Acid-base catalysis and crystal structures of a least evolved ancestral GFP-like protein undergoing green-to-red photoconversion

Hanseong Kim, Timothy J. Grunkemeyer, Chintan Modi, Liqing Chen, Raimund Fromme, Mikhail V. Matz, Rebekka Wachter

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)8048-8059
Number of pages12
JournalBiochemistry
Volume52
Issue number45
DOIs
StatePublished - Nov 12 2013

Fingerprint

Chromophores
Catalysis
Crystal structure
Protons
Acids
Proteins
Histidine
Photoexcitation
Sequence Analysis
Hydrogen
Quantum yield
Catalytic Domain
Respiration
Carbon
Catalyst supports
Color
X-Rays
Light
Substitution reactions
X rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Acid-base catalysis and crystal structures of a least evolved ancestral GFP-like protein undergoing green-to-red photoconversion. / Kim, Hanseong; Grunkemeyer, Timothy J.; Modi, Chintan; Chen, Liqing; Fromme, Raimund; Matz, Mikhail V.; Wachter, Rebekka.

In: Biochemistry, Vol. 52, No. 45, 12.11.2013, p. 8048-8059.

Research output: Contribution to journalArticle

Kim, Hanseong ; Grunkemeyer, Timothy J. ; Modi, Chintan ; Chen, Liqing ; Fromme, Raimund ; Matz, Mikhail V. ; Wachter, Rebekka. / Acid-base catalysis and crystal structures of a least evolved ancestral GFP-like protein undergoing green-to-red photoconversion. In: Biochemistry. 2013 ; Vol. 52, No. 45. pp. 8048-8059.
@article{d2d0759317b44fd0a667be5d1a20c7f6,
title = "Acid-base catalysis and crystal structures of a least evolved ancestral GFP-like protein undergoing green-to-red photoconversion",
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.",
author = "Hanseong Kim and Grunkemeyer, {Timothy J.} and Chintan Modi and Liqing Chen and Raimund Fromme and Matz, {Mikhail V.} and Rebekka Wachter",
year = "2013",
month = "11",
day = "12",
doi = "10.1021/bi401000e",
language = "English (US)",
volume = "52",
pages = "8048--8059",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "45",

}

TY - JOUR

T1 - Acid-base catalysis and crystal structures of a least evolved ancestral GFP-like protein undergoing green-to-red photoconversion

AU - Kim, Hanseong

AU - Grunkemeyer, Timothy J.

AU - Modi, Chintan

AU - Chen, Liqing

AU - Fromme, Raimund

AU - Matz, Mikhail V.

AU - Wachter, Rebekka

PY - 2013/11/12

Y1 - 2013/11/12

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84887578927&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84887578927&partnerID=8YFLogxK

U2 - 10.1021/bi401000e

DO - 10.1021/bi401000e

M3 - Article

VL - 52

SP - 8048

EP - 8059

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 45

ER -