This grant application seeks to study the biochemical and biomedical implications of modulating fundamental protein biophysical properties (e.g., structure, dynamics, stability, and function) through chemical glycosylation. The main hypothesis is based on the fact that chemical glycosylation effectively reduces protein structural dynamics thereby altering other relevant protein properties (e.g., enzyme kinetics and thermodynamic stability). The development of this novel technology will allow the exploration of a multitude of fundamental scientific questions related to protein structural dynamics. Since the protein dynamics can be shifted gradually by increasing the glycosylation level, we will be able to determine for the first time how protein structural dynamics influences these other properties. Multiple biophysical properties will be determined as a function of glycosylation and their changes statistically correlated. Additionally, molecular modeling and dynamics simulation techniques will be employed to explore the molecular mechanisms by which glycans achieve such effects. These experiments will thus provide fundamental insights regarding the influence of dynamics on the so-called structure-function and structure-stability relationships in proteins. Furthermore, we will study fundamental aspects regarding the stabilization of proteins by chemical glycosylation within biomedically relevant applications, namely, liquid- and solid-state formulations, sustained release devices, and interactions with bio- and nano-materials (SA2-4). Functional stability parameters (e.g., inactivation, aggregate formation) will be correlated with the innate biophysical properties (e.g., structural dynamics, thermodynamic stability) of the glycoconjugates and with the chemical glycosylation variables (e.g., glycan's size and chemical nature, glycosylation level). Experiments are designed to establish the mechanisms of enhanced functional stability by chemical glycosylation. The direct biomedical relevance of the work thus consists in furthering the understanding of the mechanisms by which protein thermodynamic, kinetic, and colloidal stabilities can be increased within therapeutic applications. The long-term goals of this research consist on furthering the development of chemical glycosylation for the study of protein structural dynamics and for the prevention of protein instabilities (e.g., inactivation, aggregation) during storage and delivery of protein therapeutics. Project Relevance. The clinical applicability of protein therapeutics critically depends on preserving their functional and structural intactness. Increasing protein stability by chemical glycosylation in solid and liquid formulations and upon exposure to interfaces (e.g., plastic tubing, surfaces of medical equipment), is essential to ascertain maximum efficiency of the treatment and preventing adverse side effects (e.g., immune reactions caused by administration of aggregated protein). Our research will provide new strategies to enhance protein thermodynamic and kinetic stability.
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