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Ligand Binding Studied by 2D IR Spectroscopy Using the Azidohomoalanine Label


Bloem, Robbert; Koziol, Klemens; Waldauer, Steven A; Buchli, Brigitte; Walser, Reto; Samatanga, Brighton; Jelesarov, Ilian; Hamm, Peter (2012). Ligand Binding Studied by 2D IR Spectroscopy Using the Azidohomoalanine Label. Journal of Physical Chemistry B, 116(46):13705-13712.

Abstract

We explore the capability of the azidohomoalanine (Aha) as a vibrational label for 2D IR spectroscopy to study the binding of the target peptide to the PDZ2 domain. The Aha label responds sensitively to its local environment and its peak extinction coefficient of 350-400 M(-1) cm(-1) is high enough to routinely measure it in the low millimolar concentration regime. The central frequency, inhomogeneous width and spectral diffusion times deduced from the 2D IR line shapes of the Aha label at various positions in the peptide sequence is discussed in relationship to the known X-ray structure of the peptide bound to the PDZ2 domain. The results suggest that the Aha label introduces only a small perturbation to the overall structure of the peptide in the binding pocket. Finally, Aha is a methionine analog that can be incorporated also into larger proteins at essentially any position using protein expression. Altogether, Aha thus fulfills the requirements a versatile label should have for studies of protein structure and dynamics by 2D IR spectroscopy.

Abstract

We explore the capability of the azidohomoalanine (Aha) as a vibrational label for 2D IR spectroscopy to study the binding of the target peptide to the PDZ2 domain. The Aha label responds sensitively to its local environment and its peak extinction coefficient of 350-400 M(-1) cm(-1) is high enough to routinely measure it in the low millimolar concentration regime. The central frequency, inhomogeneous width and spectral diffusion times deduced from the 2D IR line shapes of the Aha label at various positions in the peptide sequence is discussed in relationship to the known X-ray structure of the peptide bound to the PDZ2 domain. The results suggest that the Aha label introduces only a small perturbation to the overall structure of the peptide in the binding pocket. Finally, Aha is a methionine analog that can be incorporated also into larger proteins at essentially any position using protein expression. Altogether, Aha thus fulfills the requirements a versatile label should have for studies of protein structure and dynamics by 2D IR spectroscopy.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry

07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:570 Life sciences; biology
540 Chemistry
Language:English
Date:2012
Deposited On:21 Dec 2012 12:38
Last Modified:05 Apr 2016 16:11
Publisher:American Chemical Society
ISSN:1520-5207
Publisher DOI:https://doi.org/10.1021/jp3095209
PubMed ID:23116486

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