Outline and computational approaches of protein misfolding.

Protein misfolding is a general causation of classical conformational diseases and many pathogenic changes that are the result of structural conversion. Here I review recent progress in clinical and computational approaches for each stage of the misfolding process, aiming to present readers an outline for swift comprehension of this field.

Soluble Angiopoietin Receptor Tie-2 In Patients With Acute Myocardial Infarction And Its Detection By Optical Protein-Chip

The Tie-2 receptor has been shown to play a role in angiogenesis in atherosclerosis. The conventional method assaying the level of soluble Tie-2 (sTie-2) was ELISA. However, this method has some disadvantages. The aims of this research are to establish a more simple detection method, the optical protein-chip based on imaging ellipsomtry (OPC-IE) applying to Tie-2 assay. The sTie-2 biosensor surface on silicon wafer was prepared first, and then serum levels of sTie-2 in 38 patients with AMI were measured on admission (day 1), day 2, day 3 and day 7 after onset of chest pain and 41 healthy controls by ELISA and OPC-IE in parallel. Median level of sTie-2 increased significantly in the AMI patients when compared with the controls. Statistics showed there was a significant correlation in sTie-2 results between the two methods (r=0.923, P0.01). The result of this study showed that the level of sTie-2 increased in AMI, and OPC-IE assay was a fast, reliable, and convenient technique to measure sTie-2 in serum.

Influence Of Micro-Impurity On Protein Crystal Growth Studied By The Etch Figure Method

The investigation of the effect of micro impurity on crystal growth by optical microscopy has been validated. The results showed that the growth rate of a lysozyme crystal was affected even if the concentration of impurity of fluorescent-labeled lysozyme (abbreviation, F-lysozyme) was very small. Different concentrations of F-lysozyme had different effects on crystal growth rate. The growth rate decreased much more as F-lysozyme concentration increased. The density of incorporated F-lysozyme on different grown layers of a lysozyme crystal during crystal growth was obtained from the results of flat-bottomed etch pits density. (C) 2008 Elsevier B.V. All rights reserved.

Direct observation of adsorption sites of protein impurities and their effects on step advancement of protein crystals

We measured noninvasively step velocities of elementary two-dimensional (2D) islands on {110} faces of tetragonal lysozyme crystals, under various supersaturations, by laser confocal microscopy combined with differential interference contrast microscopy. We studied the correlation between the effects of protein impurities on the growth of elementary steps and their adsorption sites on a crystal surface, using three kinds of proteins: fluorescent-labeled lysozyme (F-lysozyme), covalently bonded dimers of lysozyme (dimer), and a 18 kDa polypeptide (18 kDa). These three protein impurities suppressed the advancement of the steps. However, they exhibited different supersaturation dependencies of the suppression of the step velocities. To clarify the cause of this difference, we observed in situ the adsorption sites of individual molecules of F-lysozyme and fluorescent-labeled dimer (F-dimer) on the crystal surface by single-molecule visualization. We found that F-lysozyme adsorbed preferentially on steps (i.e., kinks), whereas F-dimer adsorbed randomly on terraces. Taking into account the different adsorption sites of F-lysozyme and F-dimer, we could successfully explain the different effects of the impurities on the step velocities. These observations strongly suggest that 18 kDa also adsorbs randomly on terraces. Seikagaku lysozyme exhibited a complex effect that could not alone be explained by the two major impurities (dimer and 18 kDa) present in Seikagaku lysozyme, indicating that trace amounts of other impurities significantly affect the step advancement.

The adsorption of protein molecules at a crystal/solution interface observed by an improved TIRFM

We have improved the ordinary total internal reflection fluorescence microscopy (TIRFM). Two improvements have been achieved, one is the interface between opaque material and solution can be observed, another is the interface far away (usually several ten micro meters) the objective lens can be observed. By this improved TIRFM, the adsorption of protein molecules at a crystal/solution interface had been successfully observed. We have obtained the results of relationship between the amount of adsorbed protein molecules on bunched steps and the height of bunched steps of a protein crystal.