Transcript Slide 1

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
hn = DE = 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.
Intensity
I ~ |E|2
z
B | E
E || z
B || x Polarization
}
y
k || y
x
l
Wavelength
n = c/l
Frequency
Properties of light – probes of structure
• Frequency matches change in energy, type of motion
E = hn, where n = c/l (in sec-1)
• Intensity increases the transition probability—
I ~ e2 –where e is the radiation Electric Field strength
Linear Polarization (absorption) aligns with direction of
dipole change—(scattering to the polarizability)
I ~ [dm/dQ]2 where Q is the coordinate of the motion
Circular Polarization results from an interference:
Im(m • m) m and m are electric and magnetic dipole
Intensity
(Absorbance)
IR of
vegetable
oil
Absorbance
1.2
.8
.4
n
l
0
4000
3000
Frequency (cm
2000
) -1
1000
Optical Spectroscopy - Processes Monitored
UV/ Fluorescence/ IR/ Raman/ Circular Dichroism
Excited
State
(distorted
geometry)
Ground
State (equil.
geom.)
Diatomic Model
Analytical Methods
Absorption UV-vis absorp.
hn = E - E & Fluorescence.
grd
n0 nS
ex
Fluorescence
hn = Eex - Egrd
Raman: DE = hn0-hns
= hnvib
Infrared: DE = hnvib
Q
molec. coord.
move e- (change
electronic state)
high freq., intense
CD – circ. polarized
absorption, UV or IR
Raman –nuclei,
inelastic scatter
very low intensity
IR – move nuclei
low freq. & inten.
Optical Spectroscopy – Electronic,
Example Absorption and Fluorescence
Essentially a probe technique sensing changes in the local environment of fluorophores
What do you see?
(typical protein)
eg. Trp, Tyr
Change with tertiary
structure, compactness
e (M-1 cm-1)
Intrinsic fluorophores
Amide absorption broad,
Intense, featureless, far UV
~200 nm and below
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 Dichroism
CD is polarized differential absorption
DA = AL - AR
only non-zero for chiral molecules
Biopolymers are Chiral (L-amino acid, sugars, etc.)
Peptide/ Protein in uv - for amide: n-p* or p-p* in -HN-C=Opartially delocalized p-system senses structure
in IR - amide centered vibrations most important
Nucleic Acids – base p-p* in uv, PO2-, C=O in IR
Coupled transitions between amides along chain lead to
distinctive bandshapes
UV-vis Circular Dichroism Spectrometer
Slits
Sample
PMT
Xe arc
source
Double prism
Monochromator (inc. dispersion,
dec. scatter, important in uv)
PEM
quartz
This is shown
to provide a
comparison to
VCD and ROA
instruments
JASCO–quartz prisms disperse and linearly polarize light
Amino Acids - linked by Peptide bonds
 coupling yields structure sensitivity
Link is mostly planar and trans, except for Xxx-Pro
UV absorption of peptides is featureless --except aromatics
Amide
p-p* and n-p*
TrpZip peptide in water
Rong Huang, unpublished
Trp – aromatic bands
a-helix - common peptide secondary structure
(ii+4)
b-sheet cross-strand H-bonding
Anti-parallel b-sheet (extended strands)
Polypeptide Circular Dichroism
ordered secondary structure types
a-helix
De
b-sheet
turn
Brahms et al.
PNAS, 1977
l
poly-L-glu(a,____), poly-L-(lys-leu)(b,- - - -), L-ala2-gly2(turn, . . . . . )
Critical issue in CD structure studies is SHAPE of the De pattern
Large electric dipole transitions can couple over
longer ranges to sense extended conformation
Simplest representation is coupled oscillator
R



 πn  
 
T ab   m a  m b )
 2c 
De 
eL-eR
l
ma
Tab
mb
Dipole coupling
results in a
derivative shaped
circular dichroism
Real systems - more complex interactions
- but pattern is often consistent
B-DNA
Right -hand
Z-DNA
Left-hand
B- vs. Z-DNA, major success of CD
Sign change in near-UV CD suggested the helix changed handedness
Protein Circular Dichroism
DA
Myoglobin-high helix (_______), Immunoglobin high sheet (_______)
Lysozyme, a+b (_______), Casein, “unordered” (_______),
Coupling  shapes, but not isolated & modeling tough
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 components
Problem – 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 De with FC helix
 (2 2 2 n m ) v s F C h e lix
D e a t 2 2 2 n m /1 9 3 n m
 (1 9 3 n m ) v s F C h e lix
10
0
0
20
40
F C h e lix [% ]
60
80
Problem of secondary structure definition
No pure states for calibration purposes
?
?
?
helix
sheet
?
Need definition:
Where do segments begin and end?
Next step - project onto model spectra
–Band shape analysis
Peptides as models
- fine for a-helix,
-problematic for b-sheet or turns - solubility and stability
-old method:Greenfield - Fasman --poly-L-lysine, vary pH
i = aifa +bifb + cifc
--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
Electronic CD for helix to coil change in a peptide
Electronic CD spectra consistent with predicted
Note helical bands, coil has residual at 222 nm, growth of 200 nm band
helix content 0
5
4
0
Loss of order becomes a question -ECD long range sensitivity
cannot
0
determine remaining local order
3
2
0
1
High temp “coil”
0
0
-
1
2
Low
temp helix
-
3
1
190
2
2
210
2
lip tic ity
2
230
Wavelength (nm)
2
2
2 9
0
Tyr92
Tyr115
Tyr97
Tyr73
H1
Ribonuclease A
combined uv-CD
and FTIR study
H2
H3
Tyr25
Tyr76
• 124 amino acid residues, 1 domain, MW= 13.7 KDa
• 3 a-helices

• 6 b-strands
in6an

b AP b-sheet b sheet
• 6 Tyr residues (no Trp), 4 Pro residues, (2
2 cis,) 2 trans)
0 .0 6
RibonucleaseA
F T IR
0 .0 5
Absorbance
0 .0 4
0 .0 3
0 .0 2
FTIR—amide I
0 .0 1
Loss of b-sheet
0 .0 0
1720
1700
1680
1660
1640
W a v e n u m b e r (c m
-1
1620
1600
)
0
Ellipticity (mdeg)
-2
Near –uv CD
-4
-6
-8
Loss of tertiary
structure
-1 0
-1 2
-1 4
N e a r-U V C D
-1 6
260
280
300
320
W a v e le n g t h ( n m )
Far-uv CD
Ellipticity (mdeg)
5
Loss of a-helix
0
-5
Spectral Change
Temperature 10-70oC
-1 0
F a r-U V C D
-1 5
190
200
210
220
W a v e le n g t h ( n m )
230
240
250
Stelea, et al. Prot. Sci. 2001
Ribonuclease A
-6 .4
1 .0
2
C i1 (x10 )
0 .5
-7 .2
0 .0
-7 .6
-0 .5
-8 .0
-1 .0
-5
PC/FA loadings
Temp. variation
C i2 (x10)
F T IR
-6 .8
FTIR (a,b)
10
-7
5
-9
Near-uv CD
(tertiary)
C i2
-5
-1 3
-1 5
-1 0
-1 7
-1 5
-1 0
5
0
-1 1
-5
C i1
F a r-U V C D
-1 0
-1 2
-1 5
Far-uv CD
(a-helix)
C i2
C i1
0
N e a r-U V C D
-1 1
-2 0
-1 3
-2 5
-3 0
0
20
40
60
T e m p e ra tu re (oC )
80
100
Pre-transition - far-uv CD and FTIR, not near-uv
Temperature
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 b-sheet proteins
TFE and MeOH behave differently
thermal stability key to differentiating states
indicates residual partial order
3D Structure of Concanavalin A
Dimer (acidic, pH<6)
Trp40
Tetramer (pH=6-7)
Trp109
Trp182 Trp88
High b-sheet structure, flat back extended, curved front
Monomer only at very low pH, 4 Trp give fluorescence
Effect of TFE (50%) on Con A in Far and Near UV- CD
Far UV-CD
Helix induced with
TFE addition
pH=7
pH=2
Near UV-CD
Helical Content
43%
57%
Tertiary change
with TFE - loosen
Xu&Keiderling, Biochemistry 2005
Dynamics--Scheme of Stopped-flow System
- add dynamics to experiment
Denatured
protein
solution
Refolding
buffer
solution
Stopped-Flow CD for Con A Unfolding with TFE (1:1)
at Different pH Conditions
Far UV (222 nm);
Near UV (290 nm);
[Con]f=0.2mg/ml
[Con]f=2mg/ml
pH=2.0
Xu&Keiderling, Biochemistry 2005
b-lactoglobulin: a protein that goes both ways!
Native state: b-sheet dominant, but high helical propensity.
Model: intramolecular ba transition pathway as opposed
to folding pathways from a denatured state.
Zhang & Keiderling, Biochemistry 2006
Lipid-induced Conformational Transition b-Lactoglobulin
Fractional secondary structure
1. DMPG-dependent ba transition at pH 6.8
0.5
Unordered
0.4
a-Helix
0.3
0.2
b -Sheet
0.1
0
1
2
3
4
5
DMPG / mM
Zhang & Keiderling, Biochemistry 2006
Charge-induced Lipid -- b-Lactoglobulin Interaction
Fractional secondary structure
DMPC/ (DMPC+DMPG)/ %
100
80
60
40
20
0
Unordered
0.5
0.4
Helix
0.3
0.2
0.1
Sheet
0
20
40
60
80
100
DMPG / (DMPC+DMPG) / %
Zhang &
Keiderling,
Biochemistry 2006
Increase DMPG, increases helix at expense of sheet
Stopped Flow Experiments : (pH 4.60)
Vesicles (SUV)
Vesicles (SUV)
(DOPG, DMPG, DSPG)
5 Volume
+
1 Volume
BLG (1.2mg/ml)
BLG (0.2mg/ml)
CD: 222nm to monitor alpha-helix
Fluorescence: filter with a 320nm cutoff ( Trp Tertiary Structure)
10-15 kinetic traces are collected and averaged
Analysis：Multi-exponential function using Simplex Method:
S(t)=a*t+b+∑i(ci Exp(-ki*t))
Ge, Keiderling, to be submitted
Stopped-Flow CD kinetic traces
S im u la te d kin e tics tra ce s o f B L G (0 .2 m g /m l) w ith D M P G
E llip tic ity (m d e g )
-1 0
DMPG
N
0 .1 5 m M D M P G
0 .2 5 m M D M P G
0 .5 0 m M D M P G
-2 0
-3 0
1 .0 0 m M D M P G
2 .0 0 m M D M P G
5 .0 0 m M D M P G
-4 0
-5 0
0
5
10
T im e /s
15
20
Record at 222nm;
N: trace without lipid
vesicles;
Traces are fitted to
single-exponential
function
Stopped-Flow fluorescence kinetics
K in e tics tra ce s fo r B L G (0 .2 m g /m l) w ith D M P G
R e la tiv e In te n s ity
2 .4
DMPG
2 .2
5 .0 0 m M
2 .0 0 m M
1 .0 0 m M
0 .5 0 m M
2 .0
1 .8
DMPG
DMPG
DMPG
DMPG
0 .2 5 m M D M P G
1 .6
1 .4
0 .1 5 m M D M P G
1 .2
1 .0
0
5
10
T im e /s
15
20
Total fluorescence >320nm;
Each trace has been divided
by kinetic trace without lipid
vesicles;
Traces are fitted to twoexponential function
Lipid bilayer insertion of b-Lactoglobulin
b LG-DMPG, pH4.6
pH 6 .8
b LG-DMPG, pH6.8
1467
1.2
1654
1637
1.4
1731
F0 / F
1343
1328
1305
1280
1255
1229
1745
b LG
1.6
ATR-FTIR orientation
1654
1637
Fluorescence quenching
pH 4 .6
1.0
0.0
0.1
0.2
0.3
0.4
Acrylamide/M
1700
At pH 6.8 & 4.6,
4 & 6 nm blue
shift in lmax.
1600
1500
1400
W avelen gth /cm
1300
-1
a-helix Membrane surface
Zhang & Keiderling, Biochemistry 2006
Summary: Lipid - b-Lactoglobulin Interaction
Nw
Binding
Unfolding
s
N
Us
Insertion
Um
Zhang & Keiderling, Biochemistry 2006
• Continued in Part b