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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Enhancement Award (SC1)
Project #
5SC1GM086240-03
Application #
8015328
Study Section
Special Emphasis Panel (ZGM1-MBRS-X (CH))
Program Officer
Marino, Pamela
Project Start
2009-01-01
Project End
2012-12-31
Budget Start
2011-01-01
Budget End
2011-12-31
Support Year
3
Fiscal Year
2011
Total Cost
$323,166
Indirect Cost
Name
University of Puerto Rico Rio Piedras
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
143960193
City
San Juan
State
PR
Country
United States
Zip Code
00931
Hernández-Cancel, Griselle; Suazo-Dávila, Damaris; Medina-Guzmán, Johnsue et al. (2015) Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor. Anal Chim Acta 854:129-39
Pagán, Miraida; Suazo, Dámaris; Del Toro, Nicole et al. (2015) A comparative study of different protein immobilization methods for the construction of an efficient nano-structured lactate oxidase-SWCNT-biosensor. Biosens Bioelectron 64:138-46
Morales-Cruz, Moraima; Figueroa, Cindy M; González-Robles, Tania et al. (2014) Activation of caspase-dependent apoptosis by intracellular delivery of Cytochrome c-based nanoparticles. J Nanobiotechnology 12:33
Méndez, Jessica; Morales Cruz, Moraima; Delgado, Yamixa et al. (2014) Delivery of chemically glycosylated cytochrome c immobilized in mesoporous silica nanoparticles induces apoptosis in HeLa cancer cells. Mol Pharm 11:102-11
Delgado, Yamixa; Morales-Cruz, Moraima; Hernández-Román, José et al. (2014) Chemical glycosylation of cytochrome c improves physical and chemical protein stability. BMC Biochem 15:16
Montalvo-Ortiz, Brenda L; Sosa, Brian; Griebenow, Kai (2012) Improved enzyme activity and stability in polymer microspheres by encapsulation of protein nanospheres. AAPS PharmSciTech 13:632-6
Morales-Cruz, Moraima; Flores-Fernández, Giselle M; Morales-Cruz, Myreisa et al. (2012) Two-step nanoprecipitation for the production of protein-loaded PLGA nanospheres. Results Pharma Sci 2:79-85
Flores-Fernandez, Giselle M; Griebenow, Kai (2012) Glycosylation improves ýý-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic)acid microspheres. Results Pharma Sci 2:46-51
Méndez, Jessica; Monteagudo, Alina; Griebenow, Kai (2012) Stimulus-responsive controlled release system by covalent immobilization of an enzyme into mesoporous silica nanoparticles. Bioconjug Chem 23:698-704
Rodriguez-Martinez, Jose A; Rivera-Rivera, Izarys; Griebenow, Kai (2011) Prevention of benzyl alcohol-induced aggregation of chymotrypsinogen by PEGylation. J Pharm Pharmacol 63:800-5

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