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2D-IR Spectroscopy of an AHA Labeled Photoswitchable PDZ2 Domain


Stucki-Buchli, Brigitte; Johnson, Philip J M; Bozovic, Olga; Zanobini, Claudio; Koziol, Klemens L; Hamm, Peter; Gulzar, Adnan; Wolf, Steffen; Buchenberg, Sebastian; Stock, Gerhard (2017). 2D-IR Spectroscopy of an AHA Labeled Photoswitchable PDZ2 Domain. Journal of Physical Chemistry. A, 121(49):9435-9445.

Abstract

We explore the capability of the non-natural amino acid azidohomoalanine (AHA) as an IR label to sense relatively small structural changes in proteins with the help of 2D IR difference spectroscopy. To that end, we AHA-labeled an allosteric protein (the PDZ2 domain from human tyrosine-phosphatase 1E) and furthermore covalently linked it to an azobenzene-derived photoswitch as to mimic its conformational transition upon ligand binding. To determine the strengths and limitations of the AHA label, in total six mutants have been investigated with the label at sites with varying properties. Only one mutant revealed a measurable 2D IR difference signal. In contrast to the commonly observed frequency shifts that report on the degree of solvation, in this case we observe an intensity change. To understand this spectral response, we performed classical MD simulations, evaluating local contacts of the AHA labels to water molecules and protein side chains and calculating the vibrational frequency on the basis of an electrostatic model. Although these simulations revealed in part significant and complex changes of the number of intraprotein and water contacts upon trans-cis photoisomerization, they could not provide a clear explanation of why this one label would stick out. Subsequent quantum-chemistry calculations suggest that the response is the result of an electronic interaction involving charge transfer of the azido group with sulfonate groups from the photoswitch. To the best of our knowledge, such an effect has not been described before.

Abstract

We explore the capability of the non-natural amino acid azidohomoalanine (AHA) as an IR label to sense relatively small structural changes in proteins with the help of 2D IR difference spectroscopy. To that end, we AHA-labeled an allosteric protein (the PDZ2 domain from human tyrosine-phosphatase 1E) and furthermore covalently linked it to an azobenzene-derived photoswitch as to mimic its conformational transition upon ligand binding. To determine the strengths and limitations of the AHA label, in total six mutants have been investigated with the label at sites with varying properties. Only one mutant revealed a measurable 2D IR difference signal. In contrast to the commonly observed frequency shifts that report on the degree of solvation, in this case we observe an intensity change. To understand this spectral response, we performed classical MD simulations, evaluating local contacts of the AHA labels to water molecules and protein side chains and calculating the vibrational frequency on the basis of an electrostatic model. Although these simulations revealed in part significant and complex changes of the number of intraprotein and water contacts upon trans-cis photoisomerization, they could not provide a clear explanation of why this one label would stick out. Subsequent quantum-chemistry calculations suggest that the response is the result of an electronic interaction involving charge transfer of the azido group with sulfonate groups from the photoswitch. To the best of our knowledge, such an effect has not been described before.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Language:English
Date:14 December 2017
Deposited On:09 Mar 2018 12:35
Last Modified:18 Apr 2018 11:49
Publisher:American Chemical Society (ACS)
ISSN:1089-5639
Funders:European Research Council (ERC) Advanced Investigator Grant (DYNALLO), Swiss National Science Foundation (SNF) through the NCCR MUST, Swiss National Science Foundation (SNF), Deutsche Forschungsgemeinschaft
OA Status:Closed
Publisher DOI:https://doi.org/10.1021/acs.jpca.7b09675
Project Information:
  • : Funder
  • : Grant ID
  • : Project TitleEuropean Research Council (ERC) Advanced Investigator Grant (DYNALLO)
  • : FunderSNSF
  • : Grant ID
  • : Project TitleSwiss National Science Foundation (SNF) through the NCCR MUST
  • : FunderSNSF
  • : Grant ID
  • : Project TitleSwiss National Science Foundation (SNF)
  • : Funder
  • : Grant ID
  • : Project TitleDeutsche Forschungsgemeinschaft

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