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Computational Study of Two Photon Absorption in Fluorescent Protein Chromophores Open Access


Other title
Fluorescent Proteins
Two-photon absorption
Type of item
Degree grantor
University of Alberta
Author or creator
Salem, Mohammad A A A
Supervisor and department
Brown, Alex
Examining committee member and department
Klobukowski, Mariusz (Chemistry)
Tuszynski, Jack (Oncology)
Campbell, Robert (Chemistry)
Kongsted, Jacob (Physics, Chemistry and Pharmacy; University of Southern Denmark)
Department of Chemistry

Date accepted
Graduation date
2016-06:Fall 2016
Doctor of Philosophy
Degree level
Two-photon absorption (TPA) microscopy of fluorescent proteins (FPs) is a powerful bio-imaging tool. Advantages of TPA microscopy include better focus and less out-of-focus bleaching, together with absorption at longer wavelengths than in one-photon absorption (OPA), which leads to deeper penetration in tissues. However, TPA probes are usually associated with less sensitivity than OPA alternatives and thus designing fluorophores with large TPA probability (cross section) is an important area of research. A great variety of FPs have been synthesized from canonical amino acids and characterized for both their OPA and TPA properties. Although a small number of non-canonical amino acids (ncAAs) have been utilized in designing new FPs, they were not characterized for their TPA properties. In addition, incorporating ncAAs in FPs is a demanding task and thus preceding the experiment with a computational rationalization that guides the choice of ncAAs is prudent. The unique light-absorbing and fluorescence ability of FPs is due to the formation of a chromophore by a post-translational modification of three precursory amino acids within the protein shell. While the protein environment can strongly affect the photophysical properties of the chromophore, the goal of this work is to highlight ncAA-modified chromophores that have computationally large intrinsic TPA cross sections. Given the size of the chromophores, time-dependent density functional theory (TD-DFT) is the method of choice to scan their TPA properties. The TD-DFT results (using four functionals) on a given set of natural chromophores were compared to a wave-function-based method (CC2) and to averaged experimental data from the FPs; unlike OPA data, TPA measurements on isolated chromophore analogues have not been made. The comparison shows that TD-DFT with B3LYP underestimates the absolute TPA cross sections, but can be used in a semi-quantitative fashion to study the trends across various structures or the effect of conformational change on the TPA of a given structure. TD-DFT at the B3LYP/6-31+G(d,p) level of theory was further used to screen twenty-two possible chromophores that can be formed upon replacing a precursory amino acid (Tyr66) from those that form the green FP (GFP) chromophore with a ncAA. A proposed chromophore with a nitro substituent was found to have a large TPA cross section (29 GM) that is more than 7 times that of the native GFP chromophore as determined at the same level of theory. Classical molecular dynamics performed on a nitro-modified FP confirmed its stability and the large TPA cross section of the chromophore at various conformations it assumed within the protein pocket. Intrigued by the recent interest in designing ncAA-derived red FPs (RFPs), the same set of GFP-based chromophores, but with an extended structure (an acylimine moiety) that is characteristic for many RFPs, were screened for their TPA properties. In the screening of these RFP-derived chromophores, both B3LYP and CAM-B3LYP functionals were used together with re-screening the GFP-derived ones with CAM-B3LYP for completeness. Computing TPA cross-sections with B3LYP and CAM-B3LYP yield similar overall trends. Results using both functionals agree that the RFP-derived model of the gold FP (GdFP) chromophore has the largest intrinsic TPA cross section (50 GM according to B3LYP). TPA was further computed for selected chromophores following conformational changes: variation of the dihedral angle of the acylimine moiety and the tilt and twist angles between the rings of the chromophore. The TPA cross-section assumed an oscillatory trend with the rotation of the acylimine dihedral, and the TPA is maximized in the planar conformation for almost all models. One chromophore bearing a hydroxyquinoline ring is also shown to be comparable to that of the GdFP-like chromophore in terms of TPA cross section. The conformational study on the hydroxyquinoline-modified chromophore shows that the acylimine angle has a much stronger effect on the TPA than its tilt and twist angles. Having an intrinsic TPA ability that is more than 7 times that of the native RFP chromophore, the GdFP- and the hydroxyquinoline- modified chromophores are very promising for experimental investigation. The GFP-derived chromophore with a nitro substituent is likewise interesting. Overall, a number of new FP chromophores built from ncAAs, with large intrinsic TPA, have been proposed and the strong effect of conformation on TPA explored. The present work will hopefully spur complementary experimental tests in this burgeoning field.
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. 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.
Citation for previous publication
M. Alaraby Salem, Melis Gedik and Alex Brown, Handbook of Computational Chemistry, Ed. Jerzy Leszczynski. Springer Netherlands, 2016, 1-19.M. Alaraby Salem and Alex Brown, J. Chem. Theory Comput., 2014, 10, 3260-3269.M. Alaraby Salem and Alex Brown, Phys. Chem. Chem. Phys., 2015, 17, 25563-25570M. Alaraby Salem, Isaac Twelves and Alex Brown, Phys. Chem. Chem. Phys., 2016, Accepted.

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