Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Electrochemical and Structural Properties of a Protein System Designed To Generate Tyrosine Pourbaix Diagrams
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Pennsylvania, USA .
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Show others and affiliations
2011 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, no 44, 17786-17795 p.Article in journal (Refereed) Published
Abstract [en]

This report describes a model protein specifically tailored to electrochemically study the reduction potential of protein tyrosine radicals as a function of pH. The model system is based on the 67-residue alpha(3)Y three-helix bundle, alpha(3)Y contains a single buried tyrosine at position 32 and displays structural properties inherent to a protein. The present report presents differential pulse voltammograms obtained from alpha(3)Y at both:acidic (pH 5.6) and alkaline (pH 8.3) Conditions. The. observed Faradaic. response is uniquely associated. with Y32, as shown by site-directed mutagenesis. This is the first time voltammetry is successfully applied to detect a redox-active tyrosine residing in a structured protein environment. Tyrosine is a proton coupled electron transfer cofactor making voltammetry-based pH titrations a central experimental approach. A second set of experiments was performed to demonstrate that pH-dependent studies can be conducted on the redox-active tyrosine without introducing large-scale structural changes in the protein scaffold alpha(3)Y was re-engineered-with the specific aim to place the imidazole group of a histidine close to the Y32 phenol ring alpha(3)Y-K29H and alpha(3)Y-K36H each contain a histidine residue whose protonation perturbs the fluorescence of Y32. We show that these variants are stable and well-folded proteins whose helical: content, tertiary structure, solution aggregation state, and solvent-sequestered position of Y32 remain pH insensitive across a range of at least 3-4 pH units. These results confirm that the local environment of Y32 can be altered and the resulting radical site studied by voltammetry over a broad pH range without interference from long-range structural effects.

Place, publisher, year, edition, pages
2011. Vol. 133, no 44, 17786-17795 p.
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-69903DOI: 10.1021/ja206876hISI: 000296312200047OAI: oai:DiVA.org:su-69903DiVA: diva2:479734
Note

authorCount :6

Available from: 2012-01-18 Created: 2012-01-15 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Using de novo design proteins to explore tyrosine radicals and cation-π interactions
Open this publication in new window or tab >>Using de novo design proteins to explore tyrosine radicals and cation-π interactions
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Redox cofactors and amino-acid free radicals play important roles in biology. Although many of the same cofactors and amino acids that form these radicals are found across a broad range of biological systems, identical cofactors can have different reduction potentials. The local environment plays a role in defining these redox potentials. An understanding of this local-environment effect can shed more light on how redox chemistry works in nature. Our laboratory has developed a library of model proteins that are well suited to study amino-acid radicals. a3X is a de novo designed protein that is composed of 67 residues. It forms a three-helix bundle connected by two glycine loops. The radical site is located at position 32 on the central a-helix. The a3X protein is designed to be well-folded and thermodynamically stable across a broad pH range. Paper 1 describes the structural and electrochemical characterization of a3Y, a tyrosine variant of a3X. We were able to obtain a unique Faradaic response from Y32 at both low and high pH, using differential pulse voltammetry. In addition, we successfully redesigned α3Y by introducing a histidine in close proximity to Y32, creating a tyrosine/histidine pair. Our goal in creating this pair was to study proton-coupled electron transfer (PCET) in a well-structured and solvent-sequestered protein environment.  In paper 2 we illustrated the redox reversibility of Y32 and produced the first ever Pourbaix diagram for a tyrosine radical in a protein. The formal potential of the Y32-OŸ/Y32-OH redox couple was determined to be 918 ± 2 mV vs. the normal hydrogen electrode (NHE) at pH 8.40.  While at pH 5.52, the formal potential of the Y32-OŸ/Y32-OH redox couple was recorded at 1.07 V. Papers 3 and 4 utilize a3W to study cation-π interactions. In paper 3, we showed how solvation can affect the strength of these interactions by -0.9 kcal/mol. In Paper 4, we were able to monitor the disruption of the cation-π interaction with the use of high-pressure fluorescence and were able to calculate the interaction energy for a solvent exposed cation-π. The aim of the work described in this thesis was to use model proteins to study tyrosine radicals to gain a broader perspective and better understanding of the versatility of biological electron transfer and to measure cation-π interactions and how they behave in different environments.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 69 p.
Keyword
tyrosine, radicals, cation-pi interactions, de novo designed proteins, biochemistry, biophysics
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-102008 (URN)978-91-7447-885-3 (ISBN)
Public defence
2014-05-09, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2014-04-14 Created: 2014-03-20 Last updated: 2014-04-14Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Berry, Bruce W.
By organisation
Department of Biochemistry and Biophysics
In the same journal
Journal of the American Chemical Society
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 30 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf