Effects of Water Molecules and Configurations of Neighboring Amino Acid Residues Surrounding DsRed Chromophore on Its Excitation Energy

Abstract

DsRedはDiscosoma種サンゴから単離された赤色蛍光タンパク質(RFP)の一種である．私たちのグループでは，多層フラグメント分子軌道(MFMO)スキームの下，クロモフォアにCIS (D)系の励起状態計算法を用いてDsRedの励起エネルギー・発光エネルギーを算定し，実験のスペクトルの極大値との良好な対応を報告している[Mochizuki et al., Chem. Phys. Lett., 433, 360 (2007) & Taguchi et al., J. Phys. Chem. B, 113, 1153 (2009)]．しかし，色素部近傍の水分子，隣接アミノ酸残基の側鎖の配向や構造緩和についての検討は必ずしも明示的になされてはいなかった．そこで今回，励起エネルギーを指標としてこれらの影響を系統的に検証した．その結果，前者では色素部CRQ66や周辺アミノ酸残基と水素結合ネットワークを形成している水2分子の存在，および色素に隣接するSer69の側鎖のOH基の配向，さらに隣接する荷電性のLys163とGlu215の構造の緩和が重要であることが確認された．これらは，結果として既報でのタンパク質構造のモデリングの妥当性を支持するものとなった．

Figures

Figure 1.

 Graphic representation of DsRed (PDB-ID; 1ZGO [7]). (a) tetramer, (b) monomer.

Figure 2.

 Pigment parts of GFP and DsRed. The π conjugation of DsRed is elongated by an additional oxidation at peptide bond between CRQ66 and Phe65 (marked with dashed line), and thus the optical window is shifted from green to red.

Figure 3.

 Conformational differences of Ser69 (marked with dashed line) in the structure including nine water molecules (see text). The two water molecules nearest from CRQ66 (as the DsRed pigment in PDB-ID: 1ZGO [7]) are visible with stick model. The remaining seven molecules with outer positions are represented by the line model. The labels A and B specify the conformation of Ser69 in 1ZGO, and B1 and B2 differ from each other by the orientation of the OH group in the side chain.

Figure 4.

 Graphic representation of three important MOs of the central fragment (consisting of CRQ66, Phe65 and Se69) calculated at the HF/6-31G* level for the B2 structure with two water molecules. The numbering and characters are indicated for the respective MOs. Orbital phases are shown with red and blue colors.

Figure 5.

 IFIE plots for CRQ66 obtained at the FMO-MP2/6-31G*level.

Figure 6.

 Superimposed structures around the DsRed pigment. The 1ZGO crystal structure is colored with pink, and the force field-optimized structure is shown with cyan. CRQ66 is identified with yellow.

Tables

Table 1. Excitation energies (in eV) of the second excited state of ππ* type (see text).

		without PR		with PR
Structure	CIS	CIS (D)	CIS (Ds)	CIS (D)	CIS (Ds) a
No water molecule
A	3.18	1.97	2.07	1.84	1.94	(1.41)
B1	3.18	1.87	1.98	1.75	1.86	(1.45)
B2	3.19	2.05	2.14	1.93	2.01	(1.37)
2 water molecules (within 2.8Å)
A	3.23	2.14	2.21	2.01	2.08	(1.28)
B1	3.22	2.05	2.14	1.93	2.01	(1.30)
B2	3.24	2.18	2.25	2.06	2.13	(1.27)
4 water molecules (within 3Å)
A	3.24	2.07	2.16	1.94	2.03	(1.41)
B1	3.24	2.00	2.09	1.88	1.97	(1.43)
B2	3.25	2.14	2.22	2.02	2.09	(1.37)
9 water molecules (within 4Å)
A	3.25	2.10	2.18	1.98	2.05	(1.41)
B1	3.24	1.98	2.07	1.85	1.94	(1.48)
B2	3.26	2.15	2.22	2.02	2.09	(1.41)
Exp.	2.22

^a The excitation energy in parenthesis corresponds to the lowest excited state of nπ* type.

Table 2. Excitation energies (in eV) of the second excited state with and without geometry relaxations of neighboring residues.

		without PR			with PR
Relaxation	CIS	CIS (D)	CIS (Ds)		CIS (D)	CIS (Ds) b
Without a	3.24	2.18	2.25		2.06	2.13	(1.27)
With	3.28	2.29	2.34		2.16	2.22	(1.15)
Exp.	2.22

^a The same values as in Table 1, with the structure setting of two water molecules and Ser69/B2 conformation.

^b The excitation energy in parenthesis corresponds to the lowest excited state of nπ* type.

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