Direct Observation of the Protonation States in the Mutant Green Fluorescent Protein

Abstract

Neutron crystallography has been used to elucidate the protonation states for the enhanced green fluorescent protein, which has revolutionized imaging technologies. The structure has a deprotonated hydroxyl group in the fluorescent chromophore. Also, the protonation states of His148 and Thr203, as well as the orientation of a critical water molecule in direct contact with the chromophore, could be determined. The results demonstrate that the deprotonated hydroxyl group in the chromophore and the nitrogen atom ND1 in His148 are charged negatively and positively, respectively, forming an ion pair. The position of the two deuterium atoms in the critical water molecule appears to be displaced slightly toward the acceptor oxygen atoms according to their omit maps. This displacement implies the formation of an intriguing electrostatic potential realized inside of the protein. Our findings provide new insights into future protein design strategies along with developments in quantum chemical calculations.

What is the Optimal Size of the Quantum Region in Embedding Calculations of Two-Photon Absorption Spectra of Fluorescent Proteins?

“…All histidine residues are neutral and they were assigned the hydrogen atom on either δ (25, 148, 181, 199, 217) or ϵ (77, 81, 139, 169) nitrogen atom based on the visual inspection of the h-bonds network. This choice is mostly consistent with recent subatomic structures from X-ray and neutron crystallography experiments for avGFP mutants. , The most important concern is about H148 a.a. residue which interacts directly with the chromophore (Figure ) and seems to be cationic. , However, this protonation state may be characteristic for the crystal structure only because of the large steric hindrance between hydrogen bonded to ϵ nitrogen and nearby atoms . Because the reasons behind the newly discovered protonation state of H148 are not fully understood, we choose to use “traditional” H148 in our models with hydrogen bonded to the δ nitrogen atom only.…”

Mechanism of Color and Photoacidity Tuning for the Protonated Green Fluorescent Protein Chromophore

“…The qualitative trend of A state color tuning follows that of the B state (Table S3): any mutation that eliminates the positive charge of R96 − results in a blue-shift (see also ref ), while mutating T203 to an aromatic residue that π–π stacks with the chromophore’s phenol moiety red-shifts the absorption maximum. This suggests that the same direction of electron flow from phenol­(ate) to imidazolinone during excitation can be assigned to both protonation states. , Within the π–π stacking series, as we modulate the π-system’s electron density at position 203 from electron-deficient to electron-rich by using electron-withdrawing and -donating groups, respectively, a clear red-shift is observed.…”

Unusual Spectroscopic and Electric Field Sensitivity of a Chromophore with Short Hydrogen Bond: GFP and PYP as Model Systems

“…With only rotamer A, the O-O distance between E222 and T65 becomes 3.4 Å, which is slightly too long for an effective hydrogen bond; with only rotamer B, the hydrogen bond between T205 and E222 is broken. This tridentate electron density for E222 has been explicitly documented three times in the literature for EGFP without the H148D mutation [46][47] [48] and has been conjectured to cause two distinct fluorescence lifetimes for EGFP [49]. A recently reported nearly ultrahigh-resolution (0.85 Å) structure of GFP S65T (PDB: 6JGI, [50]) also shows E222 modeled with two rotamers, albeit a population ratio of 4:1.…”

“…Local structure with the electron density (2mFo -DFc contoured at 1σ) for each HhPYP mutant, including wild type (green, left, PDB: 1NWZ [48]), E46Q (cyan, middle, PDB: 1UGU [49]), and Y42F (magenta, right, PDB: 1F9I [50]). The hydrogen bond distances (in Å) are labeled in red and also listed in Table 1.…”