Fabrication and structural studies of vectorially oriented monolayers of calcium-ATPase from sarcoplasmic reticulum

Lisa Ann Prokop, University of Pennsylvania

Abstract

Detailed information concerning the structure of the Ca$\sp{2{+}}$-ATPase from the sarcoplasmic reticulum and its relation to the functional characteristics of the enzyme would provide much insight into its mechanism of active calcium transport. An ideal means of studying this enzyme would be to perform detailed structural and functional investigations simultaneously on the same system. Equally important to understanding the Ca$\sp{2{+}}$ transport mechanism by the Ca$\sp{2{+}}$-ATPase is knowledge of its atomic resolution three-dimensional structure, which is currently not available. Vectorially oriented monolayers of Ca$\sp{2{+}}$-ATPase are a significant step toward a complete characterization of both the structural and functional aspects of this enzyme. In this work, detergent-solubilized Ca$\sp{2{+}}$-ATPase has been tethered to solid substrates via bifunctional, organic self-assembled monolayers (SAMs). Amine-terminated SAMs possessing "headgroup" binding specificity for the substrate and "endgroup" binding specificity for the enzyme were employed to unidirectionally tether the enzyme to solid surfaces. The Ca$\sp{2{+}}$-ATPase was labeled with the fluorescent probe 1,5-IAEDANS prior to monolayer formation. Fluorescence measurements were then performed on amine-terminated SAM/enzyme monolayers formed on quartz substrates to assess the nature of protein binding. The interaction between the Ca$\sp{2{+}}$-ATPase and the amine-terminated SAM was found to be primarily electrostatic. Formation of the monolayers on inorganic substrates fabricated by molecular beam epitaxy (MBE) enabled the use of the techniques of X-ray interferometry and X-ray holography to determine the profile structure for the system. The profile structure established the vectorial orientation of the Ca$\sp{2{+}}$-ATPase within these monolayers, to a spatial resolution of $\sim$12 A. An atomic force microscopy (AFM) experiment on tethered Ca$\sp{2{+}}$-ATPase monolayers provided an estimate of the in-plane enzyme monolayer density. The reproducibility and relative ease with which these monolayers can be formed make this system ideal for further structural investigations by a variety of techniques. In addition, the unidirectional nature of these Ca$\sp{2{+}}$-ATPase monolayers will enable correlative structure/functions studies which would ultimately serve to elucidate the mechanism of active Ca$\sp{2{+}}$ transport by this enzyme.

Subject Area

Biochemistry|Biophysics|Cellular biology|Chemistry

Recommended Citation

Prokop, Lisa Ann, "Fabrication and structural studies of vectorially oriented monolayers of calcium-ATPase from sarcoplasmic reticulum" (1996). Dissertations available from ProQuest. AAI9627990.
https://repository.upenn.edu/dissertations/AAI9627990

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