Optical studies of a conformational change in DNA before melting

The circular dichroism of double-stranded DNA is temperature dependent prior to its melting. As the temperature is increased the spectrum becomes more nonconservative. This is certainly due to a conformational change within the framework of the double helix. To ascertain the nature of the conformational change, a series of synthetic and natural DNA's from a variety of sources was investigated. The same qualitative changes were seen for all the DNA samples, independent of base composition. However, there were definite quantitative differences, with poly [d(A-T)] manifesting the largest effect. Oligomers of the form [d(A-T)]_n with n = 10 to 21 behaved in a manner similar to the polymer. There is no observed chain-length dependence. The breadth of the pre-melt transition indicates a low ΔH (less than 5 kcal./mole); the lack of dependence on chain length indicates that the co-operative unit is smaller than eight base pairs.     

Hen egg white lysozyme fibrillation: a deep‐UV resonance Raman spectroscopic study

Amyloid fibrils are associated with numerous degenerative diseases. The molecular mechanism of the structural transformation of native protein to the highly ordered cross‐β structure, the key feature of amyloid fibrils, is under active investigation. Conventional biophysical methods have limited application in addressing the problem because of the heterogeneous nature of the system. In this study, we demonstrated that deep‐UV resonance Raman (DUVRR) spectroscopy in combination with circular dichroism (CD) and intrinsic tryptophan fluorescence allowed for quantitative characterization of protein structural evolution at all stages of hen egg white lysozyme fibrillation in vitro. DUVRR spectroscopy was found to be complimentary to the far‐UV CD because it is (i) more sensitive to β ‐sheet than to α ‐helix, and (ii) capable of characterizing quantitatively inhomogeneous and highly light‐scattering samples. In addition, phenylalanine, a natural DUVRR spectroscopic biomarker of protein structural rearrangements, exhibited substantial changes in the Raman cross section of the 1000‐cm^–1 band at various stages of fibrillation. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Online monitoring of horseradish peroxidase structural changes by Near Infrared (NIR) Spectroscopy

The development of online monitoring techniques is of great relevance for understanding the structural changes of proteins under different conditions in order to maximize their catalytic activity. This study aimed to evaluate the potential of the NIR (near-infrared spectroscopy) technique for the monitoring of alterations of secondary and tertiary structures of Horseradish peroxidase (HRP), an oxidoreductase that has several applications in the industrial environment, food industry and bioremediation. The NIR associated to the multivariate calibration, through the PLS (partial least square) method allowed the construction of a robust model for the prediction of the analysis. The values of the correlation coefficient (R²), root mean square error of calibration (RMSEC), root mean square error of prediction (RMSEP) and root mean square error of cross validation (RMSECV) for secondary structure analysis using circular dichroism (CD) data as reference (actual values) were 0.9681, 0.647 (mdeg), 0.945 (mdeg), and 1.12 (mdeg), respectively. For tertiary structure analysis, fluorescence spectroscopy (FL) data were used as reference. R², RMSEC, RMSEP and RMSECV were, respectively 0.9999, 1.95 (a.u.), 2.09 (a.u.); and 2.19 (a.u.). NIR combined multivariate calibration showed promising results for sctructural changes monitoring of HRP.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

Axial chirality in biaryl N,N-dialkylaminopyridine derivatives bearing an internal carboxy group

Axial chirality in N,N-dimethylaminopyridines as well as N,N-dipropylaminopyridines bearing an internal carboxy group were evaluated based on their racemization barriers and circular dichroism spectra. The half-life of racemization of N,N-dipropylaminopyridine derivative 2 was estimated to be 19.7 days at 20°C. Its enantiomers isolated as optically active forms showed positive-negative and negative-positive Cotton effects for (+)- 2 and (−)- 2 , respectively, from 310 to 210 nm. Furthermore, (−)- 2 was applied as a chiral nucleophilic catalyst and exhibited asymmetric induction in acylative kinetic resolution of 1-(1-naphthyl)ethane-1-ol.     

大腹园蛛管状腺丝重组蛋白的克隆表达及纺丝

TuSp1蛋白(tubuliform spidroin 1)是管状腺丝(tubuliform silkfiber)的主要组成成分。管状腺丝作为蛛丝卵袋的外层包卵丝,其结构具有很好的耐腐蚀性和良好的力学性能。目前国内外对大腹园蛛TuSp1蛋白的研究很少,仅有一条基因序列的报道。本课题首次构建含大腹园蛛N端非重复结构域、重复单元以及C端非重复结构域的重组管状腺丝蛋白TuSp1 NT-Rp-CT,并经湿法纺丝获得重组蛋白丝纤维。重组蛋白液圆二色谱分析结果显示,pH由7.0降低到5.5的过程中,始终保持稳定的α-螺旋构象;重组蛋白丝纤维的傅里叶变换红外光谱结果显示,丝纤维中主要二级结构为β-折叠及β-转角;经扫描电镜观察发现,冻干的絮状重组蛋白能自组装成丝纤维,且表面光滑纤细;湿纺后的重组蛋白丝纤维直径较粗,但表面较平整均匀,具有类似天然管状腺丝的形态特征,这些为TuSp1蛋白的成丝机理及仿生纺丝研究提供了理论依据。     

Hysteresis in poly‐2′‐deoxycytidine i‐motif folding is impacted by the method of analysis as well as loop and stem lengths

In DNA, i‐motif (iM) folds occur under slightly acidic conditions when sequences rich in 2′‐deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pH_T) between the folded and unfolded states. Two different experiments to determine pH_T values via circular dichroism (CD) spectroscopy were performed on poly‐dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pH_T values were dependent on the titration experiment performed, and (2) pH‐induced denaturing or annealing processes produced isothermal hysteresis in the pH_T values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly‐dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly‐dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pH_T parameter measured, and hysteresis was observed in the pH_T and T_m values.     

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase (MT) have been investigated at two temperatures of 20 and 30°C in 10 mM phosphate buffer solution, pHs 5.3 and 6.8. The results show that benzenethiol can inhibit both activities of mushroom tyrosinase competitively. The inhibitory effect of benzenethiol on the cresolase activity is more than the catecholase activity of MT. The inhibition constant (K_i) value at pH 5.3 is smaller than that at pH 6.8 for both enzyme activities. However, the K_i value increases in cresolase activity and decreases in catecholase activity due to the increase of temperature from 20 to 30°C at both pHs. Moreover, the effect of temperature on K_i value is more at pH 6.8 for both cresolase and catecholase activities. The type of binding process is different in the two types of MT activities. The binding process for catecholase inhibition is only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic, meanwhile the electrostatic interaction can be important for cresolase inhibition due to the enthalpy driven binding process. Fluorescence and circular studies also show a minor change in the tertiary structure, without any change in the secondary structure, of the enzyme due to the electrostatic interaction in cresolase inhibition by benzenethiol at acidic pH.     

Combined multispectroscopic and molecular docking investigation on the interaction between strictosamide and human serum albumin

The interaction between strictosamide (STM) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy, circular dichroism spectroscopy and molecular modeling under physiological pH 7.4. STM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding site number n and apparent binding constant K_a were determined at different temperatures by fluorescence quenching. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated as ?3.01 kJ/mol and 77.75 J/mol per K, respectively, which suggested that the hydrophobic force played major roles in stabilizing the HSA–STM complex. The distance r between donor and acceptor was obtained to be 4.10 nm according to Förster's theory. After the addition of STM, the synchronous fluorescence and three‐dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the circular dichroism spectral results showed that the α‐helix content of HSA decreased (from 61.48% to 57.73%). These revealed that the microenvironment and conformation of HSA were changed in the binding reaction. Furthermore, the study of molecular modeling indicated that STM could bind to site I of HSA and the hydrophobic interaction was the major acting force, which was in agreement with the binding mode study. Copyright © 2013 John Wiley & Sons, Ltd.