Building a novel vitronectin assay by immobilization of integrin on calixarene monolayer.
Membrane proteins possess significant hydrophobic domains and are likely to deplete their native activity immobilized on the solid surface relative to those occurring in a membrane environment. To investigate an efficient immobilization method, calix[4]crown-ether monolayer as an artificial protein linker system was constructed on the gold surface and characterized by Fourier transform infrared reflection absorption spectroscopy (FTIR-RAS), atomic force microscopy (AFM) and cyclic voltammetry (CV). Integrin alpha(v)beta3 was functionally immobilized onto the monolayer and the integrin-vitronectin interaction was investigated by surface plasmon resonance (SPR). It was found that calix[4]crown-ether was assembled as a monolayer on the gold surface. Orientation and accessibility of integrin alpha(v)beta3 was assessed by sensitive binding of its natural ligand, vitronectin at pg mL(-1) level. Moreover, surface coverage of integrin layer and thickness calculated through SPR curve simulation verified that integrin layer was a monolayer in activated form. In combination with the SPR method, this calix[4]crown monolayer provided a reliable and simple experimental platform for the investigation of isolated membrane proteins under experimental conditions resembling those of their native properties.
Chen
Hongxia
H
College of Pharmacy, Pusan National University, Pusan 609-735, Republic of Korea.
Lee
Minsu
M
Lee
Jaebeom
J
An
Won Gun
WG
Choi
Heung-Jin
HJ
Kim
Sung-Hoon
SH
Koh
Kwangnak
K
D047250:Calixarenes / Q000737:chemistry*; D004563:Electrochemistry; D039302:Integrin alphaVbeta3 / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D015394:Molecular Structure; D017550:Spectroscopy, Fourier Transform Infrared; D020349:Surface Plasmon Resonance / Q000379:methods; D019096:Vitronectin / Q000737:chemistry*
D039302:Integrin alphaVbeta3; D019096:Vitronectin; D047250:Calixarenes