In situ monitoring of the orientated assembly of strep-tagged membrane proteins on the gold surface by surface enhanced infrared absorption spectroscopy.
Surface enhanced infrared absorption spectroscopy (SEIRAS) has been employed to monitor the orientated assembly of a strep-tagged membrane protein on the gold surface via a streptavidin/biotin interlayer. The high surface sensitivity of SEIRAS allows for tracking the individual assembling steps on the molecular level. The sequence of surface modification steps comprises: (i) cross-linking of biotin to the self-assembled monolayer of cysteamine along the gold surface; (ii) adsorption of streptavidin to and desorption from the biotin layer; and (iii) adsorption of the strep-tagged membrane protein ecgltP (glutamate transporter of E. coli) on the streptavidin/biotin layer. The analysis of the SEIRA spectra reveals that the biotin layer undergoes a phase transition from an isotropic orientation to a densely packed layer during coupling to the cysteamine monolayer. Formation of the densely packed layer weakens the interaction between streptavidin and the biotin layer but yields a binding specificity of 80%. The specificity of strep-tagged ecgltP to the streptavidin layer is with 60% only modest. Nevertheless, the streptavidin/biotin interlayer reveals a higher regeneration propensity than the His-tag/Ni-NTA interlayer.
Jiang
Xiue
X
Bielefeld University, Department of Chemistry, Biophysical Chemistry, 33615 Bielefeld, Germany.
Zuber
Anne
A
Heberle
Joachim
J
Ataka
Kenichi
K
D000327:Adsorption; D027322:Amino Acid Transport System X-AG / Q000737:chemistry; D001692:Biological Transport; D001710:Biotin / Q000737:chemistry*; D002627:Chemistry, Physical / Q000379:methods; D003543:Cysteamine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry; D006046:Gold / Q000737:chemistry*; D008956:Models, Chemical; D011485:Protein Binding; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry*; D013499:Surface Properties; D013997:Time Factors
D027322:Amino Acid Transport System X-AG; D005973:Glutamine; D003543:Cysteamine; D001710:Biotin; D006046:Gold; D019809:Streptavidin