electronic spectroscopyramsey1.chem.uic.edu/tak/chem34407x/chem344slideselect-cd-part… ·...
TRANSCRIPT
Electronic Spectroscopy
Chem 344 final lecture topics
Time out—states and transitions
Spectroscopy—transitions between energy states of a molecule excited by absorption or emission of a photon
hν = ∆E = Ei - Ef
Energy levels due to interactions between parts of molecule (atoms, electrons and nucleii) as described by quantum mechanics, and are
characteristic of components involved, i.e. electron distributions (orbitals), bond strengths and types plus molecular geometries and atomic masses involved
Spectroscopy• Study of the consequences of the interaction of
electromagnetic radiation (light) with molecules.• Light beam characteristics - wavelength
(frequency), intensity, polarization - determine types of transitions and information accessed.
λ
E || zB || x
ν = c/λx
z
y
Wavelength Frequency
IntensityI ~ |E|2
}PolarizationB | E
k || y
Properties of light – probes of structure• Frequency matches change in energy, type of motion
E = hν, where ν = c/λ (in sec-1)• Intensity increases the transition probability—
I ~ ε2 –where ε is the radiation Electric Field strength Linear Polarization (absorption) aligns with direction of
dipole change—(scattering to the polarizability)I ~ [δµ/δQ]2 where Q is the coordinate of the motion
Circular Polarization results from an interference:Im(µ • m) µ and m are electric and magnetic dipole
0
.4
.8
1.2
4000 3000 2000 1000 Frequency (cm )
Abs
orba
nce
-1
λν
Intensity(Absorbance) IR of
vegetable oil
Optical Spectroscopy - Processes MonitoredUV/ Fluorescence/ IR/ Raman/ Circular Dichroism
IR – move nuclei low freq. & inten.
Raman –nuclei, inelastic scatter very low intensity
CD – circ. polarized absorption, UV or IR
Raman: ∆E = hν0-hνs
Infrared: ∆E = hνvib
= hνvib
Fluorescencehν = Eex - Egrd
0
Absorptionhν = Egrd - Eex
Excited State (distorted geometry)
Ground State (equil. geom.)
Q
ν0 νS
molec. coord.
UV-vis absorp.& Fluorescence.move e- (change electronic state) high freq., intense
Analytical MethodsDiatomic Model
Essentially a probe technique sensing changes in the local environment of fluorophores
Optical Spectroscopy – Electronic, Example Absorption and Fluorescence
Intrinsic fluorophores
eg. Trp, Tyr
Change with tertiary structure, compactness ε
(M-1
cm-1
)
F lu o
r es c
e nc e
Inte
nsit y
Amide absorption broad,Intense, featureless, far UV ~200 nm and below
What do you see?(typical protein)
UV absorption of peptides is featureless --except aromatics
TrpZip peptide in waterRong Huang, unpublished
Trp – aromatic bandsAmide π−π* and n-π*
Circular Dichroism
• Most protein secondary structure studies use CD
• Method is bandshape dependent. Need a different analysis
• Transitions fully overlap, peptide models are similar but not quantitative
• Length effects left out, also solvent shifts• Comparison revert to libraries of proteins• None are pure, all mixed
Circular DichroismCD is polarized differential absorption
∆A = AL - ARonly non-zero for chiral molecules
Biopolymers are Chiral (L-amino acid, sugars, etc.)Peptide/ Protein -
in uv - for amide: n-π* or π−π* in -HN-C=O-partially delocalized π-system senses structure
in IR - amide centered vibrations most importantNucleic Acids – base π−π* in uv, PO2-, C=O in IR
Coupled transitions between amides along chain lead to distinctive bandshapes
Polarization Modulation with PEM for CD(courtesy Hinds Instr.)
UV-vis Circular Dichroism Spectrometer
Xe arcsource
Double prism Monochromator (inc. dispersion, dec. scatter, important in uv)
PEM quartz
PMT
SampleSlits
This is shown to provide a comparison to VCD and ROA instruments
JASCO–quartz prisms disperse and linearly polarize light
Polypeptide Circular Dichroismordered secondary structure types
∆ε
λ
α-helix
β-sheet
turn
Brahms et al. PNAS, 1977
poly-L-glu(α,____), poly-L-(lys-leu)(β,- − - −), L-ala2-gly2(turn, . . . . . )
Critical issue in CD structure studies is SHAPE of the ∆ε pattern
Large electric dipole transitions can couple over longer ranges to sense extended conformation
Simplest representation is coupled oscillator
Tabµa
µb
( )baabTc
µµν rrrm ×⋅⎟
⎠⎞
⎜⎝⎛=±
2πR
∆ε =
εL−εR
Dipole coupling results in a derivative shaped circular dichroism
λ
Real systems - more complex interactions- but pattern is often consistent
B-DNARight -hand
Z-DNALeft-hand
B- vs. Z-DNA, major success of CD
Sign change in near-UV CD suggested the helix changed handedness
Protein Circular Dichroism
∆A
Myoglobin-high helix (_______), Immunoglobin high sheet (_______)Lysozyme, a+b (_______), Casein, “unordered” (_______),
Simplest Analyses –Single Frequency Response
Basis in analytical chemistry Beer’s law response if isolated
Protein treated as a solution % helix, etc. is the unknown
Standard in IR and Raman, Method: deconvolve to get componentsProblem – must assign component transitions, overlap
-secondary structure components disperse freq.
Alternate: uv CD - helix correlate to negative intensity at 222 nm, CD spectra in far-UV dominated by helical contribution
Problem - limited to one factor, -interference by chromophores]
Single frequency correlation of ∆ε with FC helix
FC helix [%]
0 20 40 60 80
∆ε
at 2
22nm
/193
nm
0
10
θ(222 nm) vs FC helixθ(193 nm) vs FC helix
Problem of secondary structure definition No pure states for calibration purposes
?
?
?
?
helix
sheet
Where do segments begin and end?Need definition:
Next step - project onto model spectra –Band shape analysis
Peptides as models- fine for α-helix, -problematic for β-sheet or turns - solubility and stability-old method:Greenfield - Fasman --poly-L-lysine, vary pH
θi = aiφα +biφβ + ciφc--Modelled on multivariate analyses
Proteins as models - need to decompose spectra- structures reflect environment of protein- spectra reflect proteins used as models
Basis set (protein spectra) size and form - major issue
Nature of the peptide random coil form
Tiffany and Krimm in 1968 noted similarity of Proline II and poly-lysine ECD and suggested “extended coil”Problem -- CD has local sensitivity to chiral site
--IR not very discriminating
Dukor and Keiderling 1991 with ECD, VCD, and IR showed Pron oligomers have characteristic random coil spectraSuggests -- local order, left-handed turn character
-- no long range order in random coil form
Same spectral shape found in denatured proteins, short oligopeptides, and transient forms
Dukor, Keiderling - Biopoly 1991
Reference: Poly(Lys) – “coil”, pH 7
ECD of Pron oligomers
Builds up to Poly-Pro II frequency -->tertiary amide helix
sheet
‘coil’
Single amide
Greenfield & Fasman 1969
Poly Proline mutarotate from I (right hand cis) to II (left hand trans)
Time dependent change in H2OPoly-L-Pro I
I+II
I
Conformation driven by added salt, concentrationPoly(LKKL) Poly(KL) (LKKL)5 (KL)10
Electronic CD spectra consistent with predicted helix content
- 3
- 2
- 1
0
1 0
2 0
3 0
4 0
5 0
1 92 02 2 2 2 2 2
ticity
Wavelength (nm)190 210 230
Note helical bands, coil has residual at 222 nm, growth of 200 nm bandElectronic CD for helix to coil change in a peptide
Loss of order becomes a question --ECD long range sensitivity cannot determine remaining local order
Low temp helix
High temp “coil”
BETA-LACTOGLOBULIN
• Mw 18,400 Da, 162 residues• Primarily β-sheet (42% sheet, 16% helix)• High propensity for helical conformation• Structural homolgy to retinol binding protein
wavelength (nm)180 200 220 240 260
-6
-4
-2
0
2
4
6
8∆
ε (M
-1cm
-1)
0 mM
3 mM} 5 - 50 mM
Far-UVCD spectra of BLG titrated with SDS (0-50 mM)
wavelength (nm)260 280 300 320
∆ε
(M-1
cm-1
)
-0.0020
-0.0015
-0.0010
-0.0005
0.0000
0.0005
0 mM
0.1 mM
1.0 mM
3, 5 mM
Near-UVCD spectra of BLG titrated with SDS
PC/FA determined secondary structure change
[SDS] (mM)0 10 20 30 40 50
Seco
ndar
y St
ruct
ure
(%)
10
15
20
25
30
35
40
sheet
helix
Critical Micelle Concentration (8.2 mM)
Tyr97
Tyr25
Tyr92
H1
H3H2
Tyr76
Tyr115
Tyr73Ribonuclease A combined uv-CD and FTIR study
6 β β sheet
, 2 )
• 124 amino acid residues, 1 domain, MW= 13.7 KDa
• 3 α-helices
• 6 β-strands in an AP β-sheet
• 6 Tyr residues (no Trp), 4 Pro residues (2 cis, 2 trans)
W a v e l e n g t h ( n m )2 6 0 2 8 0 3 0 0 3 2 0
Ellip
ticity
(mde
g)
- 1 6
- 1 4
- 1 2
- 1 0
- 8
- 6
- 4
- 2
0
N e a r - U V C D
W a v e n u m b e r ( c m - 1 )1 6 0 01 6 2 01 6 4 01 6 6 01 6 8 01 7 0 01 7 2 0
Abs
orba
nce
0 . 0 0
0 . 0 1
0 . 0 2
0 . 0 3
0 . 0 4
0 . 0 5
0 . 0 6
F T I R
Ellip
ticity
(mde
g)
- 1 5
- 1 0
- 5
0
5
F a r - U V C D Temperature 10-70oC
FTIR—amide ILoss of β-sheet
RibonucleaseA
Far-uv CDLoss of α-helix
Near –uv CDLoss of tertiary structure
Spectral Change1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0
Stelea, et al. Prot. Sci. 2001W a v e l e n g t h ( n m )
Ci1
(x10
2 )
- 8 . 0
- 7 . 6
- 7 . 2
- 6 . 8
- 6 . 4
- 1 . 0
- 0 . 5
0 . 0
0 . 5
1 . 0
F T I RC
i1
- 1 7
- 1 5
- 1 3
- 1 1
- 9
- 7
- 5
Ci2
- 1 5
- 1 0
- 5
0
5
1 0
N e a r - U V C D
0 2 0 4 0 6 0 8 0 1 0 0
Ci1
- 1 3
- 1 2
- 1 1
- 1 0
Ci2
- 3 0
- 2 5
- 2 0
- 1 5
- 1 0
- 5
0
5
F a r - U V C D
Ribonuclease APC/FA loadingsTemp. variation
FTIR (α,β)
Near-uv CD(tertiary)
Far-uv CD(α-helix)
Pre-transition - far-uv CD and FTIR, not near-uvTemperature
Stelea, et al. Prot. Sci. 2001
Changing protein conformational order by organic solvent
TFE and MeOH often used to induce helix formation--sometimes thought to mimic membrane--reported that the consequent unfolding can lead to aggregation and fibril formation in selected cases
Examples presented show solvent perturbation of dominantly β-sheet proteins
TFE and MeOH behave differentlythermal stability key to differentiating statesindicates residual partial order
β-lactoglobulin--pH and TFE , MeOH
TFE and MeOH both induce helix at both pH 7 and 2
FTIR vs. time MeOH mix
Factor analysis - 2nd component loading shows loss of sheet with time, double exp.
Xu&Keiderling, Adv.Prot.Chem.2002
Concanavalin A pH, TFE and MeOH
native
TFE
MeOH normalizes β-sheet ECD, FTIR indicates aggregated
TFE induces helixXu&Keiderling, Biochem, 2005
VIBRATIONAL OPTICAL ACTIVITYVIBRATIONAL OPTICAL ACTIVITYDifferential Interaction of a Chiral Molecule with Left and Right Circularly Polarized Radiation During Vibrational ExcitationVIBRATIONAL CIRCULAR DICHROISM RAMAN OPTICAL ACTIVITYDifferential Absorption of Left and Right Differential Raman Scattering of Left Circularly Polarized Infrared Radiation and Right Incident and/or Scattered
Radiation
Combining Techniques: Vibrational CD“CD” in the infrared region
Probe chirality of vibrations goal stereochemistryMany transitions / Spectrally resolved / Local probes
Technology in place -- separate talkWeak phenomenon - limits S/N / Difficult < 700 cm-1
Same transitions as IRsame frequencies, same resolution
Band Shape from spatial relationshipsneighboring amides in peptides/proteins
Relatively short length dependenceAAn oligomers VCD have ∆A/A ~ const with n
vibrational (Force Field) coupling plus dipole couplingDevelopment -- structure-spectra relationships
Small molecules – theory / Biomolecules -- empirical, Recent—peptide VCD can be simulated theoretically
G
C F
M2S
M1
PEMP
SCL
D
D Pre-Amp
DynamicNormalization
TunedFilter
ωΜLock-in
ωCLock-in
Chopper ref. ωC
PEM ref.ωM
TransmissionFeedback Lock-in
A/DInterfaceComputerInterfaceMonochromator
UIC Dispersive VCD Schematic
Electronics
Optics and Sampling
Yes it still exists and measures VCD!
UIC FT-VCDSchematic(designed for magnetic VCD commercial ones simpler)
Electronics
OpticsFTIR
Separate VCD Bench
PolarizerPEM (ZnSe)Sample
Detector (MCT)
Optional magnet
lock-in ampfilter PEM ref
detector
FT-computer
Large electric dipole transitions can couple over longer ranges to sense extended conformation
Simplest representation is coupled oscillator
Tabµa
µb
( )baabTc
µµν rrrm ×⋅⎟
⎠⎞
⎜⎝⎛=±
2πR
∆ε =
εL−εR
Dipole coupling results in a derivative shaped circular dichroism
λ
Real systems - more complex interactions- but pattern is often consistent
Selected model Peptide VCD, aqueous solution
W a v e n u m b e r s ( c m - 1 )
1 4 5 01 5 0 01 5 5 01 6 0 01 6 5 01 7 0 01 7 5 0
VCD
(A
. U.)
- 1 0
0
1 0
2 0
3 0 h e l i x
β - s t r u c t u r e
r a n d o mc o i l
Amide IAmide II
α
β
coil
∆A
Dukor, Keiderling - Biopoly 1991
Relationship to “random coil” - compare Pron and Glun
IR ~ same, VCD - same shape, half size -- partially ordered
Thermally unfolding “random coil” poly-L-Glu -IR, VCD
VCD loses magnitude
IR shifts frequency
T = 5oC (___)25oC (- - -)75oC (-.-.-)
“random coil” must have local order
Keiderling. . . Dukor, Bioorg-MedChem 1999
Salt denaturation of “random coil” polypeptidesGlun/4.5 M LiClO4 Lysn/5 M NaCl
D2O D2O
LiClO4 NaCl
Keiderling. . . Dukor, Bioorg-MedChem 1999
Lysn local order as function of length
MW ~ 1000 (- - -)25,000 (-----)
250,000 (____)
More length -more structure
pH ~ 7 -- coil(local 31-helix)
pH ~11 -- helix/coil
VCD Example: α- vs. the 310-Helix
i, i+4 ← H-bonding → i, i+3
3.6 ← Res./Turn → 3.0
2.00 ← Trans./Res (Å) → 1.50
α-Helix 310-Helix
Wavenumbers (cm-1)
140016001800
Abs
orba
nce
0
1
2
3
4
Wavenumbers (cm-1)
14016001800
∆A
(A.U
.)
-100
0
100
200
300
400
500
α-helical
(Aib-Ala)6
Ala(AibAla)3
310-helical
The VCD success example: 3The VCD success example: 31010--helix vs. helix vs. αα--helixhelix
Relative shapes of multiple bands distinguish these similar helices
Aib2LeuAib5
(Met2Leu)6 α
310
mixed
i−>i+3
i−>i+4
Silva et al. Biopolymers 2002
-1
VCD of DNA, vary A-T to G-C ratiobase deformations sym PO2
- stretches
big variation little effect
DNA VCD of PO2- modes in B- to Z-form transition
A
Z
B
B
B, A
Z
Experimental Theoretical
Triplex DNA, RNA form by adding third strand to major groove with Hoogsteen base pairing
-20CGC+
Wavenumber (cm-1)
VCD of Triplex formation—base modes