Probing single proteins in nanofabricated bioenvironment

University Of Washington, Seattle WA

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Abstract

Because most diseases are caused by the malfunctions or perturbations of enzymes, there is a great need to gain a fundamental understanding of enzyme activities and dynamics in the context of a living cell. Absence or defects in the enzymes beta-Hexosaminidase A, Glucocerebrosidase, and Galactosylceramidase, for example, are the origins of the Tay- Sachs, Gaucher, and Krabbe diseases, correspondingly. Most biochemical investigations of enzyme kinetics and behavior, however, are based on in vitro measurements made inside macroscopic containers that do not resemble the complex and highly non-classical reaction environment in a cell. We hypothesize that the rate and mechanisms of enzymatic actions and protein interactions in vivo inside cells differ significantly from the rate and mechanisms observed in vitro inside cuvettes or other macroscopic containers. Based on preliminary single- molecule experiments and computer modeling, we speculate that this difference arises from the extremely high surface-to-volume ratio of the nanoenvironment inside cells, and enzymatic activities can be affected and influenced substantially by interactions with these membrane surfaces. In this project, we will combine three areas of expertise we have developed - single molecule detection and manipulation, synthetic vesicles and planar supported lipid bilayers, micro- / nano- fluidics - for carrying out a systematic study, at the single- molecule level, the influence of membrane surfaces on the rate and mechanism of enzyme kinetics within a high surface-to-volume ratio biomimetic nanoenvironment. Real-time measurements on the spatial and temporal variations of single-protein dynamics can reveal information previously buried under the statistical averaging of the population. We believe these studies will provide fundamentally new information about enzyme kinetics inside ultrasmall subcellular compartments where surface charge and hydrophobicity can exert significant influence n the activity of the contained proteins. Gaining a better understanding of the true in vivo activities of proteins inside cells is critical for developing new drugs that target these proteins for the treatment of devastating diseases.