In vivo resolution of oligomers with fluorescence photobleaching recovery histograms.

Simple independent enzyme-catalyzed reactions distributed homogeneously throughout an aqueous environment cannot adequately explain the regulation of metabolic and other cellular processes in vivo. Such an unstructured system results in unacceptably slow substrate turnover rates and consumes inordinate amounts of cellular energy. Current approaches to resolving compartmentalization in living cells requires the partitioning of the molecular species in question such that its localization can be resolved with fluorescence microscopy. Standard imaging approaches will not resolve localization of protein activity for proteins that are ubiquitously distributed, but whose function requires a change in state of the protein. The small heat shock protein sHSP27 exists as both dimers and large multimers and is distributed homogeneously throughout the cytoplasm. A fusion of the green fluorescent protein variant S65T and sHSP27 is used to assess the ability of diffusion rate histograms to resolve compartmentalization of the 2 dominant oligomeric species of sHSP27. Diffusion rates were measured by multiphoton fluorescence photobleaching recovery. Under physiologic conditions, diffusion rate histograms resolved at least 2 diffusive transport rates within a living cell potentially corresponding to the large and small oligomers of sHSP27. Given that oligomerization is often a means of regulation, compartmentalization of different oligomer species could provide a means for efficient regulation and localization of sHsp27 activity.

Cellulose as an inert matrix for presenting cytokines to target cells: production and properties of a stem cell factor-cellulose-binding domain fusion protein.

A chimaera of stem cell factor (SCF) and a cellulose-binding domain from the xylanase Cex (CBDCex) effectively immobilizes SCF on a cellulose surface. The fusion protein retains both the cytokine properties of SCF and the cellulose-binding characteristics of CBDCex. When adsorbed on cellulose, SCF-CBDCex is up to 7-fold more potent than soluble SCF-CBDCex and than native SCF at stimulating the proliferation of factor-dependent cell lines. When cells are incubated with cellulose-bound SCF-CBDCex, activated receptors and SCF-CBDCex co-localize on the cellulose matrix. The strong binding of SCF-CBDCex to the cellulose surface permits the effective and localized stimulation of target cells; this is potentially significant for long-term perfusion culturing of factor-dependent cells. It also permits the direct analysis of the effects of surface-bound cytokines on target cells.

Surface diffusion of cellulases and their isolated binding domains on cellulose.

The surface diffusion rate of bacterial cellulases from Cellulomonas fimi on cellulose was quantified using fluorescence recovery after photobleaching analysis. Studies were performed on an exo-beta-1-4-glycanase (Cex), an endo-beta-1-4-glucanase (CenA), and their respective isolated cellulose-binding domains (CBDs). Although these cellulose-binding domains bind irreversibly to microcrystalline cellulose, greater than 70% of bound molecules are mobile on the cellulose surface. Surface diffusion rates are dependent on surface coverage and range from a low of 2 x 10(-11) to a maximum of 1.2 x 10(-10) cm2/s. The fraction of mobile molecules increases only slightly with increasing fractional surface coverage density. Results demonstrate that the packing of C. fimi cellulases and their isolated binding domains onto the cellulose surface is a dynamic process. This suggests that the exclusion of potential CBD binding sites on the cellulose due to steric effects of neighboring bound CBDs may not fully explain the apparent negative cooperativity exhibited in CBD adsorption isotherms. Comparison with the kinetics of cellulase hydrolysis of crystalline substrate suggests that surface diffusion rates do not limit cellulase activity.