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231224s2012 xx |||||o 00| ||eng c |
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|a 10.1021/la203907t
|2 doi
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|a pubmed24n0712.xml
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|a (DE-627)NLM213662469
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|a (NLM)22148958
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|a DE-627
|b ger
|c DE-627
|e rakwb
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|a eng
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|a Baio, Joe E
|e verfasserin
|4 aut
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|a Probing the orientation of electrostatically immobilized Protein G B1 by time-of-flight secondary ion spectrometry, sum frequency generation, and near-edge X-ray adsorption fine structure spectroscopy
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|c 2012
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|a Text
|b txt
|2 rdacontent
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|a ƒaComputermedien
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|2 rdamedia
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|a ƒa Online-Ressource
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|2 rdacarrier
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|a Date Completed 23.05.2012
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|a Date Revised 21.10.2021
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|a published: Print-Electronic
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|a Citation Status MEDLINE
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|a © 2011 American Chemical Society
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|a To fully develop techniques that provide an accurate description of protein structure at a surface, we must start with a relatively simple model system before moving to increasingly complex systems. In this study, X-ray photoelectron spectroscopy (XPS), sum frequency generation spectroscopy (SFG), near-edge X-ray adsorption fine structure (NEXAFS) spectroscopy, and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to probe the orientation of Protein G B1 (6 kDa) immobilized onto both amine (NH(3)(+)) and carboxyl (COO(-)) functionalized gold. Previously, we have shown that we could successfully control orientation of a similar Protein G fragment via a cysteine-maleimide bond. In this investigation, to induce opposite end-on orientations, a charge distribution was created within the Protein G B1 fragment by first substituting specific negatively charged amino acids with neutral amino acids and then immobilizing the protein onto two oppositely charged self-assembled monolayer (SAM) surfaces (NH(3)(+) and COO(-)). Protein coverage, on both surfaces, was monitored by the change in the atomic % N, as determined by XPS. Spectral features within the SFG spectra, acquired for the protein adsorbed onto a NH(3)(+)-SAM surface, indicates that this electrostatic interaction does induce the protein to form an oriented monolayer on the SAM substrate. This corresponded to the polarization dependence of the spectral feature related to the NEXAFS N(1s)-to-π* transition of the β-sheet peptide bonds within the protein layer. ToF-SIMS data demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically within Protein G B1 (methionine: 62 and 105 m/z; tyrosine: 107 and 137 m/z; leucine: 86 m/z). For a more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of secondary-ions stemming from the amino acid residues at either end of the protein. The 2-fold increase in this ratio, observed between the protein covered NH(3)(+) and COO(-) SAMs, indicates opposite orientations of the Protein G B1 fragment on the two different surfaces
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|a Journal Article
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|a Research Support, N.I.H., Extramural
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|a Research Support, Non-U.S. Gov't
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| 650 |
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|a Research Support, U.S. Gov't, Non-P.H.S.
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| 650 |
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|a Bacterial Proteins
|2 NLM
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| 650 |
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|a Immobilized Proteins
|2 NLM
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| 700 |
1 |
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|a Weidner, Tobias
|e verfasserin
|4 aut
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1 |
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|a Baugh, Loren
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Gamble, Lara J
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Stayton, Patrick S
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Castner, David G
|e verfasserin
|4 aut
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| 773 |
0 |
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|i Enthalten in
|t Langmuir : the ACS journal of surfaces and colloids
|d 1992
|g 28(2012), 4 vom: 31. Jan., Seite 2107-12
|w (DE-627)NLM098181009
|x 1520-5827
|7 nnns
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| 773 |
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|g volume:28
|g year:2012
|g number:4
|g day:31
|g month:01
|g pages:2107-12
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|u http://dx.doi.org/10.1021/la203907t
|3 Volltext
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