BIOCH 590: Biomacromolecules
!B)+B5)C'
Spring 2010
•  !B)+B5)C',D'-")$*+,-$,".'
–  E.")-'
–  F5G4*'
•  H+5)D'51*+,I0$#,1'*,'J0%1*02'2)$4%15$-'
!"#$%&'(")$*+,-$,".'
–  K,-*0&%*)-',D'J0%1*02'2)$4%15$-'
–  L1)+G.'-*%*)-'%1I'*+%1-5#,1-'
•  <=-,+"#,1'-")$*+,-$,".'
–  78'
–  :;'
–  ?@'
/0%1*02'3)$4%15$-6'
789:;'<=-,+"#,1>'?@>'A&0,+)-$)1$)'
•  L25--5,1'-")$*+,-$,".'
Key Reference:
–  A&0,+)-$)1$)'%1I'A;LEM'2,&)$0&%+'+0&)+'
van Holde Chapter 8 (theory heavy; weak in application)
Tinoco Chapter 10 (more application oriented)
Copyright: Jianhan Chen
N$O'P5%14%1'?4)1'
(")$*+,-$,".'
Q'
?,&,+'%1I'<=-,+"#,1'(")$*+02'
•  (*0I.',D'*4)'51*)+%$#,1',D')&)$*+,2%G1)#$'+%I5%#,1'C5*4'
2%R)+M'S,=-)+B%#,1',D'J0%1*02'2)$4%15$-T'
–  F5G4*'%=-,+=)I',+')25R)I'=.'%'2,&)$0&%+'01I)+G,51G'%'*+%1-5#,1'
=)*C))1'I5-$+)*)')1)+G.'&)B)&-U'
–  L1)+G.'&)B)&-'"+)I5$*)I'=.'J0%1*02'2)$4%15$-M'V).'*,'51*)+"+)*%#,1'
•  E.")-',D'-")$*+,-$,".'
–  <=-,+"#,1M'*+%1-5#,1'*,'%'45G4W)1)+G.'&)B)&'
•  78'%1I'B5-5=&)'N78OM')&)$*+,15$'-*%*)-'
•  :1D+%+)I'N:;OM'B5=+%#,1'-*%*)-'
•  ?5+$0&%+'I5$4+,5-2'N?@OM'I5X)+)1#%&'%=-,+"#,1',D'",&%+5Y)I'&5G4*-'
–  L25--5,1M'*+%1-5#,1'*,'%'&,CW)1)+G.'&)B)&'
•  A&0,+)-$)1$)M'+)W)25--5,1'C5*4'1-'
•  K4,-"4,+)-$)1$)M''Z'2-'N%1I'0"'*,'C))V-O'
•  :1D,+2%#,1'I)+5B)IM'5I)1#[$%#,1>'J0%1#[$%#,1>'2,15*,+51G'
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F5G4*'
L&)$*+,2%G1)#$'(")$*+02'
•  ?&%--5$%&&.M')&)$*+,2%G1)#$'+%I5%#,1'
E = E 0 cos(2"#t $ x 0 )
B = B0 cos(2"#t $ x 0 )
!
" = c /#
•  /0%1*02'2)$4%15$%&&.M'"4,*,1'"%+#$&)-'
E = h" = hc / #
!
N$O'P5%14%1'?4)1'
^'
N$O'P5%14%1'?4)1'
_'
!
E+%1-5#,1-',D'L1)+G.'F)B)&-'
•  F5G4*'%=-,+=)I',+')25R)I',1&.'D,+'"4,*,1-'C5*4'%1')1)+G.'
*4%*'2%*$4)-'*4)')1)+G.'I5X)+)1$)'=)*C))1'&)B)&-'
–  A+)J0)1$.>'51*)1-5*.>'I5+)$#,1'
Absorption
Energy levels
x
"E = h#
N$O'P5%14%1'?4)1'
`'
!
Emission
N$O'P5%14%1'?4)1'
a'
K,-*0&%*)-',D'/0%1*02'3)$4%15$-'
K,-*0&%*)-',D'/0%1*02'3)$4%15$-'
•  (5b'",-*0&%*)-'-)+B)'%-'*4)'D,01I%#,1',D'/3'
•  K,-*0&%*)':::'d'8M'"4.-5$%&',=-)+B%=&)-'%-'e)+25#%1',")+%*,+-'
•  K,-*0&%*)':M'-*%*)',D'-.-*)2'I)-$+5=)I'=.'C%B)D01$#,1'
"(q1,...,q3n ,t)
!
http://www.psc.edu/Packages/VFleet_Home/
•  K,-*0&%*)'::M'"+,=%=5&5*.',D'[1I51G'*4)'-.-*)2'5-'G5B)1'=.'
"* " d#
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c'
N$O'P5%14%1'?4)1'
fg'
!
K,-*0&%*)-',D'/0%1*02'3)$4%15$-'
K,-*0&%*)-',D'/0%1*02'3)$4%15$-'
•  K,-*0&%*)':8M'%B)+%G)'B%&0)',D',=-)+B%=&)'h'G5B)1'=.M'
•  K,-*0&%*)'8:M'N#2)WI)")1I)1*O'($4+iI51G)+'LJ0%#,1'
–  !1&.')5G)1B%&0)-'N%5O'C5&&'=)',=-)+B)IM'
Aˆ "i = ai"i
–  jM'e%25&*,15%1',")+%*,+'kWZ'L1)+G.''NL'l'Em8O''
$""
*
i
j #i
=0
" = % c i"i
A = % c i2 ai
ai: always real for Hermitians
!
N$O'P5%14%1'?4)1'
ff'
N$O'P5%14%1'?4)1'
fQ'
(,&B51G'($4+iI51G)+'LJ0%#,1'
fU'K%+#$&)'51'%'H,b'
•  ?)1*+%&'"+,=&)2',D'/3'
–  n)1)+%&&.'1,*'N%1%&.#$%&&.O'-,&B%=&)')b$)"*'D,+'-52"&)-*'-.-*)2-'N-))'
)b%2"&)-'*4%*'D,&&,CO'
V =0 0< x "L
= # otherwise
•  E52)W51I)")1I)1*'($4+iI51G)+'LJ0%#,1'
–  (*%#,1%+.'-*%*)M'-*%*)-'1,*'$4%1G)'C5*4'#2)'%+)'J0%1#Y)I'%*'$)+*%51'
)1)+G.'B%&0)-o'
–  :D'8'N%1I'*40-'eO'5-'51I)")1I)1*',D'#2)M'
!
"(r,t) = "(r)e #iEt / !
Hˆ "(r) = E"(r)
–  (*%#,1%+.'-*%*)M'C4%*'5-'-*%#,1%+.p'
–  !+5G51',D'J0%1#Y)I')1)+G.'&)B)&-M'$,1[1)2)1*'N,D'C%B)D01$#,1O'
!
n
N$O'P5%14%1'?4)1'
f\'
K%+#$&)'51'%'H,b'
1
N$O'P5%14%1'?4)1'
2
3
4
f]'
QU'e%+2,15$'!-$5&&%*,+'
V =0 0< x "L
= # otherwise
(n+1/2)!"
!
" = m# /! x
= L/2 for all levels!
!
<p> = 0
(home work)
N$O'P5%14%1'?4)1'
f^'
N$O'P5%14%1'?4)1'
f_'
\U'e.I+,G)1W&5V)'<*,2'
•  <*,2-'C5*4',1)')&)$*+,1'Ne>'e)mqO'
–
–
–
–
–
rgM'B%$002'")+25sB5*.'
!M'%*,2'102=)+''
"#')&)$*+,1'$4%+G)'
$M')&)$*+,1W10$&)0-U'@5-*%1$)'
())'B%1'e,&I)'E%=&)'aUQ'D,+'%*,25$'015*'N%0O'
W
m
V (r) = "
1 Ze 2 Z
=
4 #$ 0 r
r
!
•  t0$&)0-'20$4'4)%B5)+M',1&.')&)$*+,15$'e%25&*,15%1'$,1-5I)+)I'
"#
!2 2
$ + V (r)
2me r
•  ()"%+%#,1',D'+%I5%&'%1I'%1G0&%+'C%B)D01$#,1-'''
!
" ( r ) = Rl (r)Y!lm (# , $ )
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f`'
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fa'
!
1s
e.I+,G)1W&5V)'<*,2'
2s
2
=
Z
2n 2
(in a.u.)
2pz
2px
2py
!
N$O'P5%14%1'?4)1'
fc'
N$O'P5%14%1'?4)1'
Qg'
L&)$*+,15$>'B5=+%#,1%&'%1I'+,*%#,1%&'-*%*)-'
E+5$V-'D,+'<""+,%$451G'S?,2"&)bT'(.-*)2-'
•
•
•
•
<""+,b52%*)'-,&0#,1-'D,+'$4)25$%&'-.-*)2-'
()"%+%=5&5*.',D'e%25&*,15%1-'
K)+*0+=%#,1'*4),+.'
8%+5%#,1'2)*4,I'
RT ~ 0.3 kcal/mol @ 300K
Electronic levels: ~ 40,000 cm-1 or ~ 120 kcal/mol
P ~ exp(-#E/RT) ~ 10-105
•  F51)%+'$,2=51%#,1',D'%*,25$',+=5*%&-'NF?<!O'
•  H,+1W!"")14)52)+'%""+,b52%#,1M'-)"%+%#,1',D'10$&)%+'
B5=+%#,1'%1I')&)$*+,15$'-*%*)-'
Vibrational levels: ~ 2000 cm-1 or ~ 6 kcal/mol
P ~ 10-6
Rotational levels: ~ 100 cm-1 or ~ 0.3 kcal/mol
P ~ 0.4
•  @)1-5*.'A0$#,1%&'E4),+.'N@AEO'
N$O'P5%14%1'?4)1'
Qf'
N$O'P5%14%1'?4)1'
A)%*0+)-',D'<=-,+"#,1'(")$*+%'
QQ'
E+%1-5#,1':1*)1-5#)-'
•  H))+WF%2=)+*'F%C'
Total absorbance
Frequency, intensity, (linewidth)
EM'*+%1-25--5,1'
!M'%=-,+"#,1'$,)u$5)1*'
%M'%=-,+"#,1'&)1G*4'
&M'2,&%+'%=-,+"#B5*.'N,+')b#1$#,1'
$,)u$5)1*OM'&'('&)*+'
–  $M'$,1$)1*+%#,1'
–
–
–
–
–  vM'%=-,+"#,1'$+,--'-)$#,1'
•  <'",C)+D0&'C%.'D,+'J0%1#[$%#,1M' c = "
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Q\'
N$O'P5%14%1'?4)1'
!
1
logT
#l
Q]'
:1I0$)I'B-'(",1*%1),0-'L25--5,1'
/0%1*02'3)$4%15$%&':1*)1-5*.'
•  K)+*0+=%#,1'*4),+.'%&&,C-',1)'*,'$%&$0&%*)'*4)'*+%1-5#,1'"+,=%=5&5*.'
=)*C))1'%1.'*C,'-*%*)-M'
Consider a system at equilibrium: (black-body radiation) induced
transition and spontaneous transition
h" = E2 - E1
E2
B12
B21
A21
E1
!
u fi = # " *f uˆ " i = " *f uˆ " i
dN12
= B12 N1"(# )
dt
dN 21
= B21N 2 "(# ) + A21N 2
dt
A21N 2
8%h# 3
"(# ) =
= 3 hv / kB T
B12 N1 $ B21N 2 c (e
$1)
N 2 /N1 = e
!
–  E+%1-5#,1'I5",&)M'D,+'*4)'*+%1-5#,1'D+,2',''*,'-'-*%*)'
A(v) =
–  /3'51*)1-5*.M'
!
$hv / kB T
!
8"h# 3
B(v)
c3
(Ef - Ei)/h = 10 Hz
•  Electronic and vibrational
spectroscopy: B(!)
•  Fluorescence: A(!)
C f (t) =
i(E "E +hv )t / "
i(E "E "hv )t / "
1 ! ! #e f i
"1 e f i
"1 &
(
E 0 • u fj %
+
2
E f " E i " hv '
$ E f " E i + hv
–  <=-,+"#,1M'")%V'w'4*'x'#L'
$ 21
'
& sin 2 (E f # E i # hv)t /! )
C *f (t)C f (t) = E 02 D fi cos2 " &
)
2
(E f # E i # hv)
&
)
%
(
–  E+%1-5#,1'I5",&)'-*+)1G*4M'
D~
!
# "(v) /v dv
v (hz)
band
N$O'P5%14%1'?4)1'
Q^'
N$O'P5%14%1'?4)1'
!
Q_'
!
!
u fi = # " *f uˆ " i = " *f uˆ " i
E+%1-5#,1'@5",&)'%1I'()&)$#,1';0&)-'
#! = ±1 for vibrations: Can you see why?
(remember that dipole operator, u = $q r, is odd)
•  E4)'=%-5-',D'-")$*+,-$,"5$'-)&)$#,1'+0&)-M'*+%1-5#,1',1&.'"+,=%=&)'D,+'
*+%1-5#,1-'C5*4'1,1WB%15-451G'*+%1-5#,1'I5",&)-'
!
!
u fi = # " *f uˆ " i = " *f uˆ " i
•  'y4%*'*4)'4)&&'5-'*4)'I5",&)',")+%*,+p'
Transition type
!
! transforms as
note
Electric dipole
Electric quadrupole
Electric polarizability
Magnetic dipole
x, y, z (or simply r)
x2, y2, z2, xy, xz, yz
x2, y2, z2, xy, xz, yz
Rx, Ry, Rz
Optical spectra
Constraint x2 + y2 + z2 = 0
Raman spectra
Optical spectra (weak)
•  :1'"+%$#$)M'-0u$5)1*'*,'$,1-5I)+',1&.'*4)'-.22)*+.'N)B)19,II'"%+5*.O'*,'
5I)1#D.'SD,+=5II)1T'*+%1-5#,1-'
–  #(lgM'-51G&)*'*,'*+5"&)*'D,+=5II)1'N("51';0&)O''
–  #&'l'zfM'+)J05+)-'",&%+5*.'$4%1G)'51'$)1*+,-.22)*+5$'-.-*)2-6''-W->'"W"')*$'1,*'%&&,C)I6'
N!+=5*%&';0&)O'
–  {-'l'zf'D,+'B5=+%#,1->',+'{.'l'zf'D,+'+,*%#,1-'
Additional reading: http://en.wikipedia.org/wiki/Selection_rule
N$O'P5%14%1'?4)1'
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N$O'P5%14%1'?4)1'
Qa'
''''''''''''''?&%--5$%&'@5",&)'85)C'
N$O'P5%14%1'?4)1'
3,&)$0&%+'!+=5*%&-'
Qc'
N$O'P5%14%1'?4)1'
Lb")$*)I'51*)1-5#)-',D')&)$*+,15$'*+%1-5#,1-''
<+)'*4)-)'
*+%1-5#,1-'
%&&,C)Ip'
\g'
<=-,+"#,1'(")$*+,-$,".'
Transition type
Example
Typical value of "
Spin forbidden,
Laporte forbidden
[Mn(H2O)6]2+
0.1 m2 mol-1
Spin allowed (octahedral complex)
Laporte forbidden
[Ti(H2O)6]3+
1 m2 mol-1
Spin allowed (tetrahedral complex),
Laporte partially allowed
by d-p mixing
[CoCl4]2-
50 m2 mol-1
Spin allowed,
Laporte allowed
e.g. charge transfer bands
[TiCl6]2- or MnO4-
1000 m2 mol-1
•  E.")-',D'%=-,+"#,1M'+)&%#,1'*,')1)+G.'&)B)&-'
•  }).'51D,+2%#,1'D+,2'-")$*+%M'
–
–
–
–
H%1IM')1)+G.'&)B)&-'
:1*)1-5*.M'*+%1-5#,1'I5",&)-'
F51)'C5I*4M'&5D)'#2)-'
<=-,+=%1$)M'$,1$)1*+%#,1'
A,+=5II)1'5-',1&.'*+0)'*,'*4)'[+-*',+I)+M'51'+)%&5*.>'5*',1&.'2)%1-'&,C)+'"+,=%=5&5*.o'
NE4)'2,&)$0&%+9%*,25$',+=5*%&-'%+)',1&.'%""+,b52%*)'%|)+'%&&M'$,0"&51G-O''
N$O'P5%14%1'?4)1'
\f'
N$O'P5%14%1'?4)1'
\Q'
78'<=-,+"#,1'(")$*+%',D'K+,*)51-'
A+%1VW?,1I,1'K+51$5"&)'
•  @,251%*)I'=.'%=-,+"#,1'D+,2'=%$V=,1)'%25I)-M'xfc^'12'%1I'QQg'12'
•  <'D)C'-5I)$4%51-'%&-,'B5-5=&)'N%+,2%#$->'?.->'3)*>'(W(OM'xQag'12'
•  L&)$*+,15$'*+%1-5#,1-'*4%*'$,1-)+B)'*4)'10$&)%+'I5-"&%$)2)1*'4%B)'*4)'
45G4)-*'"+,=%=5&5*.M'B%+5,0-'=+,%I)151G')X)$*-'&)%I'*,'%=-,+"#,1'=%1I-'
%%*
“single molecule”
n%*
Collision broadening
%%*
Solvent broadening
N$O'P5%14%1'?4)1'
\\'
$-strand
\]'
78'(")$*+%',D't0$&)5$'<$5I-'
()$,1I%+.'(*+0$*0+)'@)")1I)1$)'
%%*
N$O'P5%14%1'?4)1'
())'B%1'e,&I)'
"G']\gW]\\'D,+'
%'I)*%5&)I'
%1%&.-5-',D'
%25I)'
$4+,2,"4,+)U'
:1*)+%$#,1-'%2,1G'~~'*+%1-5#,1-',D'
,+5)1*)I'%25I)-'%1I'4.",$4+,25-2M'
&,CW)1)+G.'*+%1-5#,1-'&,-)'51*)1-5*.'
$,2"%+)I'*,'5-,&%*)I'2,1,2)+U''
random coil
%%*
!-helix
Hypochromism: degree of
orientation of bases (or
degree of melting)
N$O'P5%14%1'?4)1'
\^'
N$O'P5%14%1'?4)1'
\_'
85=+%#,1%&'<=-,+"#,1M'AEW:;'
A,0+5)+'E+%1-D,+2'NAEO'
•  t,+2%&'2,I)-',D'10$&)5'B5=+%#,1M'1lg'WZ'1lf'
–  t02=)+',D'B5=+%#,1%&'I)G+))-',D'D+))I,2M'\t'Ä'_'N,+'\t'Ä'^'D,+'&51)%+'2,&)$0&)-O'
•  3,I)+1':;'-")$*+%'%$J05+)I'C5*4'%1'51*)+D)+,2)*)+M'A,0+5)+'*+%1-D,+2'NAEO'
0-)I'*,',=*%51'51*)1-5*.'%-'%'D01$#,1',D'D+)J0)1$.'51'&)--'%'-)$,1Io'
FT
B(&)= 'I(() cos 2(&% (&,
N$O'P5%14%1'?4)1'
\`'
:1*)+D)+,G+%2'B-U'(")$*+02'
N$O'P5%14%1'?4)1'
\a'
:;',D'K+,*)51'H%$V=,1)'
•  e.I+,G)1'=,1I51G'.5)&I-'$4%+%$*)+5-#$'-45|-'51'B5=+%#,1%&'=%1I-'
•  <25I)':'B5=+%#,1%&'=%1I'Nxf_^g'$2WfO'2,-*',|)1'D,&&,C)I'
C-N and C-C
stretch mixed
with N-H bend
IR Amide I Frequencies
N$O'P5%14%1'?4)1'
\c'
C=O stretch
Structure
!-helix
Random coil
$-sheet
N$O'P5%14%1'?4)1'
N-H stretch
Frequency (cm-1)
1648-1655
1655-1675
1620-1635,1675-1690
]g'
K+,*)51'()$,1I%+.'(*+0$*0+)'D+,2':;'
•  t))I-'*,')-*%=&5-4'%'S=%-5-'-)*T',D':;'
-")$*+%',D'<25I)':'=%1I'
•  A,0+5)+'-)&DWI)$,B&0#,1'$,0"&)I'C5*4'
5*)+%#B)'$0+B)'[s1G''
AEW:;'3,15*,+51G',D'K+,*)51'A,&I51G'
:;'%=-,+=%1$)'-")$*+%',D'*4)'%25I)':'
=%1I',D'*4)'-,&0=&)'t<@e'
I)4.I+,G)1%-)'=)D,+)'N<O'%1I'%|)+'
+)I0$#,1'C5*4't<@e'NHO'
K,4&')*'%&>'H5,$4)2'(,$'E+%1-'NQggaO'
30% )-helical
17% *-sheet
53% random
VKLKVYPLKVKLYP
25% )-helical
16% *-sheet
19% turns
40% random
Original slide from:
chsfpc5.chem.ncsu.edu/~franzen/Poland/
Poznan07/lec4_appl_spec.ppt
N$O'P5%14%1'?4)1'
]f'
N$O'P5%14%1'?4)1'
]Q'
E52)W;)-,&B)I':;'(")$*+,-$,".'
AE:;'D,+'?%1$)+'@5%G1,-5-'
•  EC,W"0&-)'N"02"W"+,=)OM'"+,B5I)'"-W1-'+)-,&0#,1'
•  35G4*'=)'&525*)I'=.'-")$*+%&',B)+&%"-'
Villin headpiece
(HP35)
Wong et al, “Distinct Infrared Spectroscopic
Patterns of Human Basal Cell Carcinoma of
the Skin”, Cancer Research (1993).
1632 cm-1 : 9.63x106 s-1, 3.96x105 s-1 (104 ns, 2.53 µs)
Baker et al., “An investigation of the RWPE
prostate derived family of cell lines using FTIR
spectroscopy”, Analyst (2010)
N$O'P5%14%1'?4)1'
1650 cm-1 : 8.19x106 s-1, 3.87x105 s-1 (122 ns, 2.58 µs)
]\'
Original slide from: chsfpc5.chem.ncsu.edu/~franzen/Poland/Poznan07/lec4_appl_spec.ppt
N$O'P5%14%1'?4)1'
]]'
EC,W@52)1-5,1%&'AEW:;'
“The burst phase unfolding of ubiquitin is
characterized by a loss of the coupling between
vibrations of the *-sheet, …”
•  ;)J05+)-'D)2*,-)$,1I'ND-O':;'"0&-)-M'-0")+5,+'#2)'+)-,&0#,1'Nx"-Oo'
•  @)*)$*-'$,0"&51G'=)*C))1'B5=+%#,1%&'2,I)-M'-*+0$*0+%&'-5G1%*0+)-'
•  (*%*)W,DW*4)W%+*M'$4%&&)1G51G')b")+52)1*-6'&%$V'-)J0)1#%&'+)-,&0#,16'
51*)+"+)*%#,1',D'-")$*+%&'-5G1%*0+)'D,+'$,2"&)b'-.-*)2-'
t2 ~ ps
(Analogous to NMR)
Typically requires molecular dynamics for interpretation!
Maekawa et al., “Different Spectral Signatures of Octapeptide 310- and )-Helices Revealed
by Two-Dimensional Infrared Spectroscopy“, J. Phys. Chem B (2006).
Chung et al., “Transient 2D IR spectroscopy of ubiquitin unfolding dynamics”, PNAS (2007).
N$O'P5%14%1'?4)1'
N$O'P5%14%1'?4)1'
]^'
F51)%+'@5$4+,5-2'NF@O'
F51)%+'%1I'?5+$0&%+'@5$4+,5-2'
]_'
LD( ") = A|| ( " ) # A$( ")
•  !+5)1*)I'2%*)+5%&-M'-0$4'%-'%$45)B)I'=.'-*+)*$451G'",&.2)+-'
•  <=-,+"#,1'I)")1I-',1'*4)'+)&%#B)'I5+)$#,1',D')&)$*+5$'[)&I',D'&51)%+&.'
!
",&%+5Y)I'&5G4*-'%1I'*+%1-5#,1'I5",&)'
•  @5$4+,5-2M'I5X)+)1*'%=-,+"#,1'D,+'I5X)+)1*'I5+)$#,1-',D'",&%+5Y%#,1'
–  F51)%+',+'$5+$0&%+'I5$4+,5-2M'&51)%+&.',+'$5+$0&%+&.'",&%+5Y)I'&5G4*'
! !
E = E 0 cos(kz " 2#$t)
–  35G4*'4)&"'*,'+)-,&B)',B)+&%""51G'=%1I-6'?,1*%51-'%II5#,1%&'-*+0$*0+%&'51D,+2%#,1'
!
!
!
–  ?5+$0&%+&.'",&%+5Y)I'&5G4*M' E = E 0 [cos(kz " 2#$t)iˆ + sin(kz " 2#$t) ˆj ]
!
Sample oriented in a Couette flow device
N$O'P5%14%1'?4)1'
]`'
Reymer et al, Structure of human Rad51 protein filament from molecular modeling and site-specific linear
dichroism spectroscopy, PNAS (2009).
N$O'P5%14%1'?4)1'
]a'
?5+$0&%+'@5$4+,5-2'N?@O'
?@',D't0$&)5'<$5I-'
•  <&&'=5,",&.2)+-'%+)',"#$%&&.'%$#B)'C5*4'$45+%&'$)1*)+-U'
•  ?@'I,)-'1,*'+)J05+)',+5)1*)I'-%2"&)M'$,1B)15)1$)'
•  <'S-*%1I%+IT'*)$415J0)'D,+'J0%1#D.51G'Q1I'-*+0$*0+%&'N4)&5$%&O'$,1*)1*'
On the basis of the measured CD curve, an assignment
to one of the structure groups can be made.
Due to the considerable number of subgroups and the
dependence of the CD spectrum on the nucleotide
sequence, no detailed information can be extracted
from the CD curve alone.
A = -log T = +lc
CD( ") = AL ( " ) # AR ( ")
•  !|)1'+)",+*)I'51'S)&&5"#$5*.T'
! " (rad cm#1 ) = 2.303(AL # AR )
4l
',+'51'S2,&%+'$5+$0&%+'I5$4+,5-2T'
!
"# = # L $ # R =
AL ( %) $ AR ( % )
lc
'%2,1G',*4)+-'"%+%2)*)+-''
''''''N-))'E%=&)'fgU_',D'E51,$,O'
!
Wavelength (nm)
N$O'P5%14%1'?4)1'
]c'
?@',D'K+,*)51-'
Requires small amount of materials (<100 µg)
Acquired in <30 minutes;
Applicable in almost any solution conditions!
N$O'P5%14%1'?4)1'
3Qn&.;W@)+5B)I'(.1*4)#$'?4%11)&-'
Extremely powerful tool for
monitoring structural changes!
p1080 KKKKPARVGLGITTVLTMTTQS
p1096 KKKKPARVGLGITTVLTMTTQW
p1130 KKKKPARVGLGITTVLTMRTQW
p1146 KKKKRVGLGITTVLTMRTQW
2.9 Å resolution x-ray structure
of the GLIC channel protein
Synchrotron Radiation |
^g'
Concentration dependence of Isc induced by
modified M2GlyR peptides on epithelial
monolayers.
UV Range
N$O'P5%14%1'?4)1'
^f'
N$O'P5%14%1'?4)1'
^Q'
(*+0$*0+)-',D'3Qn&.;W@)+5B)I'K)"#I)-'
K)"#I)'n&0)-'
K3h5mIK3
K3h5nK3
KKK-FLIVI-KKK
KKK-IVIGS-KKK
Water
40% TFE
SDS
Herrera et al. PROTEINS (in press).
N$O'P5%14%1'?4)1'
^\'
K)"#I)'n&0)-'
K3h5mIK3
K3h5nK3
Mo et al. Biophys J (2007).
N$O'P5%14%1'?4)1'
^]'
L25--5,1'(")$*+,-$,".'
K3h5mIK3
KKK-FLIVI-KKK
KKK-IVIGS-KKK
•  L25--5,1M'*+%1-5#,1'*,'%'&,CW)1)+G.'&)B)&'
–  A&0,+)-$)1$)M'+)W)25--5,1'C5*4'1-'
–  K4,-"4,+)-$)1$)M''Z'2-'N%1I'0"'*,'C))V-O6'C)%V'
K3h5nK3
Mo et al. Biophys J (2007).
N$O'P5%14%1'?4)1'
^^'
N$O'P5%14%1'?4)1'
^_'
(51G&)*'B-'E+5"&)*''
E+%1-5#,1-'%1I'F5D)#2)-'
1.  All processes compete and the one with
the shortest lifetime typically prevails
(symmetric)
2.  Phosphorescence involves intersystem
crossing to triplet states and is thus slow
& weak.
(antisymmetric)
3.  Phosphorescence is of longer
wavelength!
4.  Any excitation mechanism
•
•
•
•
L&)$*+,1'-"51M'/'l'f9Q''
@)G)1)+%$.',D'*4)')&)$*+,1'-"51'-*%*)M'Q'/'m'f'
n+,01I'-*%*)',D'2,&)$0&)-M'-51G&)*'
E+%1-5#,1'=)*C))1'I5X)+)1*'-"51'-*%*)-'51'"+51$5"&)'S"+,45=5*)IT'N5U)U>'B)+.'
52"+,=%=&)O'
N$O'P5%14%1'?4)1'
^`'
N$O'P5%14%1'?4)1'
^a'
A&0,+)-$)1$)'%-'3,&)$0&%+'(".'
Lb$5*%#,1'B-'A&0,+)-$)1$)'
•  n+))1'Å0,+)-$)1$)'"+,*)51M'nAK'NQgga't,=)&'K+5Y)O'
–  n+))1'Å0,+)-$)1$)'C4)1')b",-)I'*,'=&0)'&5G4*-'
N$O'P5%14%1'?4)1'
^c'
N$O'P5%14%1'?4)1'
_g'
:1*+51-5$'A&0,+)-$)1$)',D'E+"'
n)1)+%&'(,&B)1*'LX)$*-'
•  K,&%+5Y%=5&5*.',D'-,&B)1*M'-,&B)1*'+)&%b%#,1'+)I0$)-'*4)')1)+G.'G%"'%1I'
-45|'Å0,+)-$)1$)'*,'&,1G)+'C%B)&)1G*4-'
Most of the intrinsic fluorescence
emissions of a folded protein are due to
excitation of tryptophan residues, with
some emissions due to tyrosine and
phenylalanine.
–  E4)'45G4)+'I5)&)$*+5$'$,1-*%1*'5->'*4)'=5GG)+'*4)'-45|'5-o'
–  K+,B5I)'%'"+,=)'D,+'-,&B)1*')b",-0+)M'-0$4'%-'I0+51G'D,&I51G901D,&I51G''
KKKKPARVGLGITTVLTMRTQW
Dependence of average blue shift (nm)
of peptide p22-T19R/S22W on the
POPC concentration in liposomes
(Tomich, unpublished data)
N$O'P5%14%1'?4)1'
•  <=-,+"#,1'x'Qag'12'
•  L25--5,1'w'\ggW\^g'12M'
I)")1I51G',1'*4)'",&%+5*.',D'*4)'
&,$%&')1B5+,12)1*'N$%1'=)'0-)I'
*,'51D)+'$,1D,+2%#,1-oO'
•  /0)1$4)I'=.'<-"9n&0'
•  Lb$5*%#,1'*+%1-D)+'NA;LEM'
I5-*%1$)'d'I.1%25$-'51D,O'
•  <'V).'51*)+1%&'"+,=)'
_f'
/0)1$451G'
RAM-ANK
ANK
W4F
Exposed Trp
Bertagna et al, Biochem Soc Trans (2008)
N$O'P5%14%1'?4)1'
_Q'
(51G&)'3,&)$0&%+'A&0,+)-$)1$)'
•  30&#"&)'"+,$)--)-'25G4*'I)$+)%-)'Å0,+)-$)1$)'51*)1-5*.''
–  (*%#$'J0)1$451GM'NG+,01I'-*%*)O'$,2"&)bWD,+2%#,1'
–  @.1%25$'J0)1$451GM')b$5*)I'-*%*)'N*+5"&)*O'+)%$#,1->'$,&&5-5,1%&'J0)1$451G>'
)1)+G.'*+%1-D)+')*$'
–  ?,22,1'N-2%&&'%1I'-,&0=&)O'J0)1$4)+-'D,+'"+,*)51-M':,I5I)'N$4%+G)I>'
)u$5)1*&.'J0)1$4)-'-0+D%$)'+)-5I0)-OU'!b.G)1M'(2%&&'%1I'$%1'")1)*+%*)'*4)'
"+,*)51'*,'-,2)'I)G+))U'<$+.&%25I)>'15*+,b5I)-')*$M't)0*+%&'N"+,=51G'
%$$)--5=5&5*.OU'
Noji et al., Science (1999)
Stern-volmer relationship
Jang and el-Sayed, “Tryptophan
fluorescence quenching as a monitor for the
protein conformation changes occurring
during the photocycle of bacteriorhodopsin
under different perturbations.” PNAS (1989).
N$O'P5%14%1'?4)1'
http://www.youtube.com/watch?v=P0xHuyg9r5k&feature=related
_\'
N$O'P5%14%1'?4)1'
_]'
A+i-*)+';)-,1%1$)'L1)+G.'E+%1-D)+'NA;LEO'
A;LE'%-'3,&)$0&%+';0&)+'
kET
•  @)[15#,1',D')u$5)1$.'
D ! D*
kf "
D
+
A ! D + A*
! A
ki
•  @5",&)WI5",&)'$,0"&51G'
•  L%+&.'B5)CM'%=-,+"#,1',D')25R)I'"4,*,1-'
•  A+i-*)+'%+G0)I'51'fc]_'*4%*'5*'5-')b$5*%#,1'*+%1-D)+'N5U)U'I5+)$*'$,0"&51G',D'
J0%1*02'2)$4%15$%&'-*%*)-O'%1I'"+,B5I)I'*4)'*4),+)#$%&'D,01I%#,1'D,+'%&&'
*,I%.Ç-'A;LE'%1%&.-5-'
•  E+%1-D)+')u$5)1$.'I)")1I-',1M'I,1,+W%$$)"*,+'I5-*%1$)>'-")$*+%&',B)+&%"->'
N*+%1-5#,1O'I5",&)',+5)1*%#,1-'
N$O'P5%14%1'?4)1'
_^'
Ai+-*)+'?+5#$%&'@5-*%1$)'D,+'?,22,1';LE'@,1,+W<$$)"*,+'K%5+-'
•  ?%&$0&%#,1',D';g'$,2"&5$%*)I'=.'
%15-,*+,"5$',+5)1*%#,1-'
•  L1-)2=&)'%B)+%G)I'I5-*%1$)'
Range: ~ 3 to 7 nm
N$O'P5%14%1'?4)1'
__'
(51G&)W3,&)$0&%+'A;LE'
•  E52)W;)-,&B)I'A;LE',1'-51G&)'2,&)$0&%+'&)B)&M'45G4'-)1-5#B5*.'I)*)$*,+-'
•  <&&,C'I5+)$*>'#2)WI)")1I)1*',=-)+B%#,1',D'51I5B5I0%&'2,&)$0&%+')B)1*-'
•  :*'C5&&9-4,0&I'=)$,2)',1)',D'*4)'-*%1I%+I>'=0*')b*+)2)&.'",C)+D0&>'
*)$415J0)->'&5V)'2%1.',*4)+'-51G&)'2,&)$0&%+'*)$415J0)-'-0$4'%-',"#$%&'
*C))Y)+->'<A3'"0&&51G')*$U''
Also see: Ha et al, A practical guide to single-molecule FRET, Nat Method (2008).
N$O'P5%14%1'?4)1'
http://www.microscopy.fsu.edu/primer/techniques/fluorescence/fret/
_`'
N$O'P5%14%1'?4)1'
_a'
N$O'P5%14%1'?4)1'
http://www.nigms.nih.gov/news/reports/single_molecules.html
_c'
N$O'P5%14%1'?4)1'
http://www.nigms.nih.gov/news/reports/single_molecules.html
`g'
<1'e:8'-)$+)*'01$,B)+)I'=.'-51G&)'2,&)$0&)'A;LE'
“polymerase mode”
“RNase H mode”
(polypurine tracts retained as a primer)
“polymerase mode”
RNA-dependent
DNA polymerase
(PDB: 1HMV)
HIV Reverse
Transcriptase
N$O'P5%14%1'?4)1'
Arnold & Sarafianos, Nature (2008);
Zhuang and coworkers Nature (2008).
`f'
Zhuang and coworkers Nature (2008).
N$O'P5%14%1'?4)1'
`Q'
a. Before cleavage of the PPT RNA, HIV reverse transcriptase binds to PPT (blue
sequence) in an RNase H mode — that is, the RNase H domain of the enzyme is
close to the 3 end of PPT (circled), thus blocking the polymerase domain's access to
it and preventing premature extension. b, When the RNA nucleotides next to the
PPT sequence are cleaved, allowing PPT to function as a primer, the enzyme can
bind in a polymerase-competent mode (with its polymerase domain close to PPT's 3
end) to initiate DNA synthesis. The HIV reverse transcriptase seems to rapidly switch
orientation between these two binding modes.
N$O'P5%14%1'?4)1'
`\'