Charge-Modulated Accessibility of Tyrosine Residues for Silk-Elastin Copolymer Cross-Linking.

The modulation of reaction kinetics with horseradish peroxidase (HRP)-catalyzed cross-linking of proteins remains a useful strategy to modulate hydrogel formation. Here, we demonstrate that the presence of positively charged lysines in silk-elastin-like polymers impacts the thermal transition temperature of these proteins, while the location in the primary sequence modulates the reactivity of the tyrosines. The positively charged lysine side chains decreased π-π interactions among the tyrosines and reduced the rate of formation and number of HRP-mediated dityrosine bonds, dependent on the proximity of the charged group to the tyrosine. The results suggest that the location of repulsive charges can be used to tailor the reaction kinetics for enzymatic cross-linking, providing further control of gelation rates for in situ gel formation and the resulting protein-based gel characteristics.

An Image-Analysis-Based Method for the Prediction of Recombinant Protein Fiber Tensile Strength.

Silk fibers derived from the cocoon of silk moths and the wide range of silks produced by spiders exhibit an array of features, such as extraordinary tensile strength, elasticity, and adhesive properties. The functional features and mechanical properties can be derived from the structural composition and organization of the silk fibers. Artificial recombinant protein fibers based on engineered spider silk proteins have been successfully made previously and represent a promising way towards the large-scale production of fibers with predesigned features. However, for the production and use of protein fibers, there is a need for reliable objective quality control procedures that could be automated and that do not destroy the fibers in the process. Furthermore, there is still a lack of understanding the specifics of how the structural composition and organization relate to the ultimate function of silk-like fibers. In this study, we develop a new method for the categorization of protein fibers that enabled a highly accurate prediction of fiber tensile strength. Based on the use of a common light microscope equipped with polarizers together with image analysis for the precise determination of fiber morphology and optical properties, this represents an easy-to-use, objective non-destructive quality control process for protein fiber manufacturing and provides further insights into the link between the supramolecular organization and mechanical functionality of protein fibers.

Conducting microhelices from self-assembly of protein fibrils.

Herein we utilize insulin to prepare amyloid based chiral helices with either right or left handed helicity. We demonstrate that the helices can be utilized as structural templates for the conducting polymer alkoxysulfonate poly(ethylenedioxythiophene) (PEDOT-S). The chirality of the helical assembly is transferred to PEDOT-S as demonstrated by polarized optical microscopy (POM) and Circular Dichroism (CD). Analysis of the helices by conductive atomic force microscopy (c-AFM) shows significant conductivity. In addition, the morphology of the template structure is stabilized by PEDOT-S. These conductive helical structures represent promising candidates in our quest for THz resonators.

Controlling amyloid fibril formation by partial stirring.

Many proteins undergoe self-assembly into fibrillar structures known as amyloid fibrils. During the self-assembly process, related structures known as spherulites can be formed. Herein we report a facile method where the balance between amyloid fibrils and spherulites can be controlled by stirring of the reaction mixture during the initial stages of the self-assembly process. Moreover, we report how this methodology can be used to prepare non-covalently functionalized amyloid fibrils. By stirring the reaction mixture continuously or for a limited time during the lag phase, the fibril length, and hence the propensity to form liquid crystalline phases, can be influenced. This phenomena is utilized in order to prepare films consisting of aligned protein fibrils incorporating the laser dye Nile red. The resulting films display polarized Nile red fluorescence.

Development and application of methodology for rapid screening of potential amyloid probes.

Herein, we demonstrate that it is possible to rapidly screen hydrophobic fluorescent aromatic molecules with regards to their properties as amyloid probes. By grinding the hydrophobic molecule with the amyloidogenic protein insulin, we obtained a water-soluble composite material. When this material is dissolved and exposed to conditions promoting amyloid formation, the protein aggregates into amyloid fibrils incorporating the hydrophobic molecule. As a result, changes in the fluorescence spectra of the hydrophobic molecule can be correlated to the formation of amyloid fibrils, and the suitability of the hydrophobic molecular skeleton as an amyloid probe can thus be assessed. As a result, we discovered two new amyloid probes, of which one is the well-known laser dye DCM. The grinding method can also be used for rapid preparation of novel composite materials between dyes and proteins, which can be used in materials science applications such as organic electronics and photonics.