The heats of formation and the normalized clustering energies (NCEs) for the group 4 and group 6 transition metal oxide (TMO) trimers and tetramers have been calculated by the Feller-Peterson-Dixon (FPD) method. The heats of formation predicted by the FPD method do not differ much from those previously derived from the NCEs at the CCSD(T)/aT level except for the CrO3 nanoclusters. New and improved heats of formation for Cr3O9 and Cr4O12 were obtained using PW91 orbitals instead of Hartree-Fock (HF) orbitals. Diffuse functions are necessary to predict accurate heats of formation. The fluoride affinities (FAs) are calculated with the CCSD(T) method. The relative energies (REs) of different isomers, NCEs, electron affinities (EAs), and FAs of (MO2)n (M = Ti, Zr, Hf, n = 1-4) and (MO3)n (M = Cr, Mo, W, n = 1-3) clusters have been benchmarked with 55 exchange-correlation density functional theory (DFT) functionals including both pure and hybrid types. The absolute errors of the DFT results are mostly less than ±10 kcal/mol for the NCEs and the EAs and less than ±15 kcal/mol for the FAs. Hybrid functionals usually perform better than the pure functionals for the REs and NCEs. The performance of the two types of functionals in predicting EAs and FAs is comparable. The B1B95 and PBE1PBE functionals provide reliable energetic properties for most isomers. Long range corrected pure functionals usually give poor FAs. The standard deviation of the absolute error is always close to the mean errors, and the probability distributions of the DFT errors are often not Gaussian (normal). The breadth of the distribution of errors and the maximum probability are dependent on the energy property and the isomer.

Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics-function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.split GFP | potential energy surface | photodissociation | cis-trans isomerization | photosensory protein O ptical techniques for investigating biological processes in vivo can achieve subcellular spatial and millisecond temporal resolution by using genetically encoded light-responsive proteins (1). Photoactivatable proteins are used as either imaging tools, such as reversibly photoswitchable fluorescent proteins (RSFPs), with fluorescence that can be modulated by light (2) or optogenetic switches that convert light input into physiological outputs, such as channelrhodopsins, which can regulate ion flow through membranes in response to light (3). Split GFPs have been widely applied for imaging as fluorescent reporters of cellular processes, because they are small (∼25 kDa), are stable in cytosol, produce chromophores autocatalytically, and are amenable to mutation and circular permutation (4). Typically, the protein is expressed as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another, offering readouts of protein-protein colocalizaton with low background and high specificity (5). However, this technique can generate misleading results, because the GFP complexes, after being formed and fluorescing, are bound irreversibly, which can be highly perturbative to the processes being studied, especially if the protein interaction being probed is ordinarily reversible (6). Although split GFP complexes essentially do not dissocia...

Photoactive yellow protein (PYP) is a small photoreceptor protein that has two unusually short hydrogen bonds between the deprotonated p-coumaric acid chromophore and two amino acids, a tyrosine and a glutamic acid. This has led to considerable debate as to whether the glutamic acid-chromophore hydrogen bond is a low barrier hydrogen bond, with conflicting results in the literature. We have modified the pK a of the tyrosine by amber suppression and of the chromophore by chemical substitution. X-ray crystal structures of these modified proteins are nearly identical to the wild-type protein, so the heavy atom distance between proton donor and acceptor is maintained, even though these modifications change the relative proton affinity between donor and acceptor. Despite a considerable change in relative proton affinity, the NMR chemical shifts of the hydrogen-bonded protons are only moderately affected. QM/MM calculations were used to explore the protons’ potential energy surface and connect the calculated proton position with empirically measured proton chemical shifts. The results are inconsistent with a low barrier hydrogen bond but in all cases are consistent with a localized proton, suggesting an ionic hydrogen bond rather than a low barrier hydrogen bond.

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