Application of fluorescent proteins (FPs), e.g., as probes for biological imaging, has led to the goal of finding FPs with notable one-and twophoton absorption (OPA and TPA, respectively) features. The variables that affect the TPA cross section are many; e.g., structurally speaking, some studies have shown its magnitude is influenced by the presence of the protein backbone and the molecules of water surrounding the chromophore. However, the impact that the surroundings have on the TPA cross section has not been conclusively determined. One of the main problems that can be faced when trying to account for excited state properties is the cost associated with such computations. Among the methods chosen for this type of computations is time-dependent density functional theory (TD-DFT), commonly used on molecules with no more than 50 atoms due to its computational cost. A cheaper alternative to DFT and, moreover, to TD-DFT is the so-called time-dependent tight binding density functional theory (TD-DFTB), which within the second-order approximation is designated TD-DFTB2. In the present work, TD-DFTB2 was tested to determine whether or not it is an alternative method to TD-DFT for computing excited state properties beyond excitation energies and oscillator strengths such as TPA cross sections. Studies around the performance of TD-DFTB2 on the computation of excitation energies have been previously carried out, and the results show it is comparable to TD-DFT in terms of the computation of excitation energies and oscillator strengths. Despite the latter, what we found is that neither the magnitude nor the trend of the obtained TPA cross sections is preserved with respect to CAM-B3LYP and B3LYP TPA cross sections previously reported by other authors. The computation of TPA cross sections within the two-level model allowed us to determine that among the reasons behind such behavior is the overestimation of the excited state dipole moments. Based on the above, we conclude that TD-DFTB2 is not (yet) a viable route to obtain quantitatively TPA cross sections.

Light‐responsive proteins are widely employed in bioimaging, for example, fluorescent proteins (FPs), which are comprised of a chromophore centered within a barrel‐shaped protein. FPs exhibit remarkable one‐ and multi‐photon absorption (1PA and MPA, respectively) in addition to their emissive properties. Over the last two decades, many types of quantum mechanical, molecular dynamics, and combined quantum mechanical/molecular‐mechanical (QM/MM) approaches have been employed in the study of the photophysics of FPs. Among the latter, QM/MM approaches have proven to be capable of capturing the strong correlation between FPs' light‐responsive properties and their chromophore–environment interactions. In particular, polarizable embedding QM/MM methods are gaining attention by reason of their outstanding performance in the computation of MPA in FPs. Herein, we discuss the outcomes of some of the investigations performed on the 1PA, MPA, and emissive features of FPs using QM/MM approaches. In addition, critical aspects of the use of QM/MM approaches to study FPs' 1PA and MPA features are described. To those researchers interested in starting to perform MPA computations for FPs using QM/MM methods, this review aims to be a compass to navigate among the relevant available literature. This article is categorized under: Electronic Structure Theory > Combined QM/MM Methods Structure and Mechanism > Computational Biochemistry and Biophysics

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.

In this study, density functional theory (DFT) and time dependent density functional theory (TD-DFT) are used to investigate the stabilities and spectral properties [IR, UV-vis, and two-photon absorption (2PA)] of two sets of modified RNA nucleobase tautomers. The modifications introduce either a sulfur or selenium atom to form an isothiazolo[4,3-d]pyrimidine or isoselenazolo[4,3-d]pyrimidine heterocylic core respectively. The relative stabilities of both sets of modified tautomers determined with B3LYP/6-31++G(d,p) reveal that in water (with a polarizable continuum model) the 6-keto-2-amino tautomer of guanine and the rare 4-imino-2-keto tautomer of cytosine may be present at significant populations while the 6-enol-2-amino tautomer of guanine is more common in the gas phase. The identification of these modified tautomers due the natural differences in their vibrational modes and hence IR spectra is possible. Furthermore, the photophysical properties of both these sets of modified tautomers indicate that excitation and emission energies are shifted relative to their more abundant form in both one photon absorption and emission, and two-photon absorption (2PA) spectra as determined at the B3LYP/6-31++G(d,p) and CAM-B3LYP/aug-cc-pVDZ levels of theory, respectively. Even though the 2PA cross sections in water for all of the species are small (0.3 - 2.3 GM), the modified cytosine tautomer shows promise as its cross section is larger than the more dominant form. The spectral separation between the dominant form and the tautomers of isoselenazole and isothiazole modified cytosine and guanine are relatively similar, suggesting both modifications could be useful in elucidating the tautomers from their more abundant counterparts.

Nucleobases (adenine, cytosine, guanine, and uracil), the four molecules that forms RNA, have been found to be useful in probing in the human body when modified because they can emit light. Non-modified nucleobases do not exhibit emissive properties and cannot be used as probes. Some of the modifications include the substitution of nitrogen atoms with sulfur and selenium, and the resulting modified nucleobases give place to the so-called tz- and ts- RNA alphabets, respectively. Therefore, the aim of this project was to provide insights about the viability, from a computational perspective, of using the modified nucleobases as probes, evaluating the differences in thermochemical, structural and emissive properties of the modified nucleobases with respect to the non-modified ones. Nucleobases can coexist with other modified nucleobases or tautomers, molecules that differ due to the change in position of hydrogen atoms in a molecule’s structure and as a result have different physical and chemical properties. The thermochemical properties evaluation mainly consisted in the computation of the relative Gibbs Free Energy (G), which is related to the fraction F, an index of the relative population among tautomers. This was done using Gaussian 09 software by performing geometry analysis and frequency computations on each one of the tautomers. By comparing the equilibrium fractions, it was determined that in both cases, tz- and ts- guanine and cytosine exist principally in the form of one of their tautomers (Cytosine 2 and Guanine 2) as in the case of the non-modified cases. After confirming which tz- and ts- tautomers were the ones with the largest probable population, infrared (IR) and ultraviolet-visible (UV-vis) spectra were obtained. The IR spectra of selenium and sulfur tautomers of guanine and cytosine indicated that the tautomers had peaks at similar frequencies with respect to each other, however, the intensities varied, implying slight structural changes between the tautomers. On the other hand, the UV-vis spectra showed a change in peak positions between the tautomers with sulfur and selenium, suggesting that the change between sulfur and selenium has an effect on the spectra by shifting the peaks from the original molecules’ λmax values. Their relative population fractions show that only the canonical forms of the modified nucleobases exist in a larger extent than the rest of their tautomer forms. In addition, the features in their UV-vis and IR spectra allow these tautomers to be differentiated from each other.

The cover image is based on the Focus Article Quantum mechanical/molecular mechanical studies of photophysical properties of fluorescent proteins by Maria Rossano‐Tapia and Alex Brown., https://doi.org/10.1002/wcms.1557.