structural of biomolecules using radiation ultraviolet...
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Structural Analysis of Biomolecules using Synchrotron‐Radiation
Vacuum‐Ultraviolet Circular Dichroism Spectroscopy
Koichi Matsuo
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.
Left circularly‐polarized light
Right circularly‐polarized light
=L−R
190 200 210 220 230 240 250 260-60
-40
-20
0
20
40
60
80
Wavelength / nm
・
・
×
[ ]
10
-3 /
deg
cm
2 dm
ol -1
CD spectra of Poly‐L‐Lysine
Coil
‐helix
‐sheet
Polypeptide
Protein
Amino‐acid sequence
‐helix
‐sheetTurn
Circular dichroism of biomolecules
Peptide bond
Circular dichroism of biomolecules
Saccharide (sugar)
190 200 210 220 230 240‐20
‐16
‐12
‐8
‐4
0
4
Chondroitin(CH) Chondroitin sulfate A(CS‐A) Chondroitin sulfate B(CS‐B) Chondroitin sulfate C(CS‐C)
Wavelength / nm
[ ] X 10
‐3 / deg
. cm
2 . dmol
‐1
DNA
A form B form Z form
A
B
Z
Riazance and Johnson 1992, Biopolymers 32, 271‐276
Matsuo et al. Biosci. Biotechnol. Biochem. (2009) 73, 551
Sulfate group
Carboxyl group
Synchrotron‐radiation vacuum‐ultraviolet circular dichroism(VUVCD) Wavelength (nm)
120 140 150 200 250 300 320
Far‐UVVacuum‐ultraviolet (VUV)
Synchrotron‐Radiation VUVCD spectrophotometer
Near‐UV
Conventional CD spectrophotometer
190nm
Amino acid in thin film
Uwe et al. Angew. Chem. Int. Ed. (2010) 49, 7799
Coil
-helix
-sheetPolypeptide in water
190nm160 180 200 220 240 260‐60‐40‐200
20406080
Wavelength / nm
[ ] x 10
‐3 / deg
. cm
2 . dmol ‐1
HiSOR
Hiroshima University
Science
Engineering Education
Letter
VUVCD spectrophotometer
Chem. Lett. (2000) 29, 832
SR PM
Ethylene Glycol
Sample chamber
Temperature control unitTop view of sample chamber
Anal. Sci. (2003) 19, 129
Temperature range from –30 to 70 oC
Assembled‐type optical cell
Anal. Sci. (2003) 19, 129
CaF2
O‐ring
Cylindrical Screw
Stainless steel container
Spacer: 100, 50, 25, 10, and 5 m No spacer: 1.4 ‐ 1.8 m
160 170 180 190 200 210 220-8-6-4-202468
101214
[ ]×
10-3 /
deg・
cm2 ・
dmol
-1
Wavelength / nm
D‐Glucose
D‐Galactose
D‐Mannose
VUVCD spectra of five mono‐saccharides
D‐XyloseD‐Lyxose
Carbohydr. Res. (2004) 339, 591
n‐* transitions (hydroxy group and acetal bond)Conc.: 10%, Path length: 1.4m
O‐5
‐anomer
C‐5
C‐6
C‐1
O‐6
C‐4
C‐1
‐anomer
O‐5
C‐5
C‐6
O‐6
C‐4
Gauche‐Gauche (GG) Gauche‐Trans (GT) Trans‐Gauche (TG)
Six conformers of D‐glucose in aqueous solution
‐GG, ‐GT, ‐TG, ‐GG, ‐GT, and ‐TG conformers
Three rotamers
Two anomers
160 180 200 220 240‐6
‐4
‐2
0
2
4
160 180 200 220 240‐4
‐2
0
2
4
Experimental spectrum
[ ] X 10
‐3/deg . cm
2 . dm
ol‐1
Wavelength / nm
Theoretical spectrum
• L‐Alanine with nine water molecules Fukuyama et al. (2005) J. Phys. Chem. A 109, 6928
• D‐Lactic acid with six water molecules Fukuyama et al. (2011) Chirarity 23, E52
• Di‐ and tri‐Peptides Šebek et al (2007) J. Phys. Chem. A 111, 2750
• DNA Nielsen et al (2009) J. Phys. Chem. B 113, 9614
Theoretical calculation of CD spectra of small biomolecule using a time‐dependent density functional theory (TDDFT)
n*
n*, *
VUVCD spectra of D‐lactic acidFour hydrated structures of D‐lactic acid
Water molecules
160 170 180 190 200‐6‐4‐202468
[]
× 10‐3
/ deg
・ cm
2
・ dmol
‐1
Wavelength / nm
VUVCD spectrum and chemical structure of methyl ‐D‐glucopyranoside (methyl ‐D‐Glc)
Methyl ‐D‐Glc is composed of ‐GT, ‐GG, ‐TG rotamers
‐type
J. Phys. Chem. A (2012) 116, 9996
Conc.: 10%, Path length: 1.4m
Relative population = ‐GT : ‐GG : ‐TG = 38 : 58 : 4
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
Native VUVCD 14.4 3 37.7 10 19.3 28.8Modeler 11.5 3 36.6 10 23.5 28.4
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.