Two-Photon Absorption Cross-Sections in Fluorescent Proteins Containing Non-canonical Chromophores Using Polarizable QM/MM

Abstract

Multi-photon absorption properties, particularly two-photon absorption (2PA), of fluorescent proteins (FPs) have made them attractive tools in deep-tissue clinical imaging. Although the diversity of photophysical properties for FPs is wide, there are some caveats predominant among the existing FP variants that need to be overcome, such as low quantum yields and small 2PA cross-sections. From a computational perspective, Salem et al. (2016) suggested the inclusion of non-canonical amino acids in the chromophore of the red fluorescent protein DsRed, through the replacement of the tyrosine amino acid. The 2PA properties of these new non-canonical chromophores (nCCs) were determined in vacuum, i.e., without taking into account the protein environment. However, in the computation of response properties, such as 2PA cross-sections, the environment plays an important role. To account for environment and protein-chromophore coupling effects, quantum mechanical/molecular mechanical (QM/MM) schemes can be useful. In this work, the polarizable embedding (PE) model is employed along with time-dependent density functional theory to describe the 2PA properties of a selected set of chromophores made from non-canonical amino acids as they are embedded in the DsRed protein matrix. The objective is to provide insights to determine whether or not the nCCs could be developed and, thus, generate a new class of FPs. Results from this investigation show that within the DsRed environment, the nCC 2PA cross-sections are diminished relative to their values in vacuum. However, further studies toward understanding the 2PA limit of these nCCs using different protein environments are needed.

Quantum mechanical/molecular mechanical studies of photophysical properties of fluorescent proteins

“…In addition to composing benchmark data on MPA of FPs, efforts have been made using Olsen et al's model for describing the roles of the residues closest to the chromophore on the 2PA cross‐section and excitation energy of the RFP DsRed 85 . More recently, this polarizable embedding model was employed to determine the 2PA of a curated set of non‐canonical chromophores within the RFP protein environment 104 …”

Quantum chemistry study of the multiphoton absorption in enhanced green fluorescent protein at the single amino acid residue level

“…Moreover, the 2PA intensity as a function of chromophore structure in vacuo [14] may not be recovered in the complex protein environment. [15] This diversity must be attributed to complex CRO -environment interactions (Figure S1 in the Supporting Information). Understanding the elements of spectral properties modulation mechanism is important for a directed design of novel markers with desired spectral characteristic.…”

“…Similar wide spectral tuning [1–4,10,13] is observed also for other FPs families and also in terms of one‐photon and two‐photon absorption (1PA and 2PA) intensity as well as emission wavelength, brightness and many other photophysical properties. Moreover, the 2PA intensity as a function of chromophore structure in vacuo [14] may not be recovered in the complex protein environment [15] …”

E/Z Molecular Photoswitches Activated by Two-Photon Absorption: Comparison between Different Families

“…For these reasons, the design of efficient E / Z photoswitches with a relatively high TPA cross section is highly desirable for practical purposes. Diverse studies have been already published focused on the TPA properties of different photoswitches families, such as azobenzene [ 54 , 55 , 56 , 57 , 58 ], stilbene [ 56 , 59 , 60 ], fluorescent protein chromophores [ 61 , 62 , 63 ], and retinal [ 64 , 65 , 66 ]. In addition, related compounds have been also explored under TPA conditions: retinal (chromophore of photoactive proteins) [ 64 ], retinoic acid derivatives [ 67 ], carotenoids and other pigments [ 68 ], and several caged compounds [ 69 ].…”