Green Fluorescent Protein (GFP) is a widely used fluorescent marker exhibiting two excitation peaks – a strong peak at 398 nm and a second at 475 nm, with the fluorescence at ca. 510 nm. Its molecular structure consists of a β-barrel composed of 11 β-strands and a central helix containing the fluorophore. Two different forms of the fluorophore – a protonated/neutral fluorophore and a de-protonated/anionic fluorophore – are thought to be responsible for the two distinct spectroscopic states. Notably, the isolated fluorophore in solution is efficiently quenched. Conformational flexibility within the protein cavity is an implicitly important factor that governs the photochemistry of GFP. However, the literature contains accounts of studies that reach conflicting conclusions, claiming that either the fluorophore's barrier to internal rotation is negligibly small or that the protein cavity is not complementary to a planar fluorophore. In this work, we calculate the torsional potential of one of the two exocyclic bonds that connect the two rings in the fluorophore, taking into account its immediate environment by applying a quantum mechanics/molecular mechanics method, with the ultimate aim of evaluating the protein-environment effects on the fluorescence.

An approach is developed to study the dynamics of protein conformational transitions in depth. A computational (Monte Carlo) approach based on a united residue model is used. Unbiased transitions between the Apo and Holo conformations/states of calmodulin are observed at the rate of 1 per day per processor. A series of models of increasing complexity is studied, accounting for hydrophobic interactions and calcium binding. Details of the transitional region and structural information about intermediate states are obtained. Statistically converged ensembles of transitions are obtained in a reasonable real time period.

Microtubules (MT) are dynamic protein-based polymers with numerous applications in materials science including the active transport of nanomaterials and as templates for biomimetic materials synthesis. Some of these applications require that the dynamic nature of the MT be suppressed, and in this report we will discuss the preparation and stability of chemically crosslinked microtubules (CLMTs). MT reaction with gluteraldehyde results in the formation of protein dimers and higher molecular weight oligimers as observed by gel electrophoresis, confirming the formation of covalent inter-protein linkages. While extensive crosslinking was found to destabilize MTs and inactivate them with regards to kinesin binding, moderate amounts of crosslinking lead to CLMTs that had functional lifetimes of at least twice that of uncrosslinked MTs. Further studies demonstrated that CLMTs exhibited a wider thermal stability window and were far more resistant to metal-ion induced depolymerization than uncrosslinked MTs.

We use terahertz (THz) spectroscopy as a biomaterials characterization tool. Previously we have shown a strong contrast between the THz dielectric response for wild type (WT) and D96N mutant of bacteriorhodopsin. In those studies we observed a large increase in the THz absorbance of WT with excitation to thermally captured photo-intermediates whereas no such increase in absorbance was observed for the mutant D96N. These results suggest that the THz response is sensitive to structural changes and relative flexibility of biomolecules. However the photo-intermediate populations of the WT and D96N samples were not equivalent in those measurements. While the WT samples had relaxed (bR), M and P state intermediates present, the D96N samples had only bR and M states. Here we present terahertz absorbance measurements of D96N as a function of M and P state populations at room temperature. The THz response is constant for intermediate states populations up to 23% M state and up to 30% P state. These results verify that there is a fundamental difference in the conformational dynamics as measured by THz dielectric response for a single residue mutation.

Biomimetic surfaces were prepared by chemisorption of oxidized dextran (Mw = 110 kDa) onto SiO2 substrates that were previously modified with aminopropyl-tri-ethoxy silane (APTES). The kinetics of dextran oxidation by sodium metaperiodate (NaIO4) were quantified by 1H NMR and pH measurements. The extent of oxidation was then used to control the morphology of the biomimetic surface. Oxidation times of 0.5, 1, 2, 4, and 24 hours resulted in <20, ∼30, ∼40, ∼50 and 100% oxidation, respectively. The surfaces were characterized by contact angle analysis and atomic force microscopy (AFM). Surfaces prepared with low oxidation times revealed a more densely packed “brushy” layer when imaged by AFM than those prepared at low oxidation times. Finally, the contact angle data revealed, quite unexpectedly, that the surface with the greatest entropic freedom (0.5 h) wetted the fastest and to the greatest extent (θAPTES>θ1h>θ2,4h>θ0.5h).