Membrane Biophysics

1
Membrane Biophysics

• ???
• ?????????????

2
3. ??????
3
??? (membrane protein)

• ??????????
• ????- ?? (?????),??(K??),???(Na/K-ATP?)
• ????- ?? (G-??????),?(???C)
• ????- ?(ATP???),??????(????C)
• ????- ???(?????)

4
3.1 ??????????

• ?????
• ???? (molecular cloning)
• - ????
• ???? (site-directed mutagenesis)
• - ??
• ???? (molecular hybridization)
• - ??????

5
(No Transcript)
6
3.1.1 ???????????

• ???????????
• cDNA???
• ?
• mRNA???
• ?
• ??????

7
???????????

• ??
• ?????mRNAs
• ?mRNA???,??cDNAs
• ???? (??????)????(????),??cDNA??
• ??cDNA
• ??????????cDNA
• ??cDNA??
• ??mRNA??
• ???????

8
3.1 ??????????

• ?????
• ?????? (secondary structure prediction)
• - ????
• X-????? (X-ray crystallography)
• - ???????
• ????? (electron microscopy)
• - ???????

9
3.1.2 X-?????????

• X-????????
• ???????????,?????????????????
• ???????????????????????
• ???????????,?????????????????
• ?????????????????,?????????????N????????

10
3.1.2.1 ??????

• ??????
• ?
• f0?????
• ??????
• ????
• ??????????????,?????????
• ????????????,????2?

11
??????

• ??????,???????????
• ????,?????????,????????-??????

12
???? I
k

• ?????
• ??r?k??????
• ??????????
• r,k?????r?k???
• ?k?????(k 1),?
• ???r?k????????

?
r
13
???? II

• ?????(Fourier transform)
• ???(r)?????????
• ????(??????(inverse Fourier transform)
• ??v?????

14
3.1.2.2 ????????
k
k0

• ????O??????????????
• ?r????????????????????
• k0???????????
• k???????????

rk0
k
r
k0
rk
o
15
????????
k
k0
rk0

• r??????????????????
• ? ,?
• r??????????
• fe?????????

k
r
k0
rk
o
16
3.1.2.3 ????????

• ???????,????????????????
• ????????(r)???,???r???????dr???????(r)dr,
  ?????????
• ? ,?

17
????(structure factor)

• F(s)?????,???????????????????????
• ???,F(s)???????(r)??????

18
3.1.2.4. ????-??????

• ??????????, F(s)????????
• v??????
• ????,????????????????F(s),???????????????????
  ?(r)
• ?????????,?????????????,??????

19
X-???????????
?? diffraction ?????
?? structure ??????
?? crystal
20
3.3 ??????

• ???????
• ???? (integral protein)
• ? intrinsic protein
• ???? (peripheral protein)
• ? extrinsic protein
• ????? (lipid-anchored protein)
• ????????????????
• ??????????????

21
4.3.1 ???????
?-?? ????
?-?? ????
?-?? ????
?-???
22
????

• ????????????????
• ??????????
• ???????????
• ????
• -?
• -??
• -??
• -??
• -???
• -????

23
3.3.2 ????????

• ?????????? (hydrophilic)
• ???????????(hydrophobic)
• ????????,?????????
• ????????????
• ?-??
• ?-??

24
?-??
25
Ribbon C and N backbone
Arrangement of atoms showing H bonds
Ribbon illustration of a-helix
The N-H of every peptide bond is hydrogen-bonded
to the carbonyl group of the peptide bond 4
amino acids away
26
????-??????

• ?????????????
• ????????????
• ????????????
• ???????????????????
• ?????????????????
• ?????????????

27
????????I- ????
28
???????

• ?-??????5.4 Å, ???3.6????
• ?-????????????1.5Å(5.4?3.6)
• ??????????30Å
• ????20????(30?1.5)

29
?-??
30
Antiparallel ß sheet (peptide chains run in
opposite directions)
Peptide chains held together by hydrogen
bonds between peptide bonds of adjacent
peptide chains
31
????-??????

• ?????????????
• ?????????????
• ?????????
• ???????????
• ????????????????
• ????????????

32
???????II- ????
33
???????1-???

• ??????????????-???(water channel)
• ?????????
• ???? P.Agre ?1990??
• ???????????????X-???????
• P.Agre ??2003?Nobel???

34
Agre???
35
???1(AQP1) ????? (Sui et al, Nature 414, 872,
2001)
36
??????
37
??????? -??????????

• ???????(constriction area)????2.8Å
• ?????????
• ?????????????,?????????
• H2O????????? ? ?????
• ????????????? ? ??????

38
??????? -?????(H3O)?????

• ?????????
• ????
• ?????????
• ???H3O??
• ?????????
• ???
• ????????
• ???????

39
??????????? -?????????????!
Lodish et al, Molecular Cell Biology, 4th ed, 2000
40
???????2- K Channel

• ??????K Channel
• ????????
• ???????????????????
• ???Rockefeller??R.Mackinnon? X-???????
• R.Mackinnon ??2003? Nobel ?????

41
K Channel?????

• ??????????????
• K Channel????????,???????
• ?????6????? (S1-S6)
• S5?S6?????????H5, ??????????
• S4????,???????
• S6??Channel???

42
??K Channel

• ??Streptomyces lividans
• ??K??KcsA, ?4?????
• ?????2??-??,???K?????5?6
• KcsA?????????????,
• ??X-??????????????

43
??K Channel????? (Doyle et al, Science
280, 69,1998)
44
??K Channel?????
45
K Channel?????

• ?????????????????????
• ?????2???????-???????
• ??????????,???????????????
• ??????????????????-??-pore helix
• ????pore helix???????, ??????? (selectivity
  filter)
• ?4??????????????????????,????0.3nm
• ??????3???????

46
K Channel?????
47
K Channel???? -???????????

• ???????(??)??????????????
• ?????
• ????? (cations)
• ????? (anions)

48
K Channel???? -???Na?????

• ???????????,????
• ?K??,??????????
• K?????,???????
• ????????????????
• ?????????
• ?Na ??,??????Na ?
• Na??????????
• ????????????
• Na??????,??????

49
???K Channel?????
50
K Channel?????

• ??selectivity filter???????
• ???????????????
• ??????????????????
• ??????????????

51
??K Channel????? (Jiang et al, Nature 417,
523, 2002)
????????????
52
K Channel??????

• ??????????????????
• ????????,?????????????????

53
???????3- ATP???

• ATP??? (ATP synthase)
• ?????????
• ?????????????ATP??
• ?????MRC????????J.Walker?X-???????
• J.Walker??1997?Nobel???

54
ATP ??????

• F1- the catalytic subunit
• ??F1????????,??F1??? ATP ??
• Fo- a proton channel
• ??F1??????,H???F0?????
• ??F1?????,??H????????????

F1
F0
55
ATP???????-F1

• ?????F1 ?5????
• a3, b3, g, d, e
• a b subunits ????????(homologous)

• ?????
• a b subunits ????,
• ?????

56
??????????

• ? ?? ??????3??????/????(nucleotide-binding/catalyt
  ic sites)
• ??????????? ? subunits.
• ??? ? subunit ?????????ATP, ????????

57
ATP???????-F0

• Fo ??????????
• E. coli Fo ????????a, b2, c10.
• ??? Fo ?????

58
ATP????????
???
????
59
F1-ATPase????? (Abrahams JP et al, Nature
370, 621, 1994)
g subunit ?a b subunits ?????????"shaft"
(Colours a, b, g, d, e.)
60
F1-ATPase?????

• ?? ? subunit (? TP)?? ATP ??? (tight)
• ??? ? subunit (? DP)??ADP (loose)
• ??? ? subunit (?E)?????? (open)

61
ATP??????? -Banding Change Mechanism
62
Binding change mechanism

• Paul Boyer ??
• ?Fo???????????????3? ? subunits????
• ?????Fo ?H ??
• ?????3?? subunits???,??ATP??

63
Binding change mechanism

• ???????????,??? subunit ?????????
• ????????,?? ? subunit ???????????

64
Binding change mechanism

• ?????????????????
• ?? ( loose) ??,?????????ADP Pi
• ?? (tight)??, ?????????????ATP
• ?? (open)??, ???ATP???????

65
ATP??????? -Banding Change Mechanism
66
ATP???????-Banding Change Mechanism

• F1???????
• ???????????????
• ???????????????
• ???binding change model????, Walker ?Boyer???1997
  Nobel ???