Electronic Spectroscopy

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Electronic Spectroscopy

• Chem 344 final lecture topics

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Time outstates and transitions
Spectroscopytransitions 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
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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 E2
B E

Polarization
k y
Frequency
Wavelength
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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/dQ2 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
n
l
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Optical Spectroscopy - Processes MonitoredUV/
Fluorescence/ IR/ Raman/ Circular Dichroism
Analytical Methods
Diatomic Model
Excited State (distorted geometry)
Absorption hn Egrd - Eex
UV-vis absorp. Fluorescence. move e- (change
electronic state) high freq., intense
Ground State (equil. geom.)
CD circ. polarized absorption, UV or IR
n0
nS
Fluorescence hn Eex - Egrd
Raman nuclei, inelastic scatter very low
intensity
Raman DE hn0-hns
hnvib
IR move nuclei low freq. inten.
Infrared DE hnvib
Q
molec. coord.
0
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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)
Intrinsic fluorophores eg. Trp, Tyr Change with
tertiary structure, compactness
Amide absorption broad, Intense, featureless, far
UV 200 nm and below
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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

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Circular Dichroism
CD is polarized differential absorption DA AL
- AR only non-zero for chiral molecules Biopoly
mers are Chiral (L-amino acid, sugars,
etc.) Peptide/ Protein - in uv - for amide
n-p or p-p in -HN-CO- partially
delocalized p-system senses structure in IR -
amide centered vibrations most important Nucleic
Acids base p-p in uv, PO2-, CO in IR Coupled
transitions between amides along chain lead to
distinctive bandshapes
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UV-vis Circular Dichroism Spectrometer
Sample
Slits
PMT
PEM quartz
Xe arc source
This is shown to provide a comparison to VCD and
ROA instruments
Double prism Monochromator (inc. dispersion,
dec. scatter, important in uv)
JASCOquartz prisms disperse and linearly
polarize light
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Amino Acids - linked by Peptide bonds ? coupling
yields structure sensitivity
Link is mostly planar and trans, except for
Xxx-Pro
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UV absorption of peptides is featureless --except
aromatics
Amide p-p and n-p
Trp aromatic bands
TrpZip peptide in water Rong Huang, unpublished
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a-helix - common peptide secondary structure
(i?i4)
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b-sheet cross-strand H-bonding
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Anti-parallel b-sheet (extended strands)
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Polypeptide Circular Dichroism ordered secondary
structure types
a-helix
b-sheet
turn
Brahms et al. PNAS, 1977
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
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Large electric dipole transitions can couple over
longer ranges to sense extended conformation
Simplest representation is coupled oscillator
De eL-eR
Dipole coupling results in a derivative shaped
circular dichroism
l
Real systems - more complex interactions - but
pattern is often consistent
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DNA
B-DNA Right -hand
Z-DNA Left-hand
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B- vs. Z-DNA, major success of CD
DNA
Sign change in near-UV CD suggested the helix
changed handedness
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Protein Circular Dichroism
DA
Myoglobin-high helix (_______), Immunoglobin high
sheet (_______) Lysozyme, ab (_______), Casein,
unordered (_______),
Coupling ? shapes, but not isolated modeling
tough
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Simplest Analyses Single Frequency
Response   Basis in analytical chemistry ?Beers
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
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Single frequency correlation of De with FC helix
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Problem of secondary structure definition No pure
states for calibration purposes
?
?
?
helix
sheet
?
Need definition
Where do segments begin and end?
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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 methodGreenfield
- Fasman --poly-L-lysine, vary pH qi 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
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Electronic CD for helix to coil change in a
peptide
Electronic CD spectra consistent with predicted
helix content
Note helical bands, coil has residual at 222 nm,
growth of 200 nm band
Loss of order becomes a question -- ECD long
range sensitivity cannot determine remaining
local order
High temp coil
Low temp helix
190
210
230
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Tyr92
Ribonuclease A combined uv-CD and FTIR study
Tyr115
Tyr97
Tyr73
H1
H2
H3
Tyr76
Tyr25

• 124 amino acid residues, 1 domain, MW 13.7 KDa
• 3 a-helices
• 6 b-strands in an AP b-sheet
• 6 Tyr residues (no Trp), 4 Pro residues (2 cis, 2
  trans)

Ø
6
b
b
sheet
Ø
, 2
)
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RibonucleaseA
FTIRamide I Loss of b-sheet
Near uv CD Loss of tertiary structure
Far-uv CD Loss of a-helix
Spectral Change
Temperature 10-70oC
Stelea, et al. Prot. Sci. 2001
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Ribonuclease A
PC/FA loadings Temp. variation
FTIR (a,b)
Near-uv CD (tertiary)
Far-uv CD (a-helix)
Temperature
Stelea, et al. Prot. Sci. 2001
Pre-transition - far-uv CD and FTIR, not near-uv
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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
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3D Structure of Concanavalin A
Dimer (acidic, pHlt6)
Tetramer (pH6-7)
Trp40
Trp109
Trp88
Trp182
High b-sheet structure, flat back extended,
curved front Monomer only at very low pH, 4 Trp
give fluorescence
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Effect of TFE (50) on Con A in Far and Near UV-
CD
Far UV-CD
Near UV-CD
Tertiary change with TFE - loosen
Helix induced with TFE addition
XuKeiderling, Biochemistry 2005
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Dynamics--Scheme of Stopped-flow System
- add dynamics to experiment
Denatured protein solution
Refolding buffer solution
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Stopped-Flow CD for Con A Unfolding with TFE
(11) at Different pH Conditions
Far UV (222 nm) Conf0.2mg/ml
Near UV (290 nm) Conf2mg/ml
pH2.0
XuKeiderling, Biochemistry 2005
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?-lactoglobulin a protein that goes both ways!
Native state ?-sheet dominant, but high
helical propensity. Model intramolecular ???
transition pathway as opposed to folding pathways
from a denatured state.
Zhang Keiderling, Biochemistry 2006
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Lipid-induced Conformational Transition
?-Lactoglobulin
1. DMPG-dependent ??? transition at pH 6.8
Zhang Keiderling, Biochemistry 2006
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Charge-induced Lipid -- ?-Lactoglobulin
Interaction
Zhang Keiderling, Biochemistry 2006
Increase DMPG, increases helix at expense of
sheet
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Stopped Flow Experiments (pH 4.60)
Vesicles (SUV) (DOPG, DMPG, DSPG)
Vesicles (SUV) BLG (0.2mg/ml)
5 Volume
1 Volume
BLG (1.2mg/ml)
CD 222nm to monitor alpha-helix Fluorescence
filter with a 320nm cutoff ( Trp Tertiary
Structure) 10-15 kinetic traces are collected and
averaged
AnalysisMulti-exponential function using Simplex
Method
S(t)atb?i(ci Exp(-kit))
Ge, Keiderling, to be submitted
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Stopped-Flow CD kinetic traces
DMPG
Record at 222nm N trace without lipid
vesicles Traces are fitted to single-exponential
function
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Stopped-Flow fluorescence kinetics
DMPG
Total fluorescence gt320nm Each trace has been
divided by kinetic trace without lipid
vesicles Traces are fitted to two-exponential
function
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Lipid bilayer insertion of ?-Lactoglobulin
ATR-FTIR orientation
Fluorescence quenching
At pH 6.8 4.6, 4 6 nm blue shift in ?max.
?-helix ?Membrane surface
Zhang Keiderling, Biochemistry 2006
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Summary Lipid - b-Lactoglobulin Interaction
Zhang Keiderling, Biochemistry 2006
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• Continued in Part b