An investigation into silk fibroin conformation in composite materials intended for drug delivery

Regenerated silk fibroin (SF) is a promising biomaterial to design drug delivery systems. To guarantee satisfactory prolonged release of loaded drugs, the native β-sheet conformation of SF is generally induced by a final curing which can determine instability of the loaded drug. This work aimed to investigate the influence on SF conformation of the addition of hydrophilic polymers, namely poloxamer 188 (PEO), a range of poly(ethylenglycol) (PEG)and poly(vinyl pyrrolidone) (PVP) and drying conditions, namely spray-drying or evaporation at 60 °C. DSC data on spray-dried products indicated that SF in composite materials was in the random coil conformation. ATR-FTIR spectroscopy with Fourier self-deconvolution of the amide I band revealed that SF in spray dried products was partially organized in the β-sheet structure only in presence of PEG4000. Both DSC and ATR-FTIR spectra registered on composite materials obtained by the slowest evaporation method indicated that all hydrophilic polymers favoured the β-sheet conformation. This feature was attributed to the formation of H-bonds with the tyrosine residues of the semicrystalline region in SF. In conclusion, this approach to prepare of SF/hydrophilic polymer composites at slow evaporation rate leads to water insoluble materials which could be used in the development of drug delivery systems.     

Influence of secondary structure of polyglycine on some conformational properties

Values of four conformational properties, namely unperturbed dimension < r²>₀, dipole moment < μ² >, mean squared optical anisotropy < γ² >, and molar Kerr constant < _mK >, have been calculated for polyglycine chains allowing several combinations of the secondary structure with the aim of studying the dependence of these magnitudes on the secondary structure of the chain. Two different approaches to the secondary structure have been used. In the first, chains with all their units in a given conformation (random coil, α-helix or β-sheet) are interrupted at several positions by one unit in a different conformation. In the second, chains with varying composition of two conformations α-helix/β-sheet and β-sheet/random coil were allowed and the results obtained compared with previous work for α-helix/random coil chains.     

重组HPV病毒样颗粒二级结构的圆二色谱法预测

目的 分析重组HPV病毒样颗粒（virus-like particle,VLP）的二级结构。方法 取重组HPV16、18、52、58型VLP各1批,均调整蛋白浓度至250〜500 μg/ml,采用圆二色谱远紫外扫描,预测重组HPV VLP的二级结构。结果 重组HPV16 VLP二级结构的预测结果为α螺旋11.7%,反平行β折叠37.1%,平行β折叠4.5%,β转角19.2%,不规则卷曲30.3%;重组HPV18 VLP二级结构的预测结果为α螺旋11.9%,反平行β折叠36.9%,平行β折叠4.6%,β转角19.0%,不规则卷曲30.7%;重组HPV52 VLP二级结构的预测结果为α螺旋12.3%,反平行β折叠37.1%,平行β折叠4.7%,β转角19.0%,不规则卷曲30.2%;重组HPV58 VLP二级结构的预测结果为α螺旋11.7%,反平行β折叠35.3%,平行β折叠4.4%,β转角20.4%,不规则卷曲31.3%。结论 圆二色谱法预测了重组HPV16/18/52/58型VLP的二级结构,该技术可用于重组HPV VLP的结构确证研究。     

Conformation similarities of ricin A-chain and trichosanthin

The conformation of ricin A-chain from castor bean was studied by circular dichroism at pH 4.7, 7 and 9 and compared with that of trichosanthin from the Chinese herb Tianhuafen. The CD spectra of ricin A-chain and trichosanthin were nearly identical at each of the three pHs. Analysis of the data indicated that, like trichosanthin, ricin A-chain had about 29%α-helix and 42%β-sheet but no β-turn. However, there was a subtle difference in the CD spectra in 20 mm sodium dodecyl sulfate, the addition of which at pH 7 slightly increased the helicity and decreased the content of β-sheet of ricin A-chain in contrast to a larger increase in helicity at the expense of β-sheet for trichosanthin, thus indicating a different stability against the surfactant. Native ricin A-chain and trichosanthin had about the same amount of secondary structure, which supports the belief that a high degree of sequence homology of the two proteins [Zhang & Wang (1986) Nature 321, 477–478] may lead to a conformational similarity between them, even though the two proteins are not taxonomically related.     

CONFORMATIONAL ANALYSIS OF CIRCULAR DICHROISM SPECTRA OF INSULIN,PROINSULIN AND C-PEPTIDES BY NON-LINEAR REGRESSION

A method of resolving CD spectra in α-helix, β-structure and random coil conformations is described. The residue ellipticities for α-helix and β-structure given by Greenfield & Fasman or by Chen, Yang & Martinez are used together with CD spectra from at least two similar peptides to determine, by an iterative least-squares method, the number of amino acids in the three reference conformations as well as a set of residue ellipticities characteristic of the random coils of the family of peptides in question, but not necessarily of other peptides. The fits between computed and experimental spectra improve significantly and systematic deviations disappear by allowing the random coil coefficients to vary from one family of proteins to another, a liberty justified by the different types of random coils that have been encountered. The method of analysis showed that 5 M urea did not change the conformations of C-peptides of proinsulin from ox, pig and duck, all being mainly in the random coil conformation and all having 3–4 amino acids in β-structure. Bovine insulin and proinsulin showed a transfer of amino acids from α-helix to β-structure with increasing concentrations of urea, the latter at a higher concentration, indicating a stabilizing effect of the connecting peptide. The numbers of amino acids found in the α-helical conformation in insulin and proinsulin were equal and in agreement with the X-ray crystallographic data for insulin when the Greenfield & Fasman coefficients for α-helix and β-structure were employed, whereas the Chen, Yang & Martinez coefficients yielded too few amino acids in α-helix in proinsulin. Both sets of coefficients estimate more β-structure in proinsulin than in insulin.     

Studies on mechanism of thermal crystal transformation of sitafloxacin hydrates through melting and recrystallization, yielding different anhydrates depending on initial crystalline forms

The polymorphic and pseudopolymorphic forms of sitafloxacin, a novel fluoroquinolone antibiotic, were characterized by infrared spectroscopy, X-ray diffractometry, and thermal analysis. Hydrates of sitafloxacin underwent thermal transformation during the course of heating to 300°C. Monohydrate melted at 130°C and crystallized at 147°C to yield α-form (anhydrate) while sesquihydrate melted at 127°C and crystallized at 146°C to yield β-form (anhydrate). The crystal structural analysis revealed that monohydrate and sesquihydrate had opposite torsion at quinolone ring and the conformation of quinolone ring tended to be retained during hydrates to anhydrates crystal conversion. The infrared spectroscopy showed that hydrates and anhydrate α-from exists in zwitterion while β-from is consist of neutral molecule. Detail investigation of thermal behavior of hydrates suggested that water vapor also affected anhydrous crystal forms obtained by heating hydrates, though promoting ionization at carboxyl group and amine group.     

Fourier-transform infrared conformational study of bovine insulin in surfactant solutions

To obtain conformational data on the monomeric form of insulin, which is believed to be the physiologically active form of the hormone, insulin in sodium dodecyl sulfate solution was studied by Fourier-transform infrared (FTIR) spectroscopy and circular dichroism, and results were compared with those obtained with des(B23-30)octapeptide insulin (DOI) and dimeric insulin in buffer. The FTIR amide I band (1600–1700 cm^?1) was examined, and a quantitative evaluation of the secondary structure fractions of the various conformations showed less of a β-sheet component for both insulin in SDS and DOI in buffer than for insulin in buffer, corresponding to a lack of monomers binding to form dimers. At the concentrations used for FTIR (≥ 2 mg/mL), the CD spectra of insulin in SDS and DOI in buffer were qualitatively identical but different from that of insulin in buffer, which is associated at these concentrations. The CD spectrum pattern of insulin in very dilute solution (80 nM), where it is prevalently monomeric, is very similar to that of monomeric insulin in SDS, which suggests that the conformation of the hormone in the two cases is very similar. © Munksgaard 1996     

Stabilization of amphipathic α-helical and β-helical conformations in synthetic peptides in the presence and absence of ionic interactions

The role of ionic interactions in stabilizing amphipathic α-helices was studied in the synthetic peptide Ac-NLEELKKKLEELKG-NH₂ (NLEKG14), potentially stabilized by attraction between complementary ions in successive turns of the helix, and in the peptide Ac-NLEELEEELEELEG-NH₂ (NLEG14), in which no side-chain ionic attractions are possible. At a pH below the pK_a of glutamate, NLEG14 had a higher helix content than NLEKG14. At pH 3 to pH 10, the helicity of NLEKG14 did not change, whereas NLEG14 was converted to random coil at pH 7. The role of ionic interactions in stabilizing the conformation of β-structures was studied in the synthetic peptides Ac-KLKLKLELELELG-NH₂ (KLEG13) and Ac-ELELELELELELG-NH₂ (ELG13). At a pH below the pK_a of glutamate, ELG13 had a higher β-content than KLEG13, as judged by their dichroic spectra, but at higher pH, ELG13 was converted to random coil, whereas KLEG13 retained a predominantly β-conformation. At pH 7, high NaCl concentration produced a significant increase in the α-helix content of NLEKG14, converted NLEG14 from random coil to α-helix and converted ELG13 from random coil to β-conformation. Overall, the results demonstrate that ionic attraction between side-chains plays a lesser role than hydrogen bonding and hydrophobic effects in stabilizing the α- and β-conformations exhibited by these amphipathic peptides.     

Conformational and thermodynamic impact of bulky aminofluorene adduction on simulated translesion DNA synthesis

We report a systematic spectroscopic investigation on the conformational evolution during primer extension of a bulky fluoroaminofluorene-modified dG adduct (FAF-dG) in chemically simulated translesion synthesis. FAF-dG was paired either with dC or dA (dC-match and dA-mismatch series, respectively). Dynamic (19)F NMR/CD results showed that the FAF-adduct exists in a syn/anti equilibrium and that its conformational characteristics are modulated by the identity of an inserted nucleotide at the lesion site and the extent of primer elongation. At the pre-insertion site, the adduct adopted preferentially a syn conformation where FAF stacked with preceding bases. Insertion of the correct nucleotide dC at the lesion site and subsequent elongation resulted in a gradual transition to the anti conformation. By contrast, the syn conformer was persistent along with primer extension in the dA-mismatch series. In the dC-match series, FAF-induced thermal (T(m)) and thermodynamic (-ΔG°(37 °C)) stabilities were significantly reduced relative to those of the controls. However, the corresponding T(m) and -ΔG°(37 °C) values were increased in the FAF-modified mismatched dA series. The lesion impact persisted up to three 5'-nucleotides from the lesion. Occupation of the minor groove of the W-conformer with the bulky carcinogenic fluorene moiety not only would limit the DNA mobility but also would impose a serious difficulty for the active site of a polymerase throughout the replication process. Our spectroscopic results are consistent with reported data on AF, which showed dramatic (~10(4)-fold) differences in the nucleotide insertion rates between the dC-match and dA-mismatch series. The results emphasize the importance of adduct-induced steric constraints for determining the replication fidelity of a polymerase.     

Human β-endorphin

¹H spectra at 270MHz of the β_h-endorphin glycyl residues in aqueous solution are reported. The chemical shifts, coupling constants and temperature coefficients are compared with those of the glycyl residues in Met-enkephalin and in a random coil model peptide. The local conformation of Tyr-Gly-Gly-Phe-segment observed in Met-enkephalin is maintained in β_h-endorphin.     

Implementation of an FTIR calibration curve for fast and objective determination of changes in protein secondary structure during formulation development

The aim of this study was to develop a quick and objective method for the determination of changes in protein secondary structure by Fourier transform infrared spectroscopy (FTIR). Structural shifts from native regions (α-helix, intramolecular β-sheet) to aggregated strands (intermolecular β-sheet) were used to evaluate protein damage. FTIR spectra of 16 different proteins were recorded and quantified by peak fitting of the non-deconvolved and baseline corrected amide I bands. The resulting percentile secondary structures were correlated with the shape and intensity of the area normalized amide I bands using an interval partial least squares algorithm (iPLS). Structural elements were focused on the following regions: α-helix 1660–1650 cm⁻¹, intramolecular β-sheet 1695–1683 cm⁻¹ and 1644–1620 cm⁻¹, intermolecular β-sheet 1620–1595 cm⁻¹. Three calibration curves were created from the data sets. Calculated α-helix content ranged from 0% to 79.59%, intramolecular β-sheet from 10.64% to 63.89% and intermolecular β-sheet from 0.23% to 9.70%. The linear relationship between actual values (as determined by peak fitting) and calculated values was evaluated by correlation coefficient and root mean square error of calibration while cross-validation was performed to detect possible outliers. Results were verified by including two proteins as validation standards and comparing the calculated values to peak fitting and X-ray data.     

Integrity of crystalline lysozyme exceeds that of a spray-dried form

The development of proteins as therapeutic agents is challenging partly due to their inherent instabilities. Consequently, crystallisation and spray drying techniques were assessed to determine their effects on protein integrity using lysozyme as a model protein. Unprocessed, crystallised and spray-dried lysozyme were characterised by: thermal analysis using hot stage microscopy (HSM), differential scanning calorimetry (DSC), high sensitivity differential scanning calorimetry (HSDSC) and thermogravimetry (TGA); and spectroscopic analysis employing Fourier transform Raman (FT-Raman). Moisture contents were determined by TGA and Karl Fisher titration (KFT). Enzymatic assay measured biological activity. HSM showed no changes in crystals until complete melting. TGA and KFT indicated that spray-dried lysozyme contained a lower moisture content than crystals, hence the higher apparent thermal stability was shown by DSC. HSDSC revealed that crystallisation and spray drying did not affect the denaturation temperature of lysozyme in solution when compared with unprocessed material. However, in the solid state, FT-Raman spectra showed perturbation of the conformational structure of spray-dried sample, whereas crystal conformation remained intact. Enzymatic assay revealed increased activity retention of crystals compared with spray-dried powder. Hence, crystals maintained the conformational integrity and activity of lysozyme in solution.     

Conformationally stabilized gramicidin S analog containing dehydrophenylalanine in place of d-phenylalanine4,4′

Unsaturated gramicidin S analog, [ΔPhe^4,4′]gramicidin S, was synthesized by conventional solution method in order to evaluate the role of the dehydrophenylalanine residues replacing d -phenylalanine^4,4′ in stabilizing the bioactive β-shect conformation. The dehydrophenylalanine (ΔPhe) moiety was introduced by dehydroazlactonization of the β-phenylserine residue. The [ΔPhe^4,4′]gramicidin S prepared by this method showed very strong antimicrobial activities against Gram-positive bacteria, but not against Gram-negative ones. Several lines of spectroscopic evidence indicated that [ΔPhe^4,4′] gramicidin S has a reinforced β-sheet backbone conformation necessary for a full biological activity of gramicidin S. These results suggested that :α,β-dehydrogenation of the amino acid residue in a cyclic peptide can stabilize the turn structure.     

The development of modulated, quasi-isothermal and ultraslow thermal methods as a means of characterizing the α to γ indomethacin polymorphic transformation

While polymorphism remains a key issue within the pharmaceutical and related industries, the understanding of the transformation process itself remains relatively poorly understood. In this study we use a combination of conventional and modulated temperature differential scanning calorimetry (MTDSC), quasi-isothermal MTDSC (Qi-MTDSC) and ultraslow heating rate MTDSC as a novel means of investigating the temperature-induced α to γ transformation in indomethacin, using hot stage microscopy and variable temperature attenuated total reflectance FTIR spectroscopy as supportive techniques. In particular, we utilize the ability of MTDSC to measure subtle heat capacity changes through the transformation, we examine the use of Lissajous analysis of the modulated heating signal itself (both scanning and quasi-isothermal) and finally we investigate the use of ultraslow heating rates (down to 0.04 °C/min) so as to facilitate examination of the melt-crystallization process at a scanning rate whereby kinetic hindrance becomes negligible. Indomethacin was prepared in the metastable α and stable γ forms using standard approaches. Samples were studied using conventional DSC, Qi-MTDSC (involving holding and modulating the sample at a series of incremental temperature steps) and ultraslow MTDSC. All studies were conducted using a Q-1000 MTDSC using crimped pans, following standard calibration procedures. Conventional DSC at 10 °C/min showed the expected single melting responses for the α and γ forms, while MTDSC at slower rates indicated the presence of a melt-crystallization process. Quasi-isothermal studies allowed the heat capacity to be estimated as a function of time, while the associated Lissajous analysis demonstrated distortion of the elliptical response as a result of the kinetic events involved. Ultraslow heating resulted in superimposition of the melting and crystallization processes, resulting in a discrete thermal event that was enthalpically equivalent to the difference between the two processes. It is suggested that these combined thermal methods allow the conversion to be profiled in a manner which facilitates both kinetic and thermodynamic analysis of the transformation.     

Structure and Dynamics of Neurotoxins from Snake Venoms as Revealed by Nuclear Magnetic Resonance and Circular Dichroism

Abstract

Short and long neurotoxins from snake venoms were studied by nuclear magnetic resonance (NMR) and circular dichroism (CD). From the analysis of amide and a proton resonances of Laticauda semifasciata III, triple stranded antiparallel pleated β-sheet structure was found to be stable even in aqueous solution. The NMR analysis showed that this α-sheet is common structural feature to erabutoxin b, α-nigricolis, α-cobratoxin and α-bungarotoxin. Since amino acid rersidues on this β-sheet are essential for neurotoxicity, this α-sheet is supposed to be the binding surface to the acetylcholine receptor proteins. In contrast to the crystal structure of proteins, proteins in aqueous solution are more mobile and respond to the change of the environment. pH and temperature dependence of NMR spectra of α-cobratoxin was studied by NMR and CD. Detailed analysis of NMR spectra showed that protonation of His-18 disrupts the hydrophobic core which induces slow conformation change, while the loop part is sensitive to temperature change, suggesting that the loop part is more mobile. Actually, the analysis of the relaxation time of methyl carbons showed that the side chains of methyl bearing amino acid residues on the loop have large amplitude motion, suggesting that there is concerted motion in the loop part. The present results indicate that the importance of the mobile surface for the binding of neurotoxins to the acetylcholine receptor proteins.     

Weak dependence of octapeptide solubility in organic solvents on the amino acid sequence*

The six kinds of the octapeptides 1-6 to be conceivable from permutation of three peptide fragments, i.e. Gln-Val-Gly, Asn-Ala-Ile, and Phe-Leu, were prepared to confirm that their solubility was not strongly dependent on their amino acid sequences. The solubility of the octapeptides 1-6 resembled each other and they were insoluble in MeOH, EtOH, THF, and CH₂Cl₂, and soluble in HMPA and DMSO . I.r. absorption spectroscopic analysis of the octapeptides 1-6 indicated that they mainly had a β-sheet structure in the solid state. The results of the solubility and conformation of the octapeptides 1-6 strongly suggested the adequacy of the hypothesis that their solubility and their potential for β-sheet formation in the solid state were not strongly dependent on their amino acid sequences but on their amino acid compositions.     

Experimental studies and potential energy calculations of the blocked tetrapeptide Ac-Lys-Pro-Gly-Ile-NMA from the third loop of short-chain snake venom neurotoxins

The conformational space of the tetrapeptide Ac-Lys-Pro-Gly-Ile-NMA from the β-bend present in the third loop of short-chain snake venom neurotoxins was investigated with the aid of energy calculations, resulting in the identification of an ensemble of β-turn conformations. These results were compared with the experimentally determined conformations, as observed using NMR and CD spectroscopy. A random coil conformation of the peptide is indicated in polar hydrogen-bonding solvents. In less polar solvents the peptide backbone assumed a more rigid conformation, as reflected by the existence of at least a type II β-turn conformation.     

Characterisation and physical stability of PEGylated glucagon

Glucagon was mono-PEGylated with PEG 5000 at Lys-12 to examine the effect on conformation and physical stability during purification and freeze-drying. The model peptide glucagon is highly unstable and readily forms fibrils in solution. Secondary structure was determined by FTIR and far-UV CD and physical stability was assessed by the Thioflavin T assay.

Glucagon samples were included, which underwent the same RP-HPLC purification and/or freeze-drying as glucagon–PEG 5000. After purification and freeze-drying glucagon samples showed formation of intermolecular β-sheet by FTIR, this correlated with shorter lag-times for fibrillation in the Thioflavin T assay. Formation of intermolecular β-sheet was less apparent for glucagon–PEG 5000 and no fibrillation was detected by Thioflavin T assay. Apparently PEGylation significantly improved the physical stability of glucagon after purification and freeze-drying, possibly by steric hindrance of peptide–peptide interactions.

Alterations in the secondary structure were observed for freeze-dried and reconstituted peptide samples by liquid FTIR. The peak for -helix shifted to 1664 cm⁻¹, which could possibly be explained by formation of 3₁₀-helix. Neither 3₁₀-helix nor intermolecular β-sheet could be detected by far-UV CD, where all peptide samples showed similar spectra.

In conclusion, glucagon–PEG 5000 showed a significantly improved physical stability during purification and freeze-drying compared to glucagon.     

平颏海蛇神经毒组份X的纯化及性质特点

经CM—Sephadex C 25,Sephadex G 50及CM—Sephadex C 50三步柱层析,从平颏海蛇(Lapemis hardwickii)毒腺提取物中分离出神经毒组份X—2—4(Fr.X—2—4)。经三种不同类型的电泳鉴定,Fr.X—2—4为单一组份,其分子量为8000,等电点为pH 8.45。小鸡颈二腹肌及电生理实验证明,此组份为突触后神经毒,放射性配基结合实验表明,该毒份抑制~(125)I标记的眼镜蛇神经毒素与胆碱受体结合,其IC_(50)为7.45 nmol/L。拉曼光谱提示该神经毒的二级结构主要是β折叠和无规卷曲。     

Investigation of physicochemical factors affecting the stability of a pH-modulated solid dispersion and a tablet during storage

The stability of solid dispersions (SD) during storage is of concern. We prepared the pH-modulated SD (pSD) and compressed tablets consisting of polyethylene glycol (PEG) 6000 as a carrier, drug and MgO (alkalizer). Telmisartan (TEL), an ionizable poorly water-soluble drug, was chosen as a model drug. The changes in physicochemical factors such as the dissolution rate, drug crystallinity, microenvironmental pH (pH(M)) and intermolecular interactions of the pSD and the tablets were investigated over 3 months under different temperature and relative humidity (RH) conditions: refrigerator (5-8 °C), 25 °C/32% RH, 25 °C/55% RH, 25 °C/75% RH, 40°C/32% RH, 40 °C/55% RH, and 40 °C/75% RH. Differential scanning calorimetry (DSC) analysis of all samples revealed no distinct changes in the drug melting point. In contrast, powder X-ray diffraction (PXRD) diffractograms revealed that samples stored at 40 °C/75% RH for 1 month, 25 °C/75% RH for 3 months and 40 °C at all humidity conditions for 3 months showed gradual recrystallization of the drug. Fourier transform infrared (FTIR) spectra indicated a reduced intensity of intermolecular interactions between TEL and MgO in the pSD and tablet. The pH(M) also gradually decreased. These altered physicochemical factors under the stressed conditions resulted in decreased dissolution profiles in intestinal fluid (pH 6.8). In contrast, the dissolution rate in gastric fluid (pH 1.2) was almost unchanged because of the high intrinsic solubility of TEL at this pH.