Unraveling A Trap-and-Trigger Mechanism in the pH-Sensitive Self-Assembly of Spider Silk Proteins

When the major ampulate spidroins (MaSp1) are called upon to form spider dragline silk, one of nature's most amazing materials, a small drop in pH must occur. Using a state-of-the-art simulation technique, constant pH molecular dynamics, we discovered a few residues that respond to the pH signal in the dimerization of the N-terminal domain (NTD) of MaSp1 which is an integral step in the fiber assembly. At neutral pH the deprotonation of Glu79 and Glu119 leads to water penetration and structural changes at the monomer-monomer binding interface. At strongly acidic pH, the protonation of Asp39 and Asp40 weakens the electrostatic attraction between the monomers. Thus, we propose a "trap-and-trigger" mechanism whereby the intermolecular salt-bridges at physiologically relevant pH conditions always act as a stabilizing "trap" favoring dimerization. As pH is lowered to about 6, Glu79 and Glu119 become protonated, triggering the dimerization and subsequent silk formation. We speculate that this type of mechanism is operative in many other pH-sensitive biological processes.     

One-pot synthesis of pH and temperature sensitive gold clusters mediated by a recombinant elastin-like polymer

Here, we described a “one-pot” synthesis of smart hybrid materials based on elastin-like recombinamers (ELRs) and gold nanoparticles. Outstandingly, the reduction of auric acid in the presence of ELR Glu15 gave biohybrid Au-Glu15. TEM analysis carried out for Au-Glu15 exhibited nano-sized gold crystals with diameter ranging from 2 to 11 nm. Furthermore, Au-Glu15 promoted the formation of linear arrangements of gold clusters in areas of low particle density. Remarkable was that analogous architectures were obtained in a control experiment carried out with Glu15 and gold nanoparticles synthesized via a citrate reduction route. Therefore, Glu15 promoted the formation of 2D linear arrangements of gold clusters that exhibited interparticle distances in the range from 10 to 40 nm. Notable were the branched nanostructures exhibited by Au-Glu15B obtained for reduced gold-ELR mixtures that exhibited a lower gold ratio. On the other hand, Au-Glu15 exhibited spectroscopic properties (UV-vis absorption) that could be modulated as function of pH and temperature of the environment as result of reversible aggregation-expansion of gold nanoparticles. Thereby, Au-Glu15 displayed remarkable features suitable for the development of stimuli responsive optical sensors and detectors for biological applications that could operate in aqueous media and under a wide range of pH’s.     

Solution equilibria in l-glutamic acid and l-serine + iron(III) systems

Complex formation equilibria in l-glutamic acid (H2Glu) and l-serine (HSer) +iron(III) ion systems have been studied by a combination of glass electrode potentiometric and visible spectrophotometric measurements in 0.5 mol dm–3 (Na)NO3 ionic medium at 25°C. In the concentration range 1.0[Fe3+]5.0; 3.0[Glu2–]30.0 mmol dm–3 ([Glu]/[Fe]=3:1 to 30:1) and pH between 1.5 and 4.5, iron(III) and glutamic acid form the Fe(Glu)–2, Fe(Glu)+, Fe(HGlu)2+, Fe(OH)Glu, Fe2(OH)2Glu2+, Fe(OH)Glu22– complexes: and several pure hydrolytic products. Iron(III) and l-serine, beside pure hydrolytic complexes of iron(III), form the Fe(HSer)3+, Fe(Ser)2+, Fe(OH)Ser+, Fe(OH)2- Ser0, Fe(OH)Ser2 and Fe2(OH)2(Ser)2+2 complexes, over a broad concentration range of serine to iron ([Ser]/[Fe]=5:1 to 500:1), from pH 1.5 to 4.0. The stability constants of the complexes are given and their formation mechanism is suggested. The possible structure of the complexes, in solution, is discussed.     

Polymer control of ligand display on gold nanoparticles for multimodal switchable cell targeting

The function of cell-specific ligands on gold nanoparticles can be selectively gated by the action of co-grafted thermosensitive polymers. Below the LCST the responsive chain-extended polymers prevent cell-surface receptors from accessing the affinity ligands while above the LCST, the polymers collapse exposing the ligands and allowing binding to receptors, which in turn promotes cell internalisation.     

Synthesis and relaxometric studies of a dendrimer-based pH-responsive MRI contrast agent

The design of effective pH responsive MRI contrast agents is a key goal in the development of new diagnostic methods for conditions such as kidney disease and cancer. A key factor determining the effectiveness of an agent is the difference between the relaxivity of the "on" state compared to that of the "off" state. In this paper, we demonstrate that it is possible to improve the pH-responsive action of a low molecular weight agent by conjugating it to a macromolecular construct. The synthesis of a bifunctional pH responsive agent is reported. As part of that synthetic pathway we examine the Ing-Manske reaction, identifying an undesirable by-product and establishing effective conditions for promoting a clean and effective reaction. Reaction of the bifunctional pH responsive agent with a G5-PAMAM dendrimer yielded a product with an average of 96 chelates per dendrimer. The relaxivity of the dendrimer conjugate rises from 10.8 mM(-1) s(-1) (pH 9) to 24.0 mM(-1) s(-1) (pH 6) per Gd(3+) ion. This more than doubles the relaxivity pH response, Deltar(1), of our agent from just 51 % for the original low molecular weight chelate to 122 % for the dendrimer.     

Terminal Modification with 1‐Adamantylamine to Endow Hyperbranched Polyamidoamine with Thermo‐/pH‐Responsive Properties

A novel terminal modification agent to endow hyperbranched polyamidoamine (HPAMAM) with thermo‐/pH‐responsive properties is reported. HPAMAM with terminal vinyl groups is first synthesized and then end‐capped by 1‐adamantylamine (ADA). The resulting hyperbranched polymer (HPAMAM‐ADA) shows interesting thermo‐responsive properties in aqueous solution, which have been investigated by UV‐vis spectroscopy, optical microscopy, and ¹H NMR spectroscopy. The lower critical solution temperature can be controlled by adjusting the end‐capping ratio of ADA. In addition, HPAMAM‐ADA exhibits a pH‐dependent water solubility. This pH‐responsive behavior is also studied.

     

Titration curves of polypeptide chains by combined isoelectric focusing-electrophoresis in 8 M urea

Titration curves of reduced and alkylated polypeptide chains can be successfully performed in 8 M urea-polyacrylamide gel plates by electrophoresis perpendicular to a stationary stack of focused carrier ampholytes. All buffers and thiol reagents with pK values in the range pH 3--10 should be removed, since their pH-dependent ionization affects the migration and apparent pI values of the protein chains. No blurring of the patterns below pH 4.5 is observed, as usually found in titration curves in the absence of urea, thus allowing the direct titration of Glu and Asp residues. It is not possible by the present technique to titrate any group below pH ca. 3 and above pH ca. 10, due to the lack of suitable carrier ampholytes and to a "flooding" phenomenon, with concomitant identical electrophoretic mobility for all protein species, irrespective of their relative pI values and amino acid composition. The "electrophoretic titration curves" thus obtained were well correlated with the overall amino acid composition of the polypeptide chains analyzed.     

Comb‐Shaped Glycopolymer/Peptide Bioconjugates by Combination of RAFT Polymerization and Thiol‐Ene “Click” Chemistry

Comb‐shaped glycopolymer/peptide bioconjugates are constructed by grafting reduced glutathione (GSH) onto acrylate‐functional block glycocopolymers via thiol‐ene click chemistry. In aqueous solution, the glycopolymer/GSH bioconjugate self‐assembles to sugar‐installed spherical micelles. The size of micelles decreases with increasing pH, demonstrating pH‐responsive character. The isoelectric point (IEP) of the PMAGlc/GSH bioconjugate is estimated to be 3.43. The micelles show a specific interaction with the protein Concanavalin A. At endosomal pH, the PMAGlc/GSH bioconjugate can gradually degrade. These pH‐responsive glycopolymer/peptide micelles with biological recognition and degradation can be used as multifunctional nanocarriers for targeted drug delivery.

     

Dual Thermo‐ and pH‐Responsive Zwitterionic Sulfobataine Copolymers for Oral Delivery System

A novel oral delivery system consisting of thermoresponsive zwitterionic poly(sulfobetaine methacrylate) (PSBMA) and pH‐responsive poly(2‐(diisopropylamino)ethyl methacrylate) (PDPA) is synthesized via free radical polymerization. This copolymer can self‐aggregate into nanoparticles via electrostatic attraction between ammonium cation and sulfo‐anion of PSBMA and successfully encapsulate anticancer drug, curcumin (CUR), with highest loading content of 2.6% in the P(SBMA‐co‐DPA) nanoparticles. The stimuli‐responsive phase transition behaviors of P(SBMA‐co‐DPA) copolymers at different pH buffer solution show pH‐dependent upper critical solution temperature (UCST) attributed to the influence of protonation/deprotonation of the pH‐responsive DPA segments. Through the delicate adjustment of the PSBMA/PDPA molar ratios, the stimuli‐responsive phase transition could be suitable for physiological environment. The kinetic drug release profiles demonstrate that P(SBMA‐co‐DPA) nanoparticles have the potential as oral delivery carriers due to their effective release of entrapped drugs in the stimulated intestinal fluid and preventing the deterioration of drug in stimulated gastric fluid.

     

Unlocking the pH‐Responsive Degradability of Fumaramic Acid Derivatives Using Photoisomerization

Fumaramic acid derivatives can be converted into their cis isomer maleamic acid derivatives under UV illumination, and these maleamic acid derivatives show pH‐responsive degradability at acidic pH only after the preceding photoisomerization. The rate of the tandem photoisomerization–degradation of fumaramic acid derivatives can be finely controlled by changing the substituents on the double bond. Photoisomerization‐based unlocking of the pH‐responsive degradability of fumaramic acid derivatives has strong potential for the development of multisignal‐responsive smart materials in biomedical applications.     

pH-triggered strand exchange in coiled-coil heterotrimers

The capacity for pH-triggered strand exchange in designed coiled-coil heterotrimers is demonstrated. Systems employing both hydrophobic core (steric matching) and hydrophilic interface (electrostatic matching) design principles assemble into specific 1:1:1 heterotrimers. Alteration of pH creates electrostatic mismatches, inducing strand exchange in the presence of a suitable replacement peptide. Complexes with one Lys/Lys interface, favored at neutral to high pH, can be transformed to ones with a Glu/Glu contact by lowering pH and adding an appropriate new binding partner. The need to simultaneously maintain matched core alignments enforces specificity in this exchange, such that only a single specific peptide is replaced. These principles have subsequently been applied to the design of dynamically triggered cross-linked structures, in which a bifunctional disulfide-tethered peptide can cross-link two heterotrimers. Both formation and disruption of the cross-link are under pH control.     

An in situ Infrared Spectroscopic Study of Glutamic Acid and of Aspartic Acid Adsorbed on TiO(2): Implications for the Biocompatibility of Titanium

In situ ATR-IR spectroscopy has been applied to the study of glutamic (Glu) and aspartic (Asp) acid adsorbed on amorphous TiO(2) particle films. Unlike Asp, which gives evidence of one major adsorbed species, Glu yields several spectroscopically distinct structures upon adsorption to TiO(2). The pH dependence of Glu and Asp adsorption is also different, with Glu adsorbing markedly to TiO(2) at pH where electrostatic interactions between the surface and adsorbate are unfavorable. Application of the Langmuir model to adsorption isotherms yields a single binding constant for Asp and two binding constants for Glu, further supporting the evidence for different adsorbed Glu species. This is the first investigation of the molecular structure of Glu and Asp species adsorbed on amorphous TiO(2) using in situ ATR-IR spectroscopy. Copyright 2000 Academic Press.     

Modulating the pH Activity Profiles of Phenylalanine Ammonia Lyase from <Emphasis Type="Italic">Anabaena variabilis</Emphasis> by Modification of Center-Near Surface Residues

Phenylalanine ammonia lyase from Anabaena variabilis (Av-PAL) is a candidate for the treatment of phenylketonuria (PKU). However, Av-PAL shows its optimal pH at 8.5 and maintains only 70% of its highest activity when pH decreases to 7.3–7.4 (the condition of human plasma). The objective of the study was to shift its optimal pH by mutating surface amino acid residues which interact with the general base Tyr78. Based on the crystal structure and the online program GETAREA, we selected five sites: Asn69, Glu72, Glu75, Asn89, and Val90. Removing negative charges or introducing positive charges near the general base Tyr78 by mutation, the pH optima were successfully shifted to more acidic range. Especially, the pH optima of E75A, E75L, and E75Q were shifted to 7.5 with 35, 30, and 24% higher specific activities than that of the wild, respectively. Half-lives of E75L and E75Q at 70 °C prolonged to 190 and 180 min from 130 min of the wild, respectively. In addition, the higher resistance to a low pH of 3.5 and protease made E75L a candidate for oral medicine of PKU. This work would improve the therapeutic prospect of Av-PAL and provide guidance in modulating optimal pH of enzymes.     

弱酸介质中类球形谷氨酸/镁铝层状双氢氧化物的制备

在弱酸性介质中,采用阴离子交换法合成了类球形的谷氨酸(Glu)/镁铝层状双氢氧化物(MgAl-LDH)复合物,研究了介质pH值对所得Glu/LDH纳米复合物性质和形貌的影响。结果表明,与碱性介质中阴离子交换法得到的不规则六角形粒子相比,该法制备的纳米粒子呈类球形,且粒径较小,最佳pH值为5.0,探讨了可能的反应机理。     

Temperature dependence of peptide carboxylic group IR spectra in the amide I′ region at neutral and acidic pH

Analysis of the temperature-dependent amide I′ bands of peptides and proteins can be complicated by their overlap with other IR bands, particularly those of carboxylic groups of amino acid side-chains and the C-terminus. Previously, we reported IR spectra of charged carboxylic side-chains in Asp and Glu amino acids, and C-terminal groups of several amino acids and dipeptides at neutral pH. To complement these results, here we investigate the IR absorptions of Asp and Glu side-chains in capped amino acids (AcAspNMe and AcGluNMe), at both neutral and acidic pH. Spectra of protonated (acidic pH) C-terminal group absorptions are also investigated, using three dipeptides (AlaGly GlyAla and GlyGly) as model compounds. Sets of temperature-dependent experimental IR spectra were analyzed using pseudo-Voigt lineshape profiles. We find that the temperature-dependent behavior of the IR bands of deprotonated (neutral pH) side-chains in AcAspNMe and AcGluNMe dipeptides are generally similar to those reported previously for Asp and Glu. Protonated carboxylic group (acidic pH) IR bands behave uniformly with respect to temperature, showing very similar magnitude frequency shifts and intensity changes. Implications for analyses of amide I′ bands of peptides and proteins are discussed.     

Breathing Pores on Command: Redox‐Responsive Spongy Membranes from Poly(ferrocenylsilane)s

Redox‐responsive porous membranes can be readily formed by electrostatic complexation between redox active poly(ferrocenylsilane) PFS‐based poly(ionic liquid)s and organic acids. Redox‐induced changes on this membrane demonstrated reversible switching between more open and more closed porous structures. By taking advantage of the structure changes in the oxidized and reduced states, the porous membrane exhibits reversible permeability control and shows great potential in gated filtration, catalysis, and controlled release.     

酸碱-氧化还原双重刺激响应型表面活性剂的合成与性能

以11-氨基十一酸为原料制备了酸碱-氧化还原双重刺激响应型表面活性剂中间体11-叔胺十一烷基羰基二茂铁(C_(11)-N-Fe);采用~1H NMR和MS等手段对产物进行了结构表征;并通过透光率、循环伏安法和紫外光谱分析等研究了其酸/碱和氧化/还原开关响应性能.研究发现,C_(11)-N-Fe的盐酸盐在水溶液中进行自组装时,可制备具有酸碱-氧化还原双重刺激响应性的泡沫,且采用2种刺激方式的消泡均可在2 min之内完成;将C_(11)-N-Fe的盐酸盐作为乳化剂用于乳化正癸烷/水体系,可以制得颗粒均匀稳定的乳状液,同时可以通过酸碱-氧化还原双重刺激可逆调控乳液的稳定与破乳.     

Enhanced Antitumor Efficacy by a Cascade of Reactive Oxygen Species Generation and Drug Release

Reactive oxygen species (ROS) can be used not only as a therapeutic agent for chemodynamic therapy (CDT), but also as a stimulus to activate release of antitumor drugs, achieving enhanced efficacy through the combination of CDT and chemotherapy. Here we report a pH/ROS dual‐responsive nanomedicine consisting of β‐lapachone (Lap), a pH‐responsive polymer, and a ROS‐responsive polyprodrug. In the intracellular acidic environment, the nanomedicine can realize pH‐triggered disassembly. The released Lap can efficiently generate hydrogen peroxide, which will be further converted into highly toxic hydroxyl radicals via the Fenton reaction. Subsequently, through ROS‐induced cleavage of thioketal linker, doxorubicin is released from the polyprodrug. In vivo results indicate that the cascade of ROS generation and antitumor‐drug release can effectively inhibit tumor growth. This design of nanomedicine with cascade reactions offers a promising strategy to enhance antitumor efficacy.