Using de novo design proteins to explore tyrosine radicals and cation-π interactions
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
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-O/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-O/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.
tyrosine, radicals, cation-pi interactions, de novo designed proteins, biochemistry, biophysics
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:su:diva-102008ISBN: 978-91-7447-885-3OAI: oai:DiVA.org:su-102008DiVA: diva2:707841
2014-05-09, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Hofer, Anders, Associate Professor
Tommos, Cecilia, Research Assistant Professor
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.2014-04-142014-03-202014-04-14Bibliographically approved
List of papers