Immobilization and AFM of single 4 x 6-mer tarantula hemocyanin molecules.
The high molecular mass respiratory protein of the tarantula Eurypelma californicum, a 4 x 6-mer hemocyanin, was investigated by atomic force microscopy (AFM). Various substrates and methods were evaluated for immobilization of individual hemocyanin molecules on a solid surface. Samples were imaged after physisorption on mica and self-assembled monolayers, and after chemisorption on Au(111) and N-hydroxy-succinimide (NHS) functionalized surfaces. AFM measurements were carried out preferable in solution and contact mode, but also in Tapping mode and on air-dried samples. Adsorption of the protein on mica followed by drying and carrying out the measurements in Tapping mode gave the best results. In the AFM images the four hexamers of the native 4 x 6-mer hemocyanin have been defined. The results were compared with independent available structural data and represent a validation case for this technique applied for the first time on such giant and complex molecules. As observable in images taken by transmission electron microscopy and also proposed from SAXS data, 4 x 6-mers could be found where the half-molecules are tilted against each other. This study is a step in resolving conformational heterogeneities, involved in oxygen binding of hemocyanins, at the single-molecule level by AFM.
Erker
Wolfgang
W
Institute for Physical Chemistry, University Mainz, Germany. erker@mail.uni-mainz.de
Scheumann
Volker
V
Möller
Marco
M
Knoll
Wolfgang
W
Rühe
Jürgen
J
Decker
Heinz
H
D000388:Air; D000538:Aluminum Silicates; D000818:Animals; D005898:Glass; D006046:Gold; D006433:Hemocyanins / Q000302:isolation & purification / Q000648:ultrastructure*; D007089:Image Enhancement / Q000379:methods; D018625:Microscopy, Atomic Force; D011309:Preservation, Biological / Q000379:methods*; D013112:Spiders / Q000737:chemistry; D013388:Succinimides
D000538:Aluminum Silicates; D013388:Succinimides; D006046:Gold; D006433:Hemocyanins; C001426:N-hydroxysuccinimide; C011934:mica