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91.
M. Skowronek B. Stopa L. Konieczny J. Rybarska B. Piekarska E. Szneler G. Bakalarski I. Roterman 《Biopolymers》1998,46(5):267-281
The supramolecular organization of Congo Red molecules was studied to approach an understanding of the unusual complexation characteristics associated with the liquid crystalline nature of this dye. Differential scanning calorimetry (DSC) and nmr data indicate that Congo Red assembly arrangements differ in water and salt solutions. Compact, highly ordered material with a distinct melting transition is created, but not below 0.3% sodium chloride concentration. The twist in the assembly arrangement of Congo Red molecules, caused in water by repulsion, decreases when the charges are shielded, allowing for more overlapping of the naphthalene rings and their engagement in stacking interaction. The crystallization transition observed in DSC analysis of Congo Red fast-assembled by cooling in salt solutions indicates that the formation of compact crystalline mesophase material is a time-consuming process in which coplanarity and a highly ordered organization must be achieved. Two different superposition variants, called “direct” and “reversed” here, were considered fundamental to compact Congo Red organization. They correspond to optimal face-to-face ring stackings, and are formed by simple direct translation or alternative imposition of reversed (180° rotated) molecules, respectively. In NaCl solution (2.8%) there is a significant downfield chemical shift alteration of the nmr signal related to proton 8, which is in the naphthalene ring on the side opposite to the charged sulfonic group. It was associated selectively with the transition of Congo Red to compact form. This effect confirms the close approach of the sulfonic groups and proton 8, and indicates that formation of the reversed arrangement is favored in the Congo Red supramolecular organization. Molecular dynamics simulation based on AMBER 4.1 force field and analysis of electrostatic field densities around the molecule were used for comparative modeling. Molecular dynamics (150 ps) were simulated for two eight-molecule micelle models constructed to reflect direct and reversed arrangements of Congo Red molecules. Although both versions generally preserved their initial assembly structure in the simulations, the reversed version proved more stable. The proximity of the sulfonic group and proton 8, confirmed by computer analysis, explains the correlation between the formation of Congo Red micellar organization and the distinct shift alteration related to this proton, as found by nmr. © 1998 John Wiley & Sons, Inc. Biopoly 46: 267–281, 1998 相似文献
92.
Burkhard Bechinger 《Proteins》1997,27(4):481-492
The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a 15N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an α-helix content of approximately 42%. The α-helix comprises an uninterrupted hydrophobic stretch of ≤12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15–75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled 15N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of 15N solid-state NMR spectroscopy (10−4 seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed. © 1997 Wiley-Liss Inc. 相似文献
93.
High‐throughput downstream process development for cell‐based products using aqueous two‐phase systems (ATPS) – A case study
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Sarah Zimmermann Christian Scheeder Philipp K Zimmermann Are Bogsnes Mattias Hansson Arne Staby Jürgen Hubbuch 《Biotechnology journal》2017,12(2)
The availability of preparative‐scale downstream processing strategies for cell‐based products presents a critical juncture between fundamental research and clinical development. Aqueous two‐phase systems (ATPS) present a gentle, scalable, label‐free, and cost‐effective method for cell purification, and are thus a promising tool for downstream processing of cell‐based therapeutics. Here, the application of a previously developed robotic screening platform that enables high‐throughput cell partitioning analysis in ATPS is reported. In the present case study a purification strategy for two model cell lines based on high‐throughput screening (HTS)‐data and countercurrent distribution (CCD)‐modeling, and validated the CCD‐model experimentally is designed. The obtained data are shown an excellent congruence between CCD‐model and experimental data, indicating that CCD‐models in combination with HTS‐data are a powerful tool in downstream process development. Finally, the authors are shown that while cell cycle phase significantly influences cell partitioning, cell type specific differences in surface properties are the main driving force in charge‐dependent separation of HL‐60 and L929 cells. In order to design a highly robust purification process it is, however, advisable to maintain constant growth conditions. 相似文献
94.
Reversed micelle solvents as tools of enzyme purification and enzyme-catalyzed conversion 总被引:2,自引:0,他引:2
Reversed micelle solvents represent nanometer-sized aqueous droplets stabilized by surfactants inside the bulk organic solvents. The aqueous cores can host various hydrophilic solutes, including bioactive substances thus revealing a challenge to the biotechnology's needs of the safe media for bioseparations and bioconversions. This review discusses the structure and the properties of reversed micelle solvents in view of the parameters that can be easily operated in technology to achieve safe liquid-liquid extraction of proteins/enzymes or bioconversion of hydrophobic substrates. The paper highlights the importance of how the reversed micelle microenvironment should be arranged with respect to the preservation of the activity of the enzyme as target product or biocatalyst. The main aspects are demonstrated with own experimental results on alpha-amylase purification and lipase-catalyzed esterification using cationic reversed micelle solvents. The trials of performing continuous processes involving reversed micellar separation and reaction media are also reviewed and the current problems are addressed. 相似文献
95.
Panagiotis Panagiotidis Catherine P. Raptopoulou Thomas Mavromoustakos 《Inorganica chimica acta》2008,361(8):2210-2224
The aqueous reaction of TiCl4 with citric acid at pH ∼ 4 (KOH), led to the surprising isolation of a species assembly K3[Ti(C6H6O7)2(C6H5O7)] · K4[Ti(C6H5O7)2(C6H6O7)] · 10H2O (1). The same system at pH ∼ 3 (neocuproine), led to the crystalline material (C14H13N2)2[Ti(C6H6O7)3] · 5H2O (2), while at pH 5.0 (NaOH), afforded Na3[Ti(C6H6O7)2(C6H5O7)] · 9H2O (3). Analytical, spectroscopic and structural characterization of 1, 2 and 3 revealed their distinct nature exemplified by mononuclear complexes bearing variably deprotonated citrates bound to Ti(IV). Solid-state 13C MAS NMR spectroscopy in concert with solution 13C and 1H NMR on 3 provided ample evidence for the existence of bound citrates of distinct coordination mode to the metal ion. Cyclic voltammetry defined the electrochemical signature of complex 2, thereby projecting the physicochemical profile of the species formulated by the aforementioned properties. Comparison of cyclic voltammetric data on available discrete Ti(IV)-citrate species depicts the electrochemical profile and an E1/2 value trend of the species in that binary system’s aqueous speciation, further substantiating the redox behavior of mononuclear Ti(IV)-citrate species in a pH-sensitive fashion. Collectively, the well-defined discrete species in 1-3 reflect and corroborate a synthetically challenging yet complex pH-specific picture of the aqueous Ti(IV) chemistry with the physiological citric acid, and shed light on the pH-dependent speciation in the binary Ti(IV)-citrate system. 相似文献
96.
Takiya J. Ahmed 《Inorganica chimica acta》2009,362(6):2039-2043
Titration curves were measured for three molybdocene dimers, [Cp2Mo(μ-OH)]2[OTs]2 (4), [Mo(μ-OH)]2[OTs]2 (4′; Cp′ = C5H4CH3), and ansa-[C2Me4Cp2Mo(μ-OH)]2[OTs]2 (4a), and for two monomeric molybdocene complexes, Cp2MoO (6) and Cp2MoCl2 (1). The titration curves for 4, 6, and 1 were identical and showed three equivalence points each. The titration curve for 4′ was also similar in appearance but the equivalence points were shifted higher by ∼0.3, as expected for the more electron-rich Mo center in this molecule. The titration curve for the ansa-[C2Me4Cp2Mo(μ-OH)]2[OTs]2 complex showed only two equivalence points. Two of the equivalence points observed in the titration of 4, 6, and 1 were previously reported in potentiometric measurements of aqueous solutions of Cp2MoCl2 and were attributed to the Cp2Mo(OH2)2+ species (pKa = 5.5 ± 0.1 and 8.3 ± 0.2). The third equivalence point (pKa = 2.2 ± 0.2) is assigned to protonation/deprotonation of the [Cp2Mo(μ-OH)]2[OTs]2/[Cp2Mo(μ-OH2)(μ-OH)MoCp2]3+ dimer. A new equilibrium scheme is proposed for the aquated molybdocenes to provide a more complete picture of the aqueous speciation of the non-ansa molybdocene complexes, specifically by accounting for the third acidic proton in the titration curves and by describing the hydrolysis of Cp2MoO. Although the titration curve of the ansa-[C2Me4Cp2Mo(μ-OH)]2[OTs]2 complex is different from that of [Cp2Mo(μ-OH)]2[OTs]2, 1H NMR data suggest that the aqueous speciation of the ansa-[C2Me4Cp2Mo(μ-OH)]2[OTs]2 complex is analogous to that of the non-ansa molybdocenes. 相似文献
97.
Aqueous two-phase extraction of 2,3-butanediol from fermentation broths by isopropanol/ammonium sulfate system 总被引:2,自引:0,他引:2
A novel aqueous two-phase system consisted of 2-propanol/ammonium sulfate was used for the extraction of 2,3-butanediol from
fermentation broths. The maximum partition coefficient and recovery of 2,3-butanediol reached 9.9 and 93.7%, respectively,
and more than 99% of the cells and about 85% of the soluble proteins were removed when 34% (w/w) 2-propanol and 20% (w/w)
ammonium sulfate were used. The separated cells could be re-used as inocula for subsequent fermentations. The aqueous two-phase
system described in this study may have potential application in the extraction of 2,3-butanediol produced by industrial fermentation
processes. 相似文献
98.
Richard C. Page Sangwon Lee Jacob D. Moore Stanley J. Opella Timothy A. Cross 《Protein science : a publication of the Protein Society》2009,18(1):134-146
The structural characterization of small integral membrane proteins pose a significant challenge for structural biology because of the multitude of molecular interactions between the protein and its heterogeneous environment. Here, the three‐dimensional backbone structure of Rv1761c from Mycobacterium tuberculosis has been characterized using solution NMR spectroscopy and dodecylphosphocholine (DPC) micelles as a membrane mimetic environment. This 127 residue single transmembrane helix protein has a significant (10 kDa) C‐terminal extramembranous domain. Five hundred and ninety distance, backbone dihedral, and orientational restraints were employed resulting in a 1.16 Å rmsd backbone structure with a transmembrane domain defined at 0.40 Å. The structure determination approach utilized residual dipolar coupling orientation data from partially aligned samples, long‐range paramagnetic relaxation enhancement derived distances, and dihedral restraints from chemical shift indices to determine the global fold. This structural model of Rv1761c displays some influences by the membrane mimetic illustrating that the structure of these membrane proteins is dictated by a combination of the amino acid sequence and the protein's environment. These results demonstrate both the efficacy of the structural approach and the necessity to consider the biophysical properties of membrane mimetics when interpreting structural data of integral membrane proteins and, in particular, small integral membrane proteins. 相似文献
99.
目的研究中药水提物对体外白念珠菌生物膜的影响。方法体外构建白念珠菌生物膜,微量稀释法测定中药提取物对白念珠菌浮游菌最低抑菌浓度(M IC),XTT减低法评价中药提取物对白念珠菌生物膜SM IC(SM IC80,SM IC50)及对白念珠菌黏附能力的影响。结果野菊花、土槿皮和决明子SM IC80为125 mg/m l,其余中药SM IC80为250或500 mg/m l;地肤子SM IC50为7.81 mg/m l,川芎、苏梗、藿香和决明子SM IC50为15.63 mg/m l,其他中药SM IC50在62.5~250 mg/m l。10味中药在100 mg/m l时均能显著抑制白念珠菌细胞的黏附,10 mg/m l时地肤子、苏梗和苦参对黏附仍具抑制作用。结论该10味中药水提物对体外白念珠菌生物膜有较强抑制效应。 相似文献
100.