Modulating the pH dependent photophysical properties of green fluorescent protein.

The photophysical properties of the β-barrel superfolder green fluorescent protein (sfGFP) arise from the chromophore that forms post-translationally in the interior of the protein. Specifically, the protonation state of the side chain of tyrosine 66 in the chromophore, in addition to the network of hydrogen bonds between the chromophore and surrounding residues, is directly related to the electronic absorbance and emission properties of the protein. The pH dependence of the photophysical properties of this protein were modulated by the genetic, site-specific incorporation of 3-nitro-l-tyrosine (mNOY) at site 66 in sfGFP.

Unraveling Complex Local Protein Environments with 4-Cyano-l-phenylalanine.

We present a multifaceted approach to effectively probe complex local protein environments utilizing the vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNPhe) in the model system superfolder green fluorescent protein (sfGFP). This approach combines temperature-dependent infrared (IR) spectroscopy, X-ray crystallography, and molecular dynamics (MD) simulations to provide a molecular interpretation of the local environment of the nitrile group in the protein. Specifically, a two-step enantioselective synthesis was developed that provided an 87% overall yield of pCNPhe in high purity without the need for chromatography.

Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein.

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group.

Paired Spectroscopic and Crystallographic Studies of Proteases.

The active sites of subtilisin and trypsin have been studied by paired IR spectroscopic and X-ray crystallographic studies. The active site serines of the proteases were reacted with 4-cyanobenzenesulfonyl fluoride (CBSF), an inhibitor that contains a nitrile vibrational reporter. The nitrile stretch vibration of the water-soluble inhibitor model, potassium 4-cyanobenzenesulfonate (KCBSO), and the inhibitor were calibrated by IR solvent studies in HO/DMSO and the frequency-temperature line-slope (FTLS) method in HO and THF.

Crystal structures of green fluorescent protein with the unnatural amino acid 4-nitro-L-phenylalanine.

The X-ray crystal structures of two superfolder green fluorescent protein (sfGFP) constructs containing a genetically incorporated spectroscopic reporter unnatural amino acid, 4-nitro-L-phenylalanine (pNOF), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNOF at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNOF sfGFP construct crystallized with two molecules per asymmetric unit in space group P321 and the crystal structure was refined to 2.

Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine.

The vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNF) was genetically incorporated individually at three sites (5, 36, and 78) in the heme protein H-NOX to probe local hydration environments. The UAA pCNF was incorporated site-specifically using an engineered, orthogonal tRNA synthetase in . The ability of all of the pCNF-containing H-NOX proteins to form the ferrous CO, NO, or O ligated and unligated states was confirmed with UV-Vis spectroscopy.

Structural and Functional Evidence Indicates Selective Oxygen Signaling in Caldanaerobacter subterraneus H-NOX.

Acute and specific sensing of diatomic gas molecules is an essential facet of biological signaling. Heme nitric oxide/oxygen binding (H-NOX) proteins are a family of gas sensors found in diverse classes of bacteria and eukaryotes. The most commonly characterized bacterial H-NOX domains are from facultative anaerobes and are activated through a conformational change caused by formation of a 5-coordinate Fe(II)-NO complex.

Probing the effectiveness of spectroscopic reporter unnatural amino acids: a structural study.

The X-ray crystal structures of superfolder green fluorescent protein (sfGFP) containing the spectroscopic reporter unnatural amino acids (UAAs) 4-cyano-L-phenylalanine (pCNF) or 4-ethynyl-L-phenylalanine (pCCF) at two unique sites in the protein have been determined. These UAAs were genetically incorporated into sfGFP in a solvent-exposed loop region and/or a partially buried site on the β-barrel of the protein. The crystal structures containing the UAAs at these two sites permit the structural implications of UAA incorporation for the native protein structure to be assessed with high resolution and permit a direct correlation between the structure and spectroscopic data to be made.

Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins.

An unnatural amino acid, 4-(2-azidoethoxy)-L-phenylalanine (AePhe, 1), was designed and synthesized in three steps from known compounds in 54% overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the azide asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites by using the amber codon suppression method.

Porphyrin π-stacking in a heme protein scaffold tunes gas ligand affinity.

The role of π-stacking in controlling redox and ligand binding properties of porphyrins has been of interest for many years. The recent discovery of H-NOX domains has provided a model system to investigate the role of porphyrin π-stacking within a heme protein scaffold. Removal of a phenylalanine-porphyrin π-stack dramatically increased O2, NO, and CO affinities and caused changes in redox potential (~40mV) without any structural changes.

Porphyrin-substituted H-NOX proteins as high-relaxivity MRI contrast agents.

Heme proteins are exquisitely tuned to carry out diverse biological functions while employing identical heme cofactors. Although heme protein properties are often altered through modification of the protein scaffold, protein function can be greatly expanded and diversified through replacement of the native heme with an unnatural porphyrin of interest. Thus, porphyrin substitution in proteins affords new opportunities to rationally tailor heme protein chemical properties for new biological applications.

Controlling conformational flexibility of an O₂-binding H-NOX domain.

Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O(2))-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket.