All Publications

  • Photosynthetic reaction center variants made via genetic code expansion show Tyr at M210 tunes the initial electron transfer mechanism. Proceedings of the National Academy of Sciences of the United States of America Weaver, J. B., Lin, C., Faries, K. M., Mathews, I. I., Russi, S., Holten, D., Kirmaier, C., Boxer, S. G. 1800; 118 (51)


    Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were engineered to vary the electronic properties of a key tyrosine (M210) close to an essential electron transfer component via its replacement with site-specific, genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and X-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild type (WT). Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via a 4-ps and a 20-ps population to produce the charge-separated state P+HA - in all variants. Global analysis indicates that in the 4-ps population, P+HA - forms through a two-step process, P* P+BA - P+HA -, while in the 20-ps population, it forms via a one-step P* P+HA - superexchange mechanism. The percentage of the P* population that decays via the superexchange route varies from 25 to 45% among variants, while in WT, this percentage is 15%. Increases in the P* population that decays via superexchange correlate with increases in the free energy of the P+BA - intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an 110-meV increase in the free energy of P+BA - along with a dramatic diminution of the 1,030-nm transient absorption band indicative of P+BA - formation. Collectively, this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.

    View details for DOI 10.1073/pnas.2116439118

    View details for PubMedID 34907018

  • Genetic Code Expansion in Rhodobacter sphaeroides to Incorporate Noncanonical Amino Acids into Photosynthetic Reaction Centers. ACS synthetic biology Weaver, J. B., Boxer, S. G. 2018


    Photosynthetic reaction centers (RCs) are the membrane proteins responsible for the initial charge separation steps central to photosynthesis. As a complex and spectroscopically complicated membrane protein, the RC (and other associated photosynthetic proteins) would benefit greatly from the insight offered by site-specifically encoded noncanonical amino acids in the form of probes and an increased chemical range in key amino acid analogues. Toward that goal, we developed a method to transfer amber codon suppression machinery developed for E.coli into the model bacterium needed to produce RCs, Rhodobacter sphaeroides. Plasmids were developed and optimized to incorporate 3-chlorotyrosine, 3-bromotyrosine, and 3-iodotyrosine into RCs. Multiple challenges involving yield and orthogonality were overcome to implement amber suppression in R.sphaeroides, providing insights into the hurdles that can be involved in host transfer of amber suppression systems from E.coli. In the process of verifying noncanonical amino acid incorporation, characterization of this membrane protein via mass spectrometry (which has been difficult previously) was substantially improved. Importantly, the ability to incorporate noncanonical amino acids in R.sphaeroides expands research capabilities in the photosynthetic field.

    View details for PubMedID 29763307