1. Electronic Spectroscopy
Chem 344 final lecture topics

2. 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

3. 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 l
E || z
B || x
n = c/l x z y
B | E }
Polarization
k || y
Frequency
Wavelength

4. 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 n l

5. Optical Spectroscopy - Processes Monitored UV/ Fluorescence/ IR/ Raman/ Circular Dichroism
Diatomic Model
Analytical Methods
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.

6. Optical Spectroscopy – Electronic, Example Absorption and Fluorescence
Essentially a probe technique sensing changes in the local environment of fluorophores
 (M-1 cm-1)
Fluorescence Intensity
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

7. 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

8. CD is polarized differential absorption
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=O-
partially 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

9. 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)
JASCO–quartz prisms disperse and linearly polarize light

10. Link is mostly planar and trans, except for Xxx-Pro
Amino Acids - linked by Peptide bonds  coupling yields structure sensitivity
Link is mostly planar and trans, except for Xxx-Pro

11. UV absorption of peptides is featureless --except aromatics
Amide
p-p* and n-p*
Trp – aromatic bands
TrpZip peptide in water
Rong Huang, unpublished

12. a-helix - common peptide secondary structure
(ii+4)

13. b-sheet cross-strand H-bonding

14. Anti-parallel b-sheet (extended strands)

15. Polypeptide Circular Dichroism
ordered secondary structure types De l
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

16. Large electric dipole transitions can couple over
longer ranges to sense extended conformation
Simplest representation is coupled oscillator
Tab ma mb
De =
eL-eR
Dipole coupling results in a derivative shaped circular dichroism l
Real systems - more complex interactions
- but pattern is often consistent

17. DNA
B-DNA
Right -hand
Z-DNA
Left-hand

18. B- vs. Z-DNA, major success of CD
Sign change in near-UV CD suggested the helix changed handedness

19. Protein Circular DichroismDA
Myoglobin-high helix (_______), Immunoglobin high sheet (_______)
Lysozyme, a+b (_______), Casein, “unordered” (_______),
Coupling  shapes, but not isolated & modeling tough

20. Single Frequency Response
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]

21. Single frequency correlation of De with FC helix

22. Problem of secondary structure definition No pure states for calibration purposes? ? ?
helix
sheet ?
Need definition:
Where do segments begin and end?

23. 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
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

24. Electronic CD spectra consistent with predicted helix content
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 9

25. Ribonuclease A combined uv-CD and FTIR study
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 Ø 6 b b
sheet
, 2 )

26. RibonucleaseA FTIR—amide I Near –uv CD Far-uv CD Loss of b-sheet
Loss of tertiary structure
Far-uv CD
Loss of a-helix
Spectral Change
Temperature 10-70oC
Stelea, et al. Prot. Sci. 2001

27. Ribonuclease A FTIR (a,b) Near-uv CD (tertiary) Far-uv CD (a-helix)
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

28. 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

29. 3D Structure of Concanavalin A
Dimer (acidic, pH<6)
Tetramer (pH=6-7)
Trp40
Trp109
Trp182
Trp88
High b-sheet structure, flat back extended, curved front
Monomer only at very low pH, 4 Trp give fluorescence

30. 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
Xu&Keiderling, Biochemistry 2005

31. Dynamics--Scheme of Stopped-flow System
- add dynamics to experiment
Denatured protein solution
Refolding buffer solution

32. Far UV (222 nm); [Con]f=0.2mg/ml Near UV (290 nm); [Con]f=2mg/ml
Stopped-Flow CD for Con A Unfolding with TFE (1:1) at Different pH Conditions
Far UV (222 nm); [Con]f=0.2mg/ml
Near UV (290 nm); [Con]f=2mg/ml
pH=2.0
Xu&Keiderling, Biochemistry 2005

33. Zhang & Keiderling, Biochemistry 2006
-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

34. Zhang & Keiderling, Biochemistry 2006
         
Lipid-induced Conformational Transition -Lactoglobulin
1. DMPG-dependent  transition at pH 6.8
Zhang & Keiderling, Biochemistry 2006

35. Charge-induced Lipid -- -Lactoglobulin Interaction
         
Charge-induced Lipid -- -Lactoglobulin Interaction
Zhang & Keiderling, Biochemistry 2006
Increase DMPG, increases helix at expense of sheet

36. 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
Analysis：Multi-exponential function using Simplex Method:
S(t)=a*t+b+∑i(ci Exp(-ki*t))
Ge, Keiderling, to be submitted

37. Stopped-Flow CD kinetic traces
DMPG
Record at 222nm;
N: trace without lipid vesicles;
Traces are fitted to single-exponential function

38. Stopped-Flow fluorescence kinetics
DMPG
Total fluorescence >320nm;
Each trace has been divided
by kinetic trace without lipid vesicles;
Traces are fitted to two-exponential function

39. Lipid bilayer insertion of -Lactoglobulin
         
Lipid bilayer insertion of -Lactoglobulin
Fluorescence quenching
ATR-FTIR orientation
At pH 6.8 & 4.6,
4 & 6 nm blue
shift in max.
-helix Membrane surface
Zhang & Keiderling, Biochemistry 2006

40. Summary: Lipid - b-Lactoglobulin InteractionNw Ns
Unfolding Us
Insertion Um
Binding
Zhang & Keiderling, Biochemistry 2006

41. Continued in Part b