Structural Analysis of Biomolecules using Synchrotron‐Radiation Vacuum‐Ultraviolet Circular Dichroism Spectroscopy Koichi Matsuo Hiroshima Synchrotron Radiation Center, Hiroshima University Annual Users’ Meeting and 20 th Anniversary of Operation, September 5 th , 2013, NSRRC
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Structural Analysis of Biomolecules using Synchrotron‐Radiation
Hiroshima Synchrotron Radiation Center, Hiroshima University
Annual Users’ Meeting and 20th Anniversary of Operation, September 5th, 2013, NSRRC
L‐Alanine
Circular dichroismCircular Dichroism (CD) is observed when certain material absorbs left‐ andright‐circular polarized light slightly differently. This is very sensitive to thestructure of chiral molecules.
Initial structures of three rotamers of methyl ‐D‐Glc
C‐1C‐2
C‐3
C‐4 C‐5
C‐6
O‐1O‐2
O‐3
O‐4O‐5
O‐6
C‐7
‐GG‐GT (X‐ray crystal structure)
‐TG
HO‐3
HO‐4
HO‐6
HO‐2
= 60° = 60° = 180°
= O-5C-5C-6O-6
Initial structures
CD calculations
Optimizations
Schemes of CD calculations
TDDFT method at the CAM‐B3LYP/6‐311++G** level (PCM)
DFT method (Onsager model)at the CAM‐B3LYP/6‐311++G** level
‐GT, ‐GG, and ‐TG rotamers
VUVCD spectra of methyl ‐D‐glucopyranoside in water (Onsager model)
Methyl ‐D‐Glc
170 180 190 200 210‐40
‐20
0
20
40
[]
×10‐3
/ deg
・cm2
・dmol
‐1
Theoretical spectrum Experimental spectrum
Wavelength / nm
Red: Oxygen atomGreen: Carbon atom White: Hydrogen atom
MD simulation of methyl ‐D‐Glc in aqueous solution
MD simulationThe initial structures in a periodic box water molecules were simulatedfor 20‐ns under the AMBER/GLYCAM force field at 298 K and 1 atm.
Initial structures in water
‐GT, ‐GG, and ‐TG rotamers
+ Water bath =
TIP3P water molecules
CD calculationsThe spectra of 40 structures extracted at 500‐ps intervals were calculated by TDDFT method at the CAM‐B3LYP/6‐311++G** level (PCM) and averaged
Hydrations of the ‐GT rotamer during the simulation
0 5 10 15 200
2
4
6
8
10
12
14
16 Total Hydroxyl group at C‐4 Ring oxygen (O‐5)
Num
ber o
f hydrated water m
olecules
Time / ns
C‐1C‐2
C‐3
C‐4 C‐5
C‐6
O‐1O‐2
O‐3
O‐4O‐5
O‐6
C‐7
‐GT
HO‐3
HO‐4
HO‐2
Methoxy group
Hydroxy group
‐90‐60‐300
306090
120150180210240
Dihe
dral Angle / de
grees
C‐7O‐1C‐1O‐5 HO‐4O‐4C‐4C‐3
0 5 10 15 20Time / ns
The configuration of the methyl ‐Glc would fluctuate accompanying the change of hydration to reflect the VUVCD spectra.
J. Phys. Chem. A (2012) 116, 9996
Theoretical and experimental CD spectra of methyl ‐D‐Glc
160 170 180 190 200 210‐10
‐5
0
5
10
Experimental spectrum Theoretical spectrum
Wavelength / nm
[]
×10‐3
/ deg
・cm2
・dmol
‐1
MD simulations
170 180 190 200 210‐40
‐20
0
20
40
[]
×10‐3
/ deg
・cm2
・dmol
‐1
Theoretical spectrum Experimental spectrum
Wavelength / nm
Optimizations
J. Phys. Chem. A (2012) 116, 9996
160 170 180 190 200 210‐40
‐20
0
20
40 ‐GT ‐GG ‐TG
[]
×10‐3
/ deg
・cm2
・dmol
‐1
Wavelength / nm
Pairwise relationships between CD and configurations of the ‐GT, ‐GG, and ‐TG rotamers
Hydrogen Bond
The differences in hydrogen bonds should be responsible for the characteristic CD spectra of the ‐GT, ‐GG, and ‐TG rotamers.
‐GG ‐GT
‐TG
HO‐6O‐5
HO‐6 O‐5
J. Phys. Chem. A (2012) 116, 9996
1. VUVCD spectra are sensitive to the structural characteristics such as ‐ and ‐anomers of hydroxy group, and trans and gauche configurations of hydroxymethyl group.
2. Theoretical calculations clarify the pairwise relationships between configurations and VUVCD of saccharides.
3. Combining VUVCD spectroscopy, MD, and TDDFT could provide important information about the conformations, interactions, and hydrations of saccharides
Summary in the VUVCD of saccharides
VUVCD spectra of typical native proteins
160 180 200 220 240 260-40
-20
0
20
40
60
80
100
[]
×10
-3 /
deg
・ c
m2 ・
dm
ol-1
Wavelength / nm
Myoglobin (:76%)Concanavalin A
(: 4%, :46%)
STI (:2%, :37%)
Lysozyme(:42%, :6%)
J. Biochem. (2004) 135, 405; J. Biochem. (2005) 138, 79
: ‐helix: ‐strand
Protein structure analysis by VUVCD
Proteins (2008) 73, 104
160 180 200 220 240 260-40-30-20-10
01020304050
[
]×10
-3 /
deg
・ c
m2 ・
dm
ol-1
Wavelength /nm
VUVCD VUVCD
Protein structure analysis using VUVCD Spectroscopy
X‐ray crystallography and NMR• 3D Structure at atomic resolution • Limited to crystallized or small protein
VUVCD Spectroscopy• Estimation of secondary‐structure contents• Available for any proteins • Useful for various solvent conditions
VUVCD spectroscopy is becoming useful in the technique for structure analysis of protein
Several ‐helix segments
Speculated conformation of alcohol denatured proteins
VUVCD spectra of six proteins in TFE‐denatured state
0
10
20
30
40
50
60
70
80
90
100 -Helix -Strand Turn Unordered
CGNBGTrxLAHSA
%
Mb
(8) (29) (7) (6) (9) (12)
Secondary structures of six proteins at 50 % TFE concentration
0%
50%
0%
50%
Proteins (2012) 80, 281
VUVCD provides the structural characteristics of proteins at denatured states
0%
50%
0%
50%
0%
50%
0%
50%
0%
50%S
SS
S
lysozyme
1SS, 0SS variants3SS variants 2SS variants
180 200 220 240-15
-10
-5
0
5
10
15
20
180 200 220 240 260180 200 220 240
Wild 2SS12 2SS13 2SS23 2SS34 0SS
[
]×10
-3 /
deg・
cm2 ・
dmol
-1 Wild
3SS1 3SS2 3SS3 3SS4 0SS
Wild 1SS1 1SS2 1SS3 1SS4 0SS
Wavelength / nm
VUVCD characterizes the roles of disulfide bridges in the structural formation of protein
Proteins (2009) 77, 191
A‐Helix B‐Helix C‐Helix D‐Helix
3SS
2SS
1SS
0SS
VUVCD spectra of thirteen disulfide‐deficient variants of lysozyme
VUVCD gives new insights for the membrane‐induced conformations of 1‐Acid Glycoprotein (AGP)
Biochemistry (2009) 48, 9103
VUVCD spectra of AGP with and without bio‐membrane
160 180 200 220 240 260‐20
‐10
0
10
20
30
40
Membrane‐binding state
Native state
Wavelength / nm
[
] ×
10-
3 / deg ・ cm
2 ・ dmol
-1
Sequence of secondary structures of AGP
Native
Membrane‐binding state
Speculated AGP conformations at native and membrane‐binding states
VUVCD evaluates the teritiary structure from a homology modeling
State‐Helix ‐Strand
Turn (%) Unordered (%)Content (%) Number Content (%) Number
Secondary structure parameters from homology modeling
Secondary structure parameters from VUVCD spectroscopy
Comparison
Biochemistry (2009) 48, 9103
Summary in the VUVCD of proteins
1. VUVCD analysis can estimate the contents, numbers of segments, and sequences of secondary structures of proteins.
2. VUVCD can be applied to the structural analysis of denatured proteins, disulfide deficient proteins, membrane‐binding proteins, and amyloid fibrils.
Summary
1. The VUVCD spectra are very sensitive to the conformations of saccharides and proteins in aqueous solution.
2. The VUVCD spectroscopy coupled with MD and TDDFT methods could provide important information about the conformation, interaction, and hydration of saccharides.
3. The VUVCD spectroscopy combined with bioinformatics technique has a great advantage for the structure analysis of not only native proteins but also non‐native proteins.
Further accumulations of VUVCD data and developments of VUVCD analysis should open a new field in the structural biology.