One-, Two-, and Three-Photon Absorption Studies of Fluorescent Proteins and Their Chromophores Using Quantum Mechanical and Quantum Mechanical/Molecular Mechanical Approaches

  • Author / Creator
    Rossano Tapia, Maria del Sagrario
  • In the present thesis, the multi-photon absorption features of different Aequorea fluorescent proteins (FPs) are explored computationally using quantum mechanical and combined quantum mechanical/molecular mechanical approaches. In chapters two and three of the present thesis, we evaluate the performance of the semi-empirical time-dependent tight-binding density functional theory and its long-range corrected version, TD-DFTB and LC-TD-DFTB, respectively, in the computation of the two-photon absorption (2PA) cross-sections for a set of canonical and non-canonical FPs chromophores previously studied using time-dependent density functional theory (TD-DFT). From these investigations, we found that through the two-level model (2LM), TD-DFTB and LC-TD-DFTB lead to 2PA cross-sections that largely deviate from the TD-DFT values obtained in previous investigations. In comparison with TD-CAM-B3LYP, a common DFT method used in the computation of 2PA cross-sections of FPs and their chromophores, the 2PA cross-sections obtained using LC-TD-DFTB are up to 125 GM larger. Such a deviation is mainly due to an overestimation of the excited state permanent dipole moment, on which the 2PA cross-sections are strongly dependent. Chapter four explores the computation of the three-photon absorption of the intrinsic probe serotonin, as well as the dyes, fluorescein and rhodamine 6g, which are used as references in multi-photon absorption studies. The results obtained here, using a series of basis sets along with the CAM-B3LYP functional, are in reasonably good agreement with experiment. Furthermore, we found that the CAM-B3LYP/aug-cc-pVDZ method is the best option as it exhibits the best accuracy with respect to experimental results to computing cost ratio among the tested basis sets. In the case of rhodamine 6g, the three-photon absorption cross-section we obtained using the CAM-B3LYP/aug-ccpvDZ method in vacuum is close to the average measurement of different experimental sources. Experimental and computational investigations have concluded that the MPA of a given FP depends on the interaction between its chromophore and the environment, which is comprised of the protein and other moieties, such as water molecules, if present. Therefore, a given FP chromophore can exhibit a 2PA cross-section of either 10 GM or 80 GM, depending on the environment in which it is embedded. In this context, chapter five discusses the 2PA of a set of non-canonical chromophores while we consider the environmental effects through the DsRed protein barrel. The 2PA cross-sections obtained for these systems using the polarizable quantum mechanical/molecular mechanical (QM/MM) approach are about four times smaller than results reported without taking into account the protein effects (in vacuum). These results reinforce the idea of including the environment effects in the computation of multi-photon absorption properties of FPs to get a more "realistic" view of their photophysical properties. Finally, chapter six assesses some of the possible paths to engineer FPs with enhanced 2PA in comparison to the existent FPs. In this chapter, the 2PA cross-section of the chromophore surrounded by a selected set of neighbouring amino acids is computed using the polarizable QM/MM approach. This investigation is not conclusive, however, as from the results obtained, it was determined that some neighbouring amino acids can enhance the 2PA of the red fluorescent protein chromophore up to 130%. The examination of the roles of specific amino acids provides information about possible positions that can be mutated in order to engineer FPs with enhanced multi-photon absorption in comparison to the existing ones. Overall, the results presented here will be useful as benchmark to those wanting to study the MPA features of FPs.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.