4DXQ

Crystal Structure of a reconstructed Kaede-type Red Fluorescent Protein, LEA Q38A

Summary for 4DXQ

• Entry DOI: 10.2210/pdb4dxq/pdb
• Related: 4DXI 4DXM 4DXN 4DXO 4DXP 4GOB
• Descriptor: LEA Q38A GFP-LIKE PROTEINS (2 entities in total)
• Functional Keywords: beta barrel, luminescent protein
• Biological source: Synthetic Construct (artificial)
• Total number of polymer chains: 1
• Total formula weight: 26434.06

Authors

Kim, H.,Wachter, R.M. (deposition date: 2012-02-27, release date: 2013-02-27, Last modification date: 2024-10-16)

Primary citation

Kim, H.,Grunkemeyer, T.J.,Modi, C.,Chen, L.,Fromme, R.,Matz, M.V.,Wachter, R.M.
Acid-Base Catalysis and Crystal Structures of a Least Evolved Ancestral GFP-like Protein Undergoing Green-to-Red Photoconversion.
Biochemistry, 52:8048-8059, 2013

PubMed Abstract: In green-to-red photoconvertible fluorescent proteins, a three-ring chromophore is generated by the light-activated incorporation of a histidine residue into the conjugated π-system. We have determined the pH-rate profile and high- and low-pH X-ray structures of a least evolved ancestor (LEA) protein constructed in the laboratory based on statistical sequence analysis. LEA incorporates the minimal number of substitutions necessary and sufficient for facile color conversion and exhibits a maximal photoconversion quantum yield of 0.0015 at pH 6.1. The rate measurements provide a bell-shaped curve, indicating that the reaction is controlled by the two apparent pKa values, 4.5 ± 0.2 and 7.5 ± 0.2, flanking the chromophore pKa of 6.3 ± 0.1. These data demonstrate that the photoconversion rate of LEA is not proportional to the A-form of the GFP-like chromophore, as previously reported for Kaede-type proteins. We propose that the observed proton dissociation constants arise from the internal quadrupolar charge network consisting of Glu222, His203, Glu148, and Arg69. Increased active site flexibility may facilitate twisting of the chromophore upon photoexcitation, thereby disrupting the charge network and activating the Glu222 carboxylate for the abstraction of a proton from a carbon acid. Subsequently, the proton may be delivered to the Phe64 carbonyl by a hydrogen-bonded network involving Gln42 or by means of His65 side chain rotations promoted by protein breathing motions. A structural comparison of LEA with the nonphotoconvertible LEA-Q42A variant supports a role for Gln42 either in catalysis or in the coplanar preorganization of the green chromophore with the His65 imidazole ring.

PubMed: 24134825
DOI: 10.1021/bi401000e

Experimental method

X-RAY DIFFRACTION (1.95 Å)