomer structures and inhibition mechanisms in two physiologically significant environments.     

Spontaneous fibrillation of the native neuropeptide hormone Somatostatin-14

Natural Somatostatin-14 is a small cyclic neuropeptide hormone with broad inhibitory effects on endocrine secretions. Here we show that natural Somatostatin-14 spontaneously self-assembles in water and in 150 mM NaCl into liquid crystalline nanofibrils, which follow characteristic structural features of amyloid fibrils. These non-covalent highly stable structures are based on the Somatostatin native backbone conformation and are formed under non-denaturing conditions. Our results support the hypothesis that self-assembly into amyloid fibrils is a generic property of the polypeptide chain under appropriate conditions. Given recent advances on the mechanisms of biological storage and sorting modes of peptide/protein hormones into secretory granules, we propose that Somatostatin-14 fibrillation could be relevant to the regulated secretion pathway of this neuropeptide hormone. Such a hypothesis is consistent with the emerging concept of the existence of non-disease related but functional amyloids.     

利钠肽家族基因与心血管疾病研究新进展

利钠肽家族是一组由心肌细胞分泌的激素, 主要包括A型、B型和C型利钠肽, 具有相似的基因结构和生理学效应, 可对心血管系统产生血压调节、抗心肌肥厚、抗心肌纤维化和抗心肌弛缓等保护作用。利钠肽受体A、B和C亦介导多种生理活性, 调节心血管稳态。利钠肽受体A选择性结合A型、B型利钠肽。利钠肽受体B结合C型利钠肽。利钠肽受体C结合各型利钠肽, 通过受体介导的内化和退化作用清除血液循环中利钠肽。对利钠肽家族及其受体基因单核甘酸多态性及功能研究显示, 其与多种心血管疾病(房颤、高血压、心力衰竭等)的易感性相关。利钠肽家族及其受体基因缺失的转基因小鼠表现为心肌肥厚、心肌纤维化, 与高血压、心肌病及心力衰竭的发生发展相关。各种导致心肌肥厚和缺血性损伤的刺激均参与利钠肽及其受体基因的表达调控。临床将脑钠肽作为左室功能障碍和心力衰竭失代偿的一个预测指标。静脉注射重组脑钠肽已经成为治疗急性心力衰竭的有效手段。深入了解利钠肽家族基因变异及其信号调控有助于探索心血管疾病的病理生理机制, 为临床诊疗开辟新思路。     

Expediting the Fmoc solid phase synthesis of long peptides through the application of dimethyloxazolidine dipeptides.

This paper describes the step-wise Fmoc solid phase synthesis of a 95-residue peptide related to FAS death domain. Attempts to prepare this peptide employing conventional amino acid building blocks failed. However, by the judicious use of dimethyloxazolidine dipeptides of serine and threonine, the peptide could be readily prepared in remarkable purity by applying single 1 h coupling reactions.     

In silico investigation on the inhibition of Aβ42 aggregation by Aβ40 peptide by potential of mean force study

Recent experimental data revealed that small, soluble Amyloid beta (Aβ₄₂) oligomers, especially dimers impair synaptic plasticity and memory leading to Alzheimer’s disease. Here, we have studied dimerization of Aβ₄₂/Aβ₄₂ homo-dimer and Aβ₄₀/Aβ₄₂ hetero-dimer in terms of free energy profile by all-atom simulations using the ff99SB force field. We have found that in the presence of Aβ₄₀ peptide, there exists a strong tendency to form a hetero-dimer with Aβ₄₂ peptide, suggesting that a possible co-oligomerization. Furthermore, we have investigated the effects of Aβ₄₀ on the Aβ₄₂ peptide. Our study also shows that in presence of Aβ₄₀, the beta-content of Aβ₄₂ monomer is reduced. Additionally, certain residues important for bending in Aβ₄₂ peptide attained an increased flexibility in the presence of Aβ₄₀. The salt-bridge destabilization also manifested the impact of Aβ₄₀ on Aβ₄₂ peptide as a whole. Based on this, one may expect that Aβ₄₀ inhibits the aggregation propensity of Aβ₄₂. Moreover, the binding free energy obtained by the molecular mechanics–Poisson–Boltzmann surface area method also revealed a strong affinity between the two isoforms thereby suggests that Aβ₄₀ binding induces conformational change in Aβ₄₂. Our results suggest that co-oligomerization of Aβ isoforms may play a substantial role in Alzheimer’s disease.     

A Novel Oxazolidine Linker for the Synthesis of Peptide Aldehydes

A reliable method for solid-phase synthesis of peptide aldehydes by using a new oxazolidine linker is described. Based on a comparative study using the usual cleavage protocol as is used for the Fmoc-based peptide synthesis, we found that this new linker is more appropriate for the synthesis of peptide aldehydes compared with the precedent acetal, semicarbazone or threonine linker. Whereas N-Acylated oxazolidines might be partially deprotected to non-N-acylated intermediates in the TFA cocktail containing several soft nucleophiles which cause significant side reactions, the new oxazolidine linker could produce the desired peptide aldehydes by simple Et₂O washing and subsequent aqueous workup in high chemical yields and purity. We demonstrate the new method is useful especially for the preparation of highly functionalized long-chain peptide aldehydes which require several scavenger chemicals in the final deprotection step. This paper is dedicated to the memory of the late Prof. R. Bruce Merrifield, who passed away May 14, 2006.     

Chitosan-dextran Sulfate Nanoparticles for Delivery of an Anti-angiogenesis Peptide

Summary A novel nanoparticle delivery system has been developed by employing the oppositely charged polymers chitosan (CS) and dextran sulfate (DS), and a simple coacervation process. Under the conditions investigated, the weight ratio of the two polymers is identified as a determining factor controlling particle size, surface charge, entrapment efficiency and release characteristics of the nanoparticles produced. Particles of 223 nm mean diameter were produced under optimal conditions with a zeta potential of approximately −32.6 mV. A maximum of 75% anti-angiogenesis peptide entrapment efficiency was achieved with a CS:DS weight ratio of 0.59∶1. The same nanoparticle formulation also showed slow and sustained peptide release over a period of 6 days. In contrast, the formulation containing a lower ratio of CS:DS (0.5∶1) was found to have reduced entrapment efficiency and more rapid peptide release characteristics. The results of this study suggest that physicochemical and release characteristics of the CS-DS nanoparticles can be modulated by changing ratios of two ionic polymers. The novel CS-DS nanoparticles prepared by the coacervation process have potential as a carrier for small peptides.     

Ordering in a glycine-rich peptide conjugate: microscopic, fluorescence, and metalation studies

Glycine residues play an intriguing role in peptide/protein structure where they can act as tightly packing amino acids with flexible bond angles. For example, structural role of glycines is highlighted in natural silk fibers where different structural polymorphs have been reported. This study deals with a glycine-rich segment from the conserved octarepeat (PHGGGWGQ) in prion protein. We have synthesized a bis-conjugate 3, containing a truncated pentapeptide segment (GGGWG), to study its time-dependent solution phase aggregation by a combination of microscopic methods and fluorescence. This discontinuous peptide conjugate 3 exhibited interesting photophysical properties upon self-assembly allowing us to propose a possible model of peptide filament formation. Taking note of the fact that prion octarepeats bind copper, we also demonstrate the ability of this conjugate to bind copper and the growth and ultrastructure of metallized fibers formed upon incubation. Enforcing peptide fiber formation in metal binding motifs offers an entry into metal impregnated fibers for possible nanobiotechnological applications.