Flexibility Analysis of Bacillus thuringiensis Cry1Aa / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the flexibility and mobility of the Bacillus thuringiensis toxin Cry1Aa.</p><p><b>METHODS</b>The graph theory-based program Constraint Network Analysis and normal mode-based program NMsim were used to analyze the global and local flexibility indices as well as the fluctuation of individual residues in detail.</p><p><b>RESULTS</b>The decrease in Cry1Aa network rigidity with the increase of temperature was evident. Two phase transition points in which the Cry1Aa structure lost rigidity during the thermal simulation were identified. Two rigid clusters were found in domains I and II. Weak spots were found in C-terminal domain III. Several flexible regions were found in all three domains; the largest residue fluctuation was present in the apical loop2 of domain II.</p><p><b>CONCLUSION</b>Although several flexible regions could be found in all the three domains, the most flexible regions were in the apical loops of domain II.</p>

Interactions between proteins and cation exchange adsorbents analyzed by NMR and hydrogen/deuterium exchange technique / 生物工程学报

In silico acquirement of the accurate residue details of protein on chromatographic media is a bottleneck in protein chromatography separation and purification. Here we developed a novel approach by coupling with H/D exchange and nuclear magnetic resonance to observe hen egg white lysozyme (HEWL) unfolding behavior adsorbed on cation exchange media (SP Sepharose FF). Analysis of 1D 1H-NMR shows that protein unfolding accelerated H/D exchange rate, leading to more loss of signal of amide hydrogen owing to exposure of residues and the more unfolding of protein. Analysis of two-dimensional hydrogen-hydrogen total correlation spectroscopy shows that lysozyme lost more signals and experienced great unfolding during its adsorption on media surface. However, for several distinct fragments, the protection degrees varied, the adsorbed lysozyme lost more signal intensity and was less protected at disorder structures (coil, bend, and turn), but was comparatively more protected against exchange at secondary structure domains (α-helix, β-sheet). Finally, the binding site was determined by electrostatic calculations using computer simulation methods in conjunction with hydrogen deuterium labeled protein and NMR. This study would help deeply understand the microscopic mechanism of protein chromatography and guide the purposely design of chromatographic process and media. Moreover, it also provide an effective tool to study the protein and biomaterials interaction in other applications.

Effect of inactivation and reactivation conditions on activity recovery of enzyme catalysts

Enzymes are labile catalysts with reduced half-life time that can be however improved by immobilization and, furthermore, already inactivated catalyst can be recovered totally or partially, therefore allowing the large scale application of enzymes as process catalysts. In recent years a few studies about reactivation of enzyme catalysts have been published as a strategy to prolong the catalyst lifetime. Reported results are very good, making this strategy an interesting tool to be applied to industrial process. These studies have been focused in the evaluation of different variables that may have a positive impact both in the rate and level of activity recovery, being then critical variables for conducting the reactivation process at productive scale. The present work summarizes the studies done about reactivation strategies considering different variables: type of immobilization, enzyme-support interaction, level of catalyst inactivation prior to reactivation, temperature and presence of modulators.

In silico evaluation of cross linking effects on denaturant meq values and delta Cp upon protein unfolding

Important thermodynamic parameters including denaturant equilibrium m values [meq] and heat capacity changes [delta Cp] can be predicted based on changes in Solvent Accessible Surface Area [SASA] upon unfolding. Cross links such as disulfide bonds influence the stability of the proteins by decreasing the entropy gain as well as reduction of SASA of unfolded state. The aim of the study was to develop mathematical models to predict the effect of cross links on delta SASA and ultimately on meq and delta Cp based on in silico methods. Changes of SASA upon computationally simulated unfolding were calculated for a set of 45 proteins with known meq and delta Cp values and the effect of cross links on delta SASA of unfolding was investigated. The results were used to predict the meq of denaturation for guanidine hydrochloride and urea, as well as delta Cp for the studied proteins with overall error of 20%, 31% and 17%, respectively. The results of the current study were in close agreement with those obtained from the previous studies

Mechanisms Underlying Trabecular Meshwork Cell Death Caused by Mutant Myocilin Expression

PURPOSE: To determine whether the expression of mutant myocilin can lead to death of human trabecular meshwork (HTM) cells and to determine whether the mechanism by which this occurs is apoptosis. METHODS: HTM cells were transduced with a recombinant adenovirus expressing human mutant (Q368X) myocilin. The apoptotic death of HTM cells caused by expression of mutant myocilin was examined using a cell proliferation assay, flow cytometry, Western blot analysis, and immunocytochemistry. RESULTS: It appeared that the expression of mutant myocilin itself was not sufficient to cause HTM cell death. Furthermore, the expression of mutant myocilin did not lead to apoptosis of HTM cells although it did elicit a protein unfolding response. CONCLUSIONS: Our data suggest that the mechanism of myocilin glaucoma is not apoptotic death of HTM cells caused by mutant myocilin expression, and that the actual mechanism remains unknown.

Immobilization of lipase labeled with fluorescent probe and its stability / 生物工程学报

The lipase labeled with the fluorescein isothiocyanat (FITC) was immobilized on the derivatives of the polyethylene glycol. The article discussed the effect of factors on the characters of lipase and analyzed the relationships among the activity of lipase, conformation, and fluorescence spectrum while the activity and the fluorescence spectrum of immobilized lipase were determined. The results demonstrated that polyethylene glycol 400-diacrylate could form appropriate network to improve the activity of enzyme. Adding ligand induced the lipase's catalytic conformation to increase the activity twice more than before. The active centre of lipase could be released by the extraction of ligand thus increasing the activity. After immobilization, the stability of labeled lipase improved greatly: immobilized lipases retained more than 70% and 60% of initial activity under conditions of 90 degrees C and strong acid or alkali, respectively. After immersing immobilized lipases into guanidine hydrochloride or urea for 15 days, the lipases retained upwards of 70% activity. The fluorescence spectrum could obviously reflect the changes of the activity and conformation of lipase. The fluorescence intensity was the minimum in the optimal pH and temperature. In the denaturing agent it declined as time passed. These results indicated that the unfolded processes of immobilized lipases are different under different conditions.

Atomic force microscopy involved in protein study / 生物医学工程学杂志

Atomic force microscopy is a rather new type of nano microscopic technology. It has some advantages, such as high resolution (sub-nano scale); avoidance of special sample preparation; real-time detection of samples under nearly physiological environment; in situ study of samples under water environment; feasibility of investigating physical and chemical properties of samples at molecular level, etc. In recent years, the application of atomic force microscopy in protein study has brought about outstanding achievements. In this paper are introduced the principle and operation modes of atomic force microscopy, also presented are its application in protein imaging, adsorption, folding-and-unfolding, assembly, and single molecular recognition. Additionally, the future application of atomic force microscopy in protein study is prospected.