??? ????? Circular Dichroism (CD)

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snl.bjmu.edu.cn/ ourse/biophysics/cover.htm
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Circular Dichroism CD
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1.????????
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??,
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2.???????,?? ??????
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structure of membrane protein
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cm-1 Secondary structure
16244 sheet
16313 sheet
16373 sheet
16454 Random coil
16534 helix
16634 turn
16713 turn
16755 sheet
16832 turn
16892 turn
16942 turn
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????????,??????? ?????,????????? ???????????????
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Crystal Structure of Mitochondrial Respiratory
Membrane Protein Complex II. Cell, 2005,121
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hybrid orbital
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L alanine
D alanine
Chiral
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Y
X
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Y
X
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????,???? ????,????
Y
X
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????,???? ?????????
?
?1
?2
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y
y
a
????,???? ?????????
L
R
?????
x
x
? ? ?
?
? tg-1(El Er )/(El Er )
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????? ????? ????? ?????? ??????
?? ?? ?? ???? ????
?? ?? ?? ? ?
?? ?? ?? ? ?
?? ?? ?? ? ?
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Optically Active Sample
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Right and Left hand circularly polarized light
Chiral
Preferential absorption of left hand polarized
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????
ExE0x cos (?tf0 )
????
ExE0x cos ?(t-L/v)f0
Or
ExEox cos ?t-(2?L/?)nf0
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CD (circular dichroism)???
L
Beer-Lanmbert Law
?
A lgI0/I(1/2,303)lnI0/Ie?CL
Optical active object
?A?Al - Ar ?e? C L
? tg-1(El Er )/(El Er )
?? 2.303 ? A?/4 (??)
Ex E0x cos ?t-(2?L /?)nf0
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yl (the specific ellipticity)
?????? l (the molar ellipticity)
???????? lMRw (the mean residue ellipticity)
C ???? L ?? (dm) ? l deg mol-1 cm-1
or deg cm2 dmol-1
? l ?? /CL
? l Mw yl /100
? lMRw MRw yl /100
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???????
L
????(Circular birefringence)
?
nl nr
Dn nl - nr
?F(2? /?)?n
Optical active object
Chiral
????ORD (optical rotatory dispersion)
? (? /?) ?n
a f (l),??
a ??LC
ExE0x cos ?t-(2?L/?)nf0
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?l (the specific rotation)
?????? jl (the molar rotation)
??????? ml (the mean residue
rotation)
yl ?l/ Cw L
C g/mL L dm Mw molecular weight MRw the
mean residue Mw
jl al ?Mw/100
ml al ?MRw/100
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????( principle )
Plane (linearly) polarized light
Preferential absorption of left hand polarized
Optically Active Sample
Right and Left hand circularly polarized light
Chiral
ExE0x cos ?t-(2?l /?)nf0
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relationship between CD and ORD and
Cotton effect
nl , nr
al f (l)
ORD
y
y
a
L
Light
Kronig-Kramers transition
x
R
x
CD
? l f (l)
?
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e
l0
Cotton effect
?

Positive cotton effect
l0
?
-

Negative cotton effect
l0
?
-
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Instrument
http//www-structure.llnl.gov/
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CD of amino acid and peptides
Amino acid
???,???,??,???,???,???
???,???,????,???,??? ?????(240-300nm)?CD???? ???2
00-210nm?????
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??????????
?????300nm???,??????,?CD????????????????????????,?
???CD?????????????????????-??????
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Secondary structure of macromolecule
a-helix 190 nm () 208nm, 222nm (-)
a-helix
b-sheet 195nm () antiparallel red shift
parallel blue shift 215-217nm (-)
b-sheet
unordered
Unordered structure (-) below 200nm ()
218nm weak band
b-turn 180-190nm (-) 200-205nm () 225nm ()
band, weak red shift
Far UV(190-240nm)
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Content of secondary structure
??,a???,ß???,? ???a???ß??????????????????

fa? fß? f ? ???a???ß????????????? ????????
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??CD???????????
???LRR(lysine-rich repeat)????CD?
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TFE
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Unfolding/folding
222nm
216nm
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Conformational varies
216nm
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???-???????-???????
? A, Bob1(1-65)?Oct/DNA????CD?
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???????????????????
???????????CD? (a??????b??????????c???
??)
?????????????C????CD? (a??????,15????b??????,??
?c?????)
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???????????
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????????????????
1a-??84,ß-??1 ????15
2 a-??27,ß-??20 ????53
????CD?
1?????????CD?2????????????CD?3?Tris-HCL??????
??CD?
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Near UV spectra to investigate the tertiary
structure
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Typical Initial Concentrations

1. Protein Concentration 0.5 mg/ml
2. Cell Path Length 0.5 mm
3. Stabilizers (Metal ions, etc.) minimum
4. Buffer Concentration 5 mM or as low as possible
   while maintaining protein stability

One may find that the protein concentration needs
to be adjusted to produce the best data. Changing
this has a profound effect on the data, so small
increments or decrements are called for. If that
does not produce reasonably good data, a change
in buffer composition may be necessary. It would
also be a good idea to check the sample for
unforeseen aggregation via Dynamic Light
Scattering (DNA repair enzymes are an especially
good example of this behavior). If buffer poses a
problem, cells with shorter path (0.1 mm) and a
correspondingly increased protein concentration
and longer scan time can help.
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? ? ? ?

1. When CD is applied to proteins what is the main
   chromophore (absorbing group) that is looked at?
   What wavelength range does it absorb in?
2. Does the CD spectrum of a protein give most
   information about the (i) primary structure, (ii)
   secondary structure or (iii) tertiary structure?
3. Draw the CD spectrum of an a-helix. Remember to
   label the axes and mark important points with
   their values.
4. Why is nitrogen gas used in CD instruments?
5. Does CD give information about where the helices
   are in a protein?
6. Membrane proteins are difficult to study by CD.
   True or false? Why?

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????

1. ????????,???,?????-??????? 2000,p330
2. ??????????????????????,??,???,????? 1987
3. Circular Dichroism and Linear Dichroism, Alison
   Rodger and Bengt Nordén, Oxford University Press
   1997
4. ?????????,???,???,??????? 1997, p 247
5. Circular Dichroism and Optical Rotary Dispersion
   of Proteins and Polypeptides, A.J.Alder,
   N.J.Greenfield and G.D.Fasman, Meth. Enzymology
   ,27, 675 (1973).
6. Computed Circular Dichroism Spectra for the
   Evaluation of Protein Conformation, N.Greenfield
   and G.D.Fasman, Biochemistry, 8 (10), 4108 (1996)
   
7. C.R.Cantor and P.R.Schimmel, Biophysical
   Chemistry, Vol.2, Chapter 8 (1980)
8. Effect of Trifluoroethanol on Protein Secondary
   Structure, F.D.Soennichsen, J.E.VanEyk,
   R.S.Hodges and B.D.Sykes, Biochemistry, 31
   (37),8791 (1992)
9. Protein Secondary Structure and Circular
   Dichroism A Practical Guide, W.C.Johnson,
   PROTEINS, 7, 205-214 (1990)