Computational and mutagenesis studies of the streptavidin native dimer interface.

Wt streptavidin forms a domain swapped tetramer consisting of two native dimers. The role of tetramerization has been studied previously and is known to contribute to biotin binding by allowing the exchange of W120 between adjacent subunits. However, the role of dimer formation in streptavidin folding and function has been largely overlooked to date, although native dimers are necessary for tetramer formation and thus for high affinity biotin binding. To understand how the side chain interactions at the dimer interface stabilize the subunit association, we studied the structural and functional consequences of introducing interfacial mutations by a combination of molecular dynamics (MD) simulation and biochemical characterization. In particular, we introduced rational mutations at the dimer interface to engineer new side chain interactions and measured the stability and function of the resulting mutants. We focused on two residues that form a "knob" and a "hole" pair, G74 and T76, since steric complementarity plays an important role at these positions. We introduced mutations that would change the polarity and side chain packing to test if the interface can be rationally redesigned. Both energy calculation and geometric parameterization were used to interpret the simulated structures and predict how the mutations affect the dimer stability. In this regard, obtaining precise energy estimates was difficult because the simulated structures have large stochastic variations and some mutants did not reach an equilibrium by the end of the simulation. In contrast, comparing the wt and mutants to one another and parameterizing the simulation using a geometric parameter, i.e. the degree of solvation of the buried interface, resulted in a testable prediction regarding which mutations would result in a stable dimer. We present experimental data (denaturation and binding measurements) to show that an intuitive parameter based on physical reasoning can be useful for characterizing simulations that are difficult to analyze quantitatively.

Flow cytometric analysis of genetic FRET detectors containing variable substrate sequences.

A genetic Fluorescence Resonance Energy Transfer (FRET) detector undergoes a post-translational modification (PTM)-induced conformational change that results in increased FRET. To test if the PTM-dependent FRET change can be quantified by flow cytometry, we purified and immobilized a genetic detector on microbeads and used flow cytometry to measure its FRET efficiency before and after Erk-2-mediated phosphorylation. The fluorescence ratio R between the acceptor and donor fluorescence, which was obtained by fitting a straight line through the data points in linear space, increases following phosphorylation, thus demonstrating that flow cytometry is capable of detecting a PTM-dependent FRET response. Furthermore, when Erk-2 and a genetic detector are coexpressed in bacteria, the measured R value changes with the substrate sequence with near single residue resolution. Similarly, the cells coexpressing the glycosylating enzyme O-GlcNAc transferase (OGT) and a genetic detector specific for OGT exhibit a PTM-induced change in FRET efficiency. Therefore, the combination of flow cytometry and a genetic detector may be useful to characterize the substrate specificity of a PTM enzyme and identify the sequences that are preferentially targeted for PTM in vivo.

Cell-free xenogenic vascular grafts fixed with glutaraldehyde or genipin: in vitro and in vivo studies.

Chronic rejection of arterial xenografts results in aneurysmal dilation, due to immune mediated processes. To minimize the immunologic degradation of the graft, a cell-extraction process employing sodium dodecyl sulfate (SDS) was used in the study to remove the cellular components in bovine carotid arteries. To further reduce their immunogenicity, the acellular arteries were fixed with glutaraldehyde (A-GA) or genipin (A-GP). The in vitro properties of all test samples were analyzed. Additionally, the in vivo performance of the heparinized A-GA and A-GP grafts (H-A-GA and H-A-GP) was evaluated in a canine model. It was found that the SDS treatment effectively removed cells from the arterial wall, but the main structures of the extracellular matrix were preserved with a portion of the water-soluble glycosaminoglycans removed. After cell extraction, the elastic lamellae in the media became straightened, and thus made the tissue less extensile. The heparinized tissues significantly reduced platelet adhesion. At retrieval, all implanted grafts were patent and not dilated. Chronic inflammatory response surrounding the implants was observed. However, fixation of acellular tissues by glutaraldehyde or genipin inhibited immune cell penetration into the media and limited tissue degradation, and therefore prevented the arterial wall from dilation. Nevertheless, the H-A-GP graft was superior to the H-A-GA graft in completeness of endothelialization on its luminal surface, and thus precluded thrombus formation.

Acellular bovine pericardia with distinct porous structures fixed with genipin as an extracellular matrix.

A cell extraction process was employed to remove the cellular components from bovine pericardia. Various porous structures of the acellular tissues were then created, using acetic acid and collagenase, and subsequently fixed with genipin. The biological response and tissue regeneration pattern for each studied group were evaluated in a growing rat model. One month postoperatively, fibroblasts, neoconnective tissue fibrils, and neocapillaries were observed in the acellular, acetic acid-treated, and collagenase-treated tissues to fill the pores within the implanted samples, indicating that these tissue samples were being regenerated. The neoconnective tissue fibrils were identified to be neocollagen fibrils and neoglycosaminoglycans. On the other hand, no tissue regeneration was observed in the cellular tissue throughout the entire course of the study; tissue regeneration was limited to the outer most layer of the acellular tissue. In contrast, the areas of tissue regeneration in the acetic acid-treated and collagenase-treated tissues were expanded with increasing duration of implantation. However, 1 year postoperatively there were still numerous inflammatory cells observed in the acetic acid-treated tissue, whereas inflammatory cells in the collagenase-treated tissue had almost disappeared. These results indicated that tissue regeneration patterns within acellular tissues were significantly affected by their porous structures.

Effects of crosslinking degree of an acellular biological tissue on its tissue regeneration pattern.

It was reported that acellular biological tissues can provide a natural microenvironment for host cell migration and may be used as a scaffold for tissue regeneration. To reduce antigenicity, biological tissues have to be fixed with a crosslinking agent before implantation. As a tissue-engineering scaffold, it is speculated that the crosslinking degree of an acellular tissue may affect its tissue regeneration pattern. In the study, a cell extraction process was employed to remove the cellular components from bovine pericardia. The acellular tissues then were fixed with genipin at various known concentrations to obtain varying degrees of crosslinking. It was shown in the in vitro degradation study that after fixing with genipin, the resistance against enzymatic degradation of the acellular tissue increased significantly with increasing its crosslinking degree. In the in vivo subcutaneous study, it was found that cells (inflammatory cells, fibroblasts, endothelial cells, and red blood cells) were able to infiltrate into acellular tissues. Generally, the depth of cell infiltration into the acellular tissue decreased with increasing its crosslinking degree. Infiltration of inflammatory cells was accompanied by degradation of the acellular tissue. Due to early degradation, no tissue regeneration was observed within fresh (without crosslinking) and the 30%-degree-crosslinking acellular tissues. This is because the scaffolds provided by these two samples were already completely degraded before the infiltrated cells began to secrete their own extracellular matrix. In contrast, tissue regeneration (fibroblasts, neo-collagen fibrils, and neo-capillaries) was observed for the 60%- and 95%-degree-crosslinking acellular tissues by the histological examination, immunohistological staining, transmission electron microscopy, and denaturation temperature measurement. The 95%-degree-crosslinking acellular tissue was more resistant against enzymatic degradation than its 60%-degree-crosslinking counterpart. Consequently, tissue regeneration was limited in the outer layer of the 95%-degree-crosslinking acellular tissue throughout the entire course of the study (1-year postoperatively), while tissue regeneration was observed within the entire sample for the 60%-degree-crosslinking acellular tissue. In conclusion, the crosslinking degree determines the degradation rate of the acellular tissue and its tissue regeneration pattern.