Vectorially oriented monolayers of detergent-solubilized Ca(2+) -ATPase from sarcoplasmic reticulum.
A method for tethering proteins to solid surfaces has been utilized to form vectorially oriented monolayers of the detergent-solubilized integral membrane protein Ca(2+) -ATPase from the sarcoplasmic reticulum (SR). Bifunctional, organic self-assembled monolayers (SAMs) possessing "headgroup" binding specificity for the substrate and "endgroup" binding specificity for the enzyme were utilized to tether the enzyme to the substrate. Specifically, an amine-terminated 11-siloxyundecaneamine SAM was found to bind the Ca(2+)-ATPase primarily electrostatically. The Ca(2+)-ATPase was labeled with the fluorescent probe 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid before monolayer formation. Consequently, fluorescence measurements performed on amine-terminated SAM/enzyme monolayers formed on quartz substrates served to establish the nature of protein binding. Formation of the monolayers on inorganic multilayer substrates fabricated by molecular beam epitaxy made it possible to use x-ray interferometry to determine the profile structure for the system, which was proved correct by x-ray holography. The profile structures established the vectorial orientation of the Ca(2+)-ATPase within these monolayers, to a spatial resolution of approximately 12 A. Such vectorially oriented monolayers of detergent-solubilized Ca(2+)-ATPase from SR make possible a wide variety of correlative structure/function studies, which would serve to elucidate the mechanism of Ca(2+) transport by this enzyme.
Prokop
L A
LA
Department of Chemistry, University of Pennsylvania, Philadelphia 19104, USA. prokop@jkb.chem.upenn.edu
Stongin
R M
RM
Smith
A B
AB
Blasie
J K
JK
Peticolas
L J
LJ
Bean
J C
JC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003902:Detergents; D005456:Fluorescent Dyes; D006696:Holography; D018482:Muscle, Skeletal / Q000201:enzymology; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D003902:Detergents; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C006422:1,8-I-AEDANS; D000252:Calcium-Transporting ATPases; D002118:Calcium