Synthesis of new peptidic glycoclusters derived from β-alanine

The synthesis of an asymmetric glycocluster 1 has been achieved by coupling of a sugar unit with the β-alanine polypeptide, the principal chain, and combining a carbohydrate chain with the side chain causing it to branch from the N terminal. The synthesis of this side chain multivalent ligands is based on the scaffolding of some ω-amino acid (glycine, β-alanine, and GABA) derivatives. This method facilitated the synthesis of the cluster, of which the length of each unit differs.     

Gas Hydrate Formation in Reversed Micelles

We describe a technique to modify protein solubility and optimize enzyme activity in reversed micellar solutions. The technique is based on the ability of hydrates of natural gas to form in the micro-aqueous phase. Clathrate hydrates are crystalline inclusions of water and gas, and their formation in bulk water has traditionally been studied with relevance to natural gas recovery. We have found that hydrates can form in the environment of the microaqueous pools of reversed micelles, and that their extent of formation can be well controlled through the thermodynamic variables of temperature and pressure. Additionally, formation of hydrates affects the size and aggregation number of the micelles, and thus influences the solubility and conformation of encapsulated proteins. We demonstrate how the concept can be used in two applications: (i) protein extraction into reversed micelles and subsequent recovery, and (ii) optimization of enzyme activity in reversed micelles.     

新冠病毒刺突蛋白及其受体的结构与相互作用

Biochemistry is a branch subject of chemistry, which is the study of chemical structures and processes associated with living organisms. COVID-19 pandemic is a problem for human beings. From the perspective of biochemistry, this paper demonstrates the chemical structure and interactions of SARS-CoV-2's spike protein and its receptor (human angiotensin converting enzyme 2), and summarizes the related research progresses. The authors hope to provide insights for the development of COVID-19 vaccine.     

Comparison of linear-scaling semiempirical methods and combined quantum mechanical/molecular mechanical methods for enzymic reactions. II. An energy decomposition analysis

QM/MM methods have been developed as a computationally feasible solution to QM simulation of chemical processes, such as enzyme-catalyzed reactions, within a more approximate MM representation of the condensed-phase environment. However, there has been no independent method for checking the quality of this representation, especially for highly nonisotropic protein environments such as those surrounding enzyme active sites. Hence, the validity of QM/MM methods is largely untested. Here we use the possibility of performing all-QM calculations at the semiempirical PM3 level with a linear-scaling method (MOZYME) to assess the performance of a QM/MM method (PM3/AMBER94 force field). Using two model pathways for the hydride-ion transfer reaction of the enzyme dihydrofolate reductase studied previously (Titmuss et al., Chem Phys Lett 2000, 320, 169-176), we have analyzed the reaction energy contributions (QM, QM/MM, and MM) from the QM/MM results and compared them with analogous-region components calculated via an energy partitioning scheme implemented into MOZYME. This analysis further divided the MOZYME components into Coulomb, resonance and exchange energy terms. For the model in which the MM coordinates are kept fixed during the reaction, we find that the MOZYME and QM/MM total energy profiles agree very well, but that there are significant differences in the energy components. Most significantly there is a large change (approximately 16 kcal/mol) in the MOZYME MM component due to polarization of the MM region surrounding the active site, and which arises mostly from MM atoms close to (<10 A) the active-site QM region, which is not modelled explicitly by our QM/MM method. However, for the model where the MM coordinates are allowed to vary during the reaction, we find large differences in the MOZYME and QM/MM total energy profiles, with a discrepancy of 52 kcal/mol between the relative reaction (product-reactant) energies. This is largely due to a difference in the MM energies of 58 kcal/mol, of which we can attribute approximately 40 kcal/mol to geometry effects in the MM region and the remainder, as before, to MM region polarization. Contrary to the fixed-geometry model, there is no correlation of the MM energy changes with distance from the QM region, nor are they contributed by only a few residues. Overall, the results suggest that merely extending the size of the QM region in the QM/MM calculation is not a universal solution to the MOZYME- and QM/MM-method differences. They also suggest that attaching physical significance to MOZYME Coulomb, resonance and exchange components is problematic. Although we conclude that it would be possible to reparameterize the QM/MM force field to reproduce MOZYME energies, a better way to account for both the effects of the protein environment and known deficiencies in semiempirical methods would be to parameterize the force field based on data from DFT or ab initio QM linear-scaling calculations. Such a force field could be used efficiently in MD simulations to calculate free energies.     

Analyses of carcinogenic aromatic amines released from harmful azo colorants by Streptomyces SP. SS07

Extracellular fluid protein (ECFP) of Streptomyces species SS07 has been used to reduce water soluble azo dyes and the carcinogenic amines released have been compared with that from chemical reduction. The effect of temperature, pH and contact time on the recovery of amines using ECFP was studied. The ECFP releases carcinogenic amines at a pH of 9.2 and a temperature of 37 degrees C for a contact period of 24 h. The reduction products were analyzed with HPLC and their structures confirmed by LC-MS and GC-MS. It was observed that both the ECFP and chemical reduction methods released similar type of amine products. In the case of dye samples, compared to chemical reduction, 5-20% increase in the release of carcinogenic amines by ECFP was observed. The percentage of amine products released by chemical reduction was higher for leather garment samples compared to ECFP treatment.     

MALDI-TOF-MS法对重组人内皮抑素蛋白分子质量测定

肿瘤的生长依赖于血管的生成,新生血管不仅为肿瘤生长提供必需的营养物质,而且为肿瘤细胞扩散提供了重要的途径。1997年哈佛大学的O'Reilly等发现了一种内源性新血管生成抑制因子内皮抑素(Endoscatin),显示出特异抑制激活的血管内皮细胞增殖和肿瘤新血管生成的生物学活性,其抗肿瘤作用具有高效、低毒、无耐药性的优点。目前,内皮抑素的研究引起了国内外广泛的兴趣,在美国已进行以安全性为目的的I期临床实验,国内也有多家公司对内皮抑素进行了抗肿瘤研究并申报一类新药。内皮抑素有望成为医治肿瘤而又没有化疗和放疗的毒副作用的一种新的治疗方法,但是否能作为药物应用于临床,尚需对内皮抑素的结构特点及抑制肿瘤和内皮细胞的作用机制等方面进行许多深入的研究。     

A comparison of the adsorption of saliva proteins and some typical proteins onto the surface of hydroxyapatite

Adsorption of protein from saliva on hydroxyapatite was compared with adsorption of several typical proteins with different electric charges, i.e. lysozyme, human serum albumin, β-lactoglobulin and ovalbumin. Adsorbed amounts of these proteins were determined and electrophoretic mobilities of protein-covered hydroxyapatite particles were measured, at different values for the adsorbed mass and, therefore, at various degrees of surface coverage. Also, adsorption kinetics were investigated by streaming potential measurements of a hydroxyapatite surface in contact with a protein solution, allowing monitoring of changes in the zeta-potential of the protein-covered hydroxyapatite surface in real time. The adsorbed amounts show that, as compared to most of the other proteins, the saliva proteins have remarkably low adsorption affinity. The measured values for the electrophoretic mobilities indicate that the positively charged proteins in the saliva mixture preferentially adsorb onto the negatively charged hydroxyapatite surface; this is most pronounced at low protein concentration in solution (i.e. at low coverage of the surface by the protein). Preferential uptake of the positively charged saliva proteins during the initial stages of the adsorption process is also concluded from the results of the kinetics experiments. Preferential adsorption of positive proteins is somewhat suppressed by the presence of Ca²⁺ ions in the medium. The results suggest that an acquired pellicle on a tooth in an oral environment contains a significant fraction of positively charged proteins. The positively charged proteins in the pellicle reduce the zeta-potential at the tooth surface to low values; consequently, electrostatic forces are expected to play only a minor role in the interaction with other components (e.g. bacterial cells).     

Synthesis and Expression in Escherichia coli of a Human Neutrophil Activating Protein-1/Interleukin-8 Gene

The complete gene coding for human neutrophilactivating protein-1/interleukin-8 was synthesized using a semi-chemical semi-enzymatic method. The synthetic gene was then overexpressed in Escherichia coli under the temperature-regulated control of the P_RP_L tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein comprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. The recombinant NAP-1/IL-8 was then purified to>95% homogeneity by gel filtration and cation exchange chromatography. The purified protein appeared as a single band on the SDS-PAGE gel and possessed potent chemotactic activity in the concentration of <10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP1/IL-8 was determined using the Edman method and was sho     

Generic method for systematic phase selection and method development of biochromatographic processes. Part I. Selection of a suitable cation-exchanger for the purification of a pharmaceutical protein

Even if the first protein therapeutics are now for more than 20 years on the market the selection of suitable adsorbents for the preparative downstream processing (DSP) of these biomolecules as well as the method development towards process conditions are still based mainly on 'trial and error'. Therefore, theses processes are not perfectly efficient, but indeed very time consuming and laborious. In this study a novel systematic method is introduced to find a suitable adsorbent (not necessarily the best one) with appropriate separation parameters for a specific separation with reduced effort. Following this strategy, the adsorbents must first be packed into columns under preparative conditions and then characterized completely with regard to, e.g. pressure drop, k'-values, plate heights (HETP curves), selectivity and capacity by using test substances, which are similar in their characteristics (molecular mass, size, charge distribution, hydrophobicity) to the target proteins. With the database once determined, a preselection of most suitable adsorbents including separation parameters is made regarding chromatographic and also economical properties. After this, preparative experiments must be conducted with a reduced number of adsorbents to figure out the individual influence of side components. This approach is demonstrated for the separation of an exemplary industrial protein mixture using cation-exchange chromatography (CEX). Characterization of different weak CEX-adsorbents is illustrated. After comparing these phases with each other, a first preselection and a prediction of suitable adsorbents is made. In the following preparative separation conditions (load, velocity, gradient) are determined for the preparative separations using the database and results of some additional experiments. The final comparison of separation performance in preparative scale confirms this selection and so the applicability of the new method.     

Synthesis of [18,19,21,22‐13C4]‐Labeled Tautomycin as an NMR Probe of Protein Phosphatase Inhibitor

We have accomplished the synthesis of ¹³C‐labeled tautomycin at the C18, C19, C21, and C22 positions starting from 100 % [¹³C]triethylphosphonoacetate for the purpose of elucidating the dynamics and conformation of the C17–C26 moiety. NMR spectroscopy of ¹³C‐labeled tautomycin revealed strong binding with protein phosphatase type 1 and new features in the ¹³C NMR spectrum, such as the very small three‐bond coupling constants (²J).