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.
Animals ; Humans ; Microscopy, Atomic Force ; Protein Conformation ; Protein Folding ; Protein Unfolding ; Proteins ; chemistry ; ultrastructure

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.
Adsorption ; Amides ; Cations ; Computer Simulation ; Deuterium ; Hydrogen ; Magnetic Resonance Spectroscopy ; Muramidase ; chemistry ; Protein Structure, Secondary ; Protein Unfolding ; Proteins ; chemistry

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.
Dextrans ; chemistry ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; metabolism ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; chemistry ; Fluorescent Dyes ; chemistry ; Lipase ; chemistry ; metabolism ; Polyethylene Glycols ; chemistry ; Protein Unfolding ; drug effects

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.
Adenoviridae ; Apoptosis ; Blotting, Western ; Cell Death ; Cell Proliferation ; Cytoskeletal Proteins ; Eye Proteins ; Flow Cytometry ; Glaucoma ; Glycoproteins ; Humans ; Protein Unfolding ; Trabecular Meshwork

OBJECTIVETo investigate the flexibility and mobility of the Bacillus thuringiensis toxin Cry1Aa.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONAlthough several flexible regions could be found in all the three domains, the most flexible regions were in the apical loops of domain II.

Bacillus thuringiensis ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Cluster Analysis ; Computer Simulation ; Endotoxins ; chemistry ; genetics ; metabolism ; Entropy ; Hemolysin Proteins ; chemistry ; genetics ; metabolism ; Models, Structural ; Mutation ; Protein Conformation ; Protein Unfolding ; Software ; Temperature

BACKGROUND: Our goal was to evaluate anti-calcification effects of decellularization and diverse fixing methods including preincubation of the bovine pericardium with ethanol. We also assessed changes in mechanical properties. MATERIAL AND METHOD: Harvested bovine pericardium was decellularized with 0.25% sodim dodecysulfate and then treated with 5 methods of fixation: (1) 0.5% glutaraldehyde (GA) for 14 days, (2) 0.5% GA for 5 days, 2% GA for 2 days and 0.25% GA for 7 days, (3) 0.5% GA for 5 days, 2% GA for 2 days, 0.25% GA for 7 days, and then 70% ethanol for 2 days, (4) 0.5% GA for 5 days, a mixture of 2% GA and 70% ethanol for 2 days, and 0.25% GA for 7 days, (5) 0.5% GA for 5 days, a mixture of 2% GA, 65% ethanol, and 5% octanediol for 2 days and then 0.25% GA for 7 days. All treated bovine pericardia were tested for histological variables, lipid content, and mechanical properties including tensile strength and thermal stability. A total 10 kinds of differently treated bovine pericardia were implanted into rat subdermis and harvested 8 weeks later. Harvested pericardia were evaluated for calcium content. RESULT: No protein denaturation was observed microscopically after decellularization. There was a 32% mean decrease in tensile strength index after decellularization in the bovine pericardium group fixed. Octanediol preincubation attenuated the decrease in tensile strength and maintained thermal stability. TG and cholesterol were not affected by decellularization but were decreased by organic solvent. Calcium content was decreased after decellularization, and organic solvent preincubation decreased calcification in the non-decellularized bovine pericardium group. CONCLUSION: Decellularization and organic solvent preincubation have anti-calcification effects but decellularization may cause mechanical instability. A method of decellularization and fixation that does not cause damage to matrices will be needed for evaluation of the next step in using tissue-engineering for replacement of cardiac valves.
Animals ; Calcium ; Cholesterol ; Ethanol ; Glutaral ; Heart Valves ; Pericardium ; Protein Denaturation ; Rats ; Tensile Strength ; Tissue Engineering

OBJECTIVETo characterize the relationship between the refolding process of recombinant bovine β-lactoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation.

METHODSThe refolding process of recombinant bovine β-lactoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro.

RESULTSRenaturation of recombinant bovine β-lactoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity.

CONCLUSIONThe degree of protein renaturation correlated with the IgE-binding capacity of the protein. Results from this study may be of help for food allergy therapy and development of vaccination in the future.

Allergens ; Animals ; Cattle ; Circular Dichroism ; Enzyme-Linked Immunosorbent Assay ; Immunoglobulin E ; Lactoglobulins ; chemistry ; metabolism ; Models, Molecular ; Protein Binding ; Protein Conformation ; Protein Denaturation ; Protein Folding ; Spectrometry, Fluorescence ; methods

As a virus-like particle, hepatitis B surface antigen (HBsAg) was the primary component of hepatitis B vaccine. HBsAg was maintained by the non-covalent interaction of proteins and lipids. The intact structure of HBsAg particle was vital to its function. However, there was no report about the effects of solvent environment on HBsAg structure. In this paper, we studied the effects of temperature, pH, ionic type and salt concentration on HBsAg structure. The results showed that HBsAg was stable at normal temperature, but began to denature above 60 degrees C. The aggregation of HBsAg at pH 3.0 and 4.0 was nearly irreversible, but partly reversible at pH 5.0. The influence of ionic type on HBsAg was generally in accordance with Hofmeister sequence, except that SO4(2-) caused more aggregation than F-. HBsAg aggregates started to be visible in 0.4 mol/L (NH4)2SO4, and the extent of aggregation increased with the salt concentration. Therefore, caution must be taken when using (NH4)2SO4 in the hydrophobic chromatography purification of HBsAg.
Ammonium Sulfate ; chemistry ; Hepatitis B Surface Antigens ; chemistry ; Hepatitis B Vaccines ; chemistry ; Hepatitis B virus ; chemistry ; Hydrogen-Ion Concentration ; Protein Denaturation ; Solvents ; Temperature

BACKGROUND: Phenol has effects like surgical neurectomy, but may evoke pain after local infiltration in nerves. Transection of peripheral nerves may induce neuropathic pain through increased spontaneous discharge and other mechanisms. Proto-oncogene, c-fos, is an indicator of neuronal activity, and its expression in the spinal cord may be related to pain development, because inhibition of c-fos expression has corresponding effects like analgesia. We evaluated the effects of local infiltration of phenol and transection injury at the sciatic nerve on c-fos expression in the spinal cords of rats. METHODS: Sixteen male Sprague-Dawley rats were divided into 2 groups; transection of the sciatic nerve was performed for group 1; phenol was infiltrated into the sciatic nerve in group 2. Three hours, 1 week, 2 weeks, and 3 weeks after the experiment, the corresponding spinal cord was stained immunohistochemically for c-fos. RESULTS: c-fos was expressed from 3 hours to 2 weeks over the laminae of the dorsal horn in each group. Phenol increased the expression of c-fos initially, but decreased 1 week later. Transection injury did not increase it initially, but showed the peak expression at 1 week, and maintained it for 2 weeks. Therefore, it seems that phenol, rather than the transection injury, stimulates c-fos expression early, but decreases later. CONCLUSIONS: Phenol treatment, caused by chemical block due to protein denaturation and nonspecific inflammation, may induce less neuropathic pain than the transection of a nerve.
Analgesia ; Animals ; Horns ; Humans ; Inflammation ; Male ; Neuralgia ; Neurons ; Peripheral Nerves ; Phenol* ; Protein Denaturation ; Proto-Oncogenes ; Rats* ; Rats, Sprague-Dawley ; Sciatic Nerve* ; Spinal Cord*

BACKGROUND: Inducible heat shock protein 70s (iHSP70) are expressed by stressful stimuli that result in protein denaturation, and are thought to assist in the maintenance of cellular integrity and viability. In addition, iHSP70 is known to be a sensitive marker of neuronal injury. To my best knowledge, no previous studies have been documented on iHSP70 induction by nociceptive impulse transmission through peripheral nerves not by direct neural damage. The purpose of this study was to examine the hypothesis that iHSP70 can be expressed in the nervous system, which is related to the dental nociceptive pathway, by tooth pulp inflammation. METHODS: The pulp of rat mandibular molars was exposed. Animals were sacrificed at 1, 4, and 7 days after pulpal exposure, and the pulps were evaluated histologically. Also, iHSP70 levels were examined in the Gasserian ganglion (GG) and the trigeminal sensory nucleus (TSN). RESULTS: At 4 days after pulpal exposure, iHSP70 was significantly more expressed in the ipsilateral GG than in the contralateral GG. In the histological study, inflammation was found in the entire pulp tissue at 4 days. There were no significant differences in iHSP70 levels between the ipsilateral TSN and the contralateral TSN. Also, there were no significant differences in iHSP70 expression of GG and TSN between both sides at 1 and 7 days after pulpal exposure. CONCLUSIONS: These results suggest that iHSP70 can be expressed in the GG at 4 days after pulpal exposure by nociceptive impulses due to pulpal inflammation.
Animals ; Heat-Shock Proteins* ; Hot Temperature* ; HSP70 Heat-Shock Proteins* ; Inflammation ; Molar ; Nervous System ; Neurons ; Peripheral Nerves ; Protein Denaturation ; Rats* ; Tooth ; Trigeminal Ganglion