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
Using de novo design proteins to explore tyrosine radicals and cation-π interactions
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
tyrosine, radicals, cation-pi interactions, de novo designed proteins, biochemistry, biophysics
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-102008ISBN: 978-91-7447-885-3 (print)OAI: oai:DiVA.org:su-102008DiVA: diva2:707841
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
List of papers
1. Electrochemical and Structural Properties of a Protein System Designed To Generate Tyrosine Pourbaix Diagrams
Open this publication in new window or tab >>Electrochemical and Structural Properties of a Protein System Designed To Generate Tyrosine Pourbaix Diagrams
Show others...
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.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-69903 (URN)10.1021/ja206876h (DOI)000296312200047 ()
Note

authorCount :6

Available from: 2012-01-18 Created: 2012-01-15 Last updated: 2017-12-08Bibliographically approved
2. Reversible voltammograms and a Pourbaix diagram for a protein tyrosine radical
Open this publication in new window or tab >>Reversible voltammograms and a Pourbaix diagram for a protein tyrosine radical
2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 25, 9739-9743 p.Article in journal (Refereed) Published
Abstract [en]

Reversible voltammograms and a voltammetry half-wave potential versus solution pH diagram are described for a protein tyrosine radical. This work required a de novo designed tyrosine-radical protein displaying a unique combination of structural and electrochemical properties. The alpha Y-3 protein is structurally stable across a broad pH range. The redox-active tyrosine Y32 resides in a desolvated and well-structured environment. Y32 gives rise to reversible square-wave and differential pulse voltammograms at alkaline pH. The formal potential of the Y32-O-center dot/Y32-OH redox couple is determined to 918 +/- 2 mV versus the normal hydrogen electrode at pH 8.40 +/- 0.01. The observation that Y32 gives rise to fully reversible voltammograms translates into an estimated lifetime of >= 30 ms for the Y32-O-center dot state. This illustrates the range of tyrosine-radical stabilization that a structured protein can offer. Y32 gives rise to quasireversible square-wave and differential pulse voltammograms at acidic pH. These voltammograms represent the Y32 species at the upper edge of the quasirevesible range. The square-wave net potential closely approximates the formal potential of the Y32-O center dot/Y32-OH redox couple to 1,070 +/- 1 mV versus the normal hydrogen electrode at pH 5.52 +/- 0.01. The differential pulse voltammetry half-wave potential of the Y32-O-center dot/Y32-OH redox pair is measured between pH 4.7 and 9.0. These results are described and analyzed.

Keyword
protein voltammetry, proton-coupled electron transfer
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-80014 (URN)10.1073/pnas.1112057109 (DOI)000306061400024 ()
Note

AuthorCount:3;

Available from: 2012-09-12 Created: 2012-09-12 Last updated: 2017-12-07Bibliographically approved
3. Environmental modulation of protein cation-pi interactions
Open this publication in new window or tab >>Environmental modulation of protein cation-pi interactions
2007 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 129, no 17, 5308+- p.Article in journal (Refereed) Published
Abstract [en]

Protein cation-pi interactions are frequently found near the protein surface with their interacting residues partly solvent exposed. The structurally characterized alpha W-3 model protein contains the W32/K36 cation-pi interaction which has properties similar to those of naturally occurring protein cation-pi interactions. alpha W-3 was studied with the following results: Cation-pi interactions formed by a buried tryptophan and a partly solvated lysine, arginine, or histidine range from -0.8 to -0.5 kcal mol(-1) and rank as: W32/K36 approximate to W32/R36 > W32/H36. The W32/K36 pair in alpha W-3 represents the first W/K cation-pi interaction for which both the structure and the bond energy have been experimentally determined. Upon increasing the solvent exposure of the cation-pi pair, the W/K interaction energy drops from -0.73 to -0.06 and +0.15 kcal mol(-1). These results suggest that solvent exposure can tune the interaction energy between a tryptophan and a lysine by at least 0.9 kcal mol(-1).

Keyword
alpha-helical peptides, aromatic side-chains, radical enzymes, model, stability
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-56576 (URN)10.1021/ja068957a (DOI)000245946400005 ()
Note

authorCount :3

Available from: 2011-04-26 Created: 2011-04-19 Last updated: 2017-12-11Bibliographically approved
4. Using high-pressure fluorescence as a method to investigate cation-π interactions in model proteins
Open this publication in new window or tab >>Using high-pressure fluorescence as a method to investigate cation-π interactions in model proteins
(English)Manuscript (preprint) (Other academic)
Abstract [en]

This study uses High Pressure fluorescence to obtain interaction energies of weak cation-πinteractions. Since cation-π interactions are believed to contain an electrostatic component; theywould be sensitive to hydrostatic pressure. It has been established that our two model proteins,W33/K37 & W34/K38, contain cation-π interactions that are located in solvated environments.With the use of high pressure Fluorescence we were able to monitor the disruption of the cation-π interaction as well as determine the interaction energy for both solvent exposed pairs,W33/K37 & W34/K38 pairs. The weak interaction energies were found to be 0.08 ± 0.02 & 0.06± 0.02, respectively.

Keyword
de novo design proteins, cation-pi interaction, high pressure fluorescence
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-102113 (URN)
Available from: 2014-03-26 Created: 2014-03-26 Last updated: 2014-03-31

Open Access in DiVA