??? ????????? Chapter 2 Structure and Function of Protein

1
??? ?????????Chapter 2 Structure and Function
of Protein

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

2

• ????
• 1.??????????---AA????????.
• 2.???????????????
• 3.??????????????????
• 4.????????????
• ????????????????????????????????
• ????14

3
Section 1 ????????

• ???(protein)?????????????????????,?????????
• ??????????????C?H?O?N?S????N?????????,??16,?????
  ????????????????6.25?????

???????????????? ??????????N?6.25
4
?????(amino acid)

• ???????????????????????????,??????????????????????
  ????,?????L-a-????

5
L-a-????????
6
Amino acids ????????

• ????????????,?
• ???(Gly,G) ???(Ala,A)
• ??????????????

7
Stereoisomers ?????(1)
?Gly?,19????????? ???????(?????), ??L-?D-????
8
Stereoisomers ?????(2)

• ??
• 1)?L-????????????,D-??
• ??????????????????
• 2)19???????(-)???()?????L-?D-?????????

9
(?)??????

• ??R???????????,?????????
• 1.?????Glu,Asp??????????????????????
• 2.?????His,Arg,Lys??????????????????????

10

• 3.????? ???????????????,??????????????
• ?????Tyr,Cys,Ser,Thr,Asn,Gln,Met,Trp
• ??????Gly,Ala,Val,Leu,Ile,Pro,Phe?

11
(No Transcript)
12
(No Transcript)
13
(?)????????

• 1.????????
• ????????,???H,?????,????H,??????????????
• ?????,?????????????,?????

14

• ? ???????pH,?????????????????
• ? ?????????,???????????,??????,???????????????pH??
  ?,?????(????)?????,?????????,?pH???????????
• ? ????????????

15
???R???????????? pI1/2(pK1pK2) pK1?a
-???????, pK2?a-????????
16
????
A0H
Ka1
A
A-H
Ka2
A0
?????????? ??????,?A0A-?,?Ka2H,??????p
Ka2pH,??pH9.60???? ???????pKa???,??????pH????????
??????? pKpKalg
????
????
17
????????????
18

• 2.??????
• ??????????20?????,????????????????????????????280n
  m??,?????????
• ????????????????????????

19

• 3.?????
• ??????????????????,???????570nm????????????????(Pr
  o,Hyr?????????)

20
?-????-?????????
??????
???-???????????????
21
?????4.?2,4-????????
(Sanger ??)
5.???????(PITC)???(Edman??)
22
???(peptide)

• (?)??(Peptide bond )???
• ???????????????????????????????????????????a-?????
  ???????a-???????????????????

23
(No Transcript)
24

• ????????????,???????????????????????
• ????????,??????(N?),??????(C?)?
• ?????????50???,????????????????????????50???,????
  ?????????

25
(No Transcript)
26
(?)?????

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

27

• 1. ????(GSH)
• ????-????????????????????,?????(GSH)????(GSSG)????
  ???

28

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

29

• 2. ?????
• ????,???????????????????????

30
Section 2 ????????
???(protein)????????????????,????????? ?????????
????,????????,???????? ?????????,??????????? 1
)???????????????,?????????????????? 2)?????????
???,?????????????,????????? 3)????????,?????????
???????????,??????

31
4)??????????????????
5)???????????????????????????????????????????????

6)????????????????????????
7)???????????????.?????
8)?????????????
9)?????????
32

• ????????????????????
• ???????????????????????????????,??????????????????
  ?,????
• ?????????????????????????????????(???)????

33
??????????
????
primary structure
????
secondary structure
????
Tertiary structure
????
quariernary structure
34
?????????????(???)

• 1. ??
• ??(hydrogen bond)???????????????????????,?????????
  ?????
• ????????????????????????
• ???????(12kJ/mol),???????

35
???????????
36

• 2. ???
• ?????????????????,???????????,???????????????
• ??????????????????????,?????????????????,?????,?Le
  u,Ile,Val,Phe,Ala???????

37

• 3. ???(??)
• ???(salt bond)?????????????????????????????
• ???????,???????????????????????,?????????????????,
  ???????????

38
????????????
39

• 4.????(van der Waals)??
• ?????????????

40
??????????

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

41

• ???(Insulin)?51????????,??A?B????A?21??????,B?30??
  ?????A?B?????????????????,A???????????

42
(No Transcript)
43
??????? 1)????????? 2)??????????????
3)?????(?Pro?????)? ???????????????????????????
??? ??(conformation)? ??(configuration)?
44

• ???(peptide plane)?????(amide
  plane)?????????????,?????????????????????,????????
  ???????

45
(No Transcript)
46

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

47

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

48
????c?????????????
49
???????????????

• ???????????????????????.
• ????
• ????
• ?????????????,???????????????????????????.????????
  ???,??????????.

50
??????????

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

51
(?)?????????

• 1. ??CO????p????N????????????????,??????????
  ??,???????
• 2.??????????????????,?????????????a-???????????
  ????,??????????????????

52
(?)??????????

• ????????????a-??,ß-??,ß-?????????????

53
1. a-??
54

• a-??????????????????????????????,??????
• ? ??????
• ? ??????3.6??????, ???5.44???????100
• ? ?????????????gtCO???????(?3?)???gtNH???????????
  ????????

55

• ??a-????????
• ? ???????(??????)???????????(Ile,Trp,Phe?)
• ? ???????????????????(?????????)
• ? ?Pro???????(??????,?a- C??????????,??Ca-N?C-N???
  ??)?

56
2. ß-??
57

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

58

• ß-????????????????

59
3. ß-??
60

• ß-??( U???,????)????180??????????????,??????
• ? ?????????180??
• ? ????????????????
• ??????????-CO???????-NH???????????
• (4) ???????I ??II???????????????1800,
• (5) ??????Pro?Gly??????-???
  II??-??????????????Gly?

61
(No Transcript)
62
4. ????

• ?????????????????????????
• ???????????????(?)????????????????

63
RNase?????
ß-??
ß-??
a-??
????
64

• ???????,???????????????????,?????????????,???(moti
  f)???????
• ???????????

??
??
???
65
???? (??-??-????)
66
????MyoD???-?-????
67
??????????

• ????????????????????????????,?????????????????
• ?????????????????(???),????????????????,??????,???
  ???

68
??????????
69
??????????
70
??????????????????
71
???????domain)
????????????,???????????????????????,??????????
?????,????????????,????????????????????????
??????-??? ?,?-?????-???????????????
72
(No Transcript)
73
??????????

• ????????(quaternary structure)????????????????,???
  ??????????????
• ??(subunit)?????????????????????????????
• ??????????????????
• ?????????????????????????????????
• ???????????????????????????????

74
???????
75
????(hemoglobin)?????
76
?????????????????

• ????????a??????????,??????????????,????????,??????
  ???????????,????????????S???????????????????????
  ????????????????

77
??????????
78
???????
79
1.?????????????????
??????????
80
2?????????????????????????????? 3???????????????C?
-C?C?-N???????????????????????????????R???????????
?????
81
?.??????

• ?????????
• 1 ??????
• 2 ?????
• 3 ???

82
??????

• ????
• ?-?????????????
• ?-????????????????????
• ??????????????,???????Gly-x-y????,?x??Pro,y??H
  yp(4-????)?
• ????
• ???
• ????????????????

83
????

• ???????????,?????????????
• ???????
• 1 ?????????????????????
• 2 ??????????????
• 3 ???????????????????????
• 4 ????????????,????????????
• 5 ???????????????????????????

84
???
85
Section 3 ?????????????

• ??????????
•  
• (?)????????????
• ??????????????????????????????,???????????????????
  ?,????????????

86
(?)????????

• ????????????1100nm,????????????,?????????????
• ??????????????????????????????????

87
(?)????????????

• ??????????????,?????????????(????????),??????????
  ????????????,????????(denaturation)?

88

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

89

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

90

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

91
???????????
92

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

93
???????????
???? ???????????????? ???????????????
1)??????? 2)???????????(?????),??,???,????
3)??(????)??,??????,??????
4)????????,??????? 5)????????????
?????????????????? ?????????????????????
94
Selection of protein source ????????

• ???????????
• ?????????????
• ???DNA??(recombinant DNA technique)??????????

Stabilization of proteins ??????
??????????? ?? 1)??pH
2)??lt250C,??40C 3) ???????
95
Assay of proteins ??????
?????,??????????????????
???????????? ?????????????,????????
96

• (?)??
• ??????????????,???????????,????????????,?????
• ????????????????????
• ???????????

• ????????????????????
• ???,???pH??????????????
• ????????,?????????????
• ????
• ????????????????,?????????????????

97
?????????????NH42SO4 A ?? NH42SO4????????????,
??????????,????????????? B NH42SO4??,??????????
???,???????????????
98

• (?)?????????
• ????????????????,??????????,??????????
• ?????? ????? ?????????,?????????
• ?????????????????,??????????(30-50),????????????
  ?

99
(?)??

• ??(chromatography)???????????????????,????????(???
  ????)????????????????????
• ??????????????,????,???????????

100
Gel filtration chromatography ??????(1)

• ??????(??????,?????) ??????????????????????,???
  ????????????????
• ????????????????????,???????,???,?????????????
  ????????,??????,???,??????

101
Size-exclusion chromatography, also called gel
filtration, separates proteins according to size.
The column matrix is a cross-linked polymer with
pores of selected size. Larger proteins migrate
faster than smaller ones, because they are
too large to enter the pores in the beads and
hence take a more direct route through the
column. The smaller proteins enter the pores
and are slowed by their more labyrinthine path
through the column.
102
Gel filtration chromatography ??????(2)

• ??????
• ???????????Ve
• ?????? Vt
• ??????? Vm
• ????????(????) Vi
• ???????(????) V0
• ???? Vt VmViV0

103
Gel filtration chromatography ??????(3)

• ????????? Ve
• ??????????????? VeV0
• ????????????????
• VeV0Vi
• ????????????????
• VeV0Vi ?Kd Kd (Ve- V0)/Vi
• ??
• 1)??????? (???????)
• 2)??
• 3)?????

104
Gel filtration chromatography ??????(4)

• ???????????
• ???????????????(Ve)??,??????????Ve?Ve/V0
  ????????
• 1)??????
• 2)???????
• ??????????
• ???Ve?V0??
• ??????

105
Ion exchange chromatography ??????(1)

• ???? ?????????????????????????????????????
• ??????????????????????????????????????????????
• 1)????????????????????????,????????????????,DEAE
  -???(diethylaminoethyl,???????)?
• -RY- X- --- ? -RX- Y- (Y?????)
• 2)????????????????????????,????????????????
  ,CM-???(carboxymethyl,???)?
• -R-Y X --- ? -R-X Y (Y?????)

106
Ion exchange chromatography ??????(2)

• ????
• ????????????????????????????????????????,?????
  ????????????????????????????,??????????????????,??
  ????????
• ????
• 1)??????(pH??)
• 2)pH?????
• ????pH???????pH???
• ????pH??????,?pH???
• 3)????????pH????

107
Ion exchange chromatography ??????(3)
108
Ion-exchange chromatography exploits differences
in the sign and magnitude of the net electric
charges of proteins at a given pH. The column
matrix is a synthetic polymer containing bound
charged groups those with bound anionic groups
are called cation exchangers, and those with
bound cationic groups are called anion
exchangers. Ion-exchange chromatography on a
cation exchanger is shown here. The affinity of
each protein for the charged groups on the column
is affected by the pH (which determines the
ionization state of the molecule) and the
concentration of competing free salt ions in the
surrounding solution. Separation can be optimized
by gradually changing the pH and/or salt
concentration of the mobile phase so as to create
a pH or salt gradient.
109
Ion exchange chromatography ??????(4)

• ??
• ?????????(?,pI5.5 ?, pI7.2 ?,
  pI8.0)?????????????,??pH6.5????????????????????Na
  Cl???pH????,????????
• 1) pH6.5?, ?-,??? ?,?,??
• 2)??????, ????????(????????),????,??????
  ????????,????,??????
• 3)pH?????,???????,?????pH?6.5??????,?????
  ?pI,???????,??????

110
Affinity chromatography ????(1)

• ??????????????????????????????
• ??(ligand)?????????????????????????
• ???????
• 1)???????????????(????)??????????
• 2)????????????,???????????????????,????????????
  ????

111
???????????

• 3)????????(??)???????????????(??)???????????pH???
  ??,???????????????????
• 4)????????????????

112
Affinity chromatography ????(2)
113
(c) Affinity chromatography separates proteins
by their binding specificities. The proteins
retained on the column are those that bind
specifically to a ligand cross-linked to the
beads. (In biochemistry, the term ligand is
used to refer to a group or molecule that binds
to a macromolecule such as a protein.) After
proteins that do not bind to the ligand are
washed through the column, the bound protein of
particular interest is eluted (washed out of the
column) by a solution containing free ligand.
114
High performance liquid chromatography
??????(HPLC)(1)
?? 1)?????????????,?????.2)????????,??????.3)????
??HPLC(reversed-phase HPLC)?????????????????????
,??????????????????????.
115
A modern refinement in chromatographic methods is
HPLC, or high-performance liquid chromatography.
HPLC makes use of high-pressure pumps that speed
the movement of the protein molecules down the
column, as well as higher-quality chromatographic
materials that can withstand the crushing force
of the pressurized flow. By reducing the transit
time on the column, HPLC can limit diffusional
spreading of protein bands and thus greatly
improve resolution.
116
(?)??

• ?????????????????(electrophoresis) ?
• ???????????????????????,??????????????????????????
  ,????????,?????????????,????????

117
Native PAGE ????????????

• ???PAGE?????,?????????????????????????????????
  ???
• ????????(??)
• ???????????
• ?????????????
• ?????????????
• ?????????,???
• ?????????????
• ?????????????

118
Electrophoresis. Different samples are loaded in
wells or depressions at the top of the
polyacrylamide gel. The proteins move into the
gel when an electric field is applied. The gel
minimizes convection currents caused by small
temperature gradients, as well as protein
movements other than those induced by the
electric field.
119
SDS-PAGE ???????-????????(1)

• SDS-PAGE ??SDS??????,???????????????????????
  ?
• ????????????(??)?
• ???????
• 1)???????,??????????????,????
• 2)?SDS?????????,????????? ?,????????????????
• 3)??????????????????,??
• ?,?????1-20????,???(????)?????????????
• 4)?????????,????????????

120
SDS-PAGE ???????-????????(2)
121
Isoelectric focusing ????(1)

• ?????????????????????pH???????????????????????
  ,????????????????????????pH???,???????????????????
  pI??,??????????????????
• ??????????????????????????????????pH????????pH
  ???????

122
(No Transcript)
123
Isoelectric focusing ????(2)

• ?????SDS-PAGE??,??????????????????
• ??????????SDS-PAGE????????????(??)??,???
  ???

124
Two-dimensional electrophoresis.
125
Visualization of proteins in gels ?????????

• ?????(coomassie brilliant blue)????????,??,???
  ???0.1-1.0 ug?
• ?(silver)?AgNO3,????
• ??????
• ?????immunoblot(western blot)
• ??(blotting)?????????????????????????
• ???????????????????????????????(????)???
  ,????????????????????????????????????

126
(?)????

1. ???Dialysis

• Dialysis?????????????????,????????????,???????????
  ?????????????????
• ??????????????????,???????????

127

• ??????????????????????????,??????????(??????????)?
  ?????
• ?????????????????

128
2.????

• ??????????????,?????????????????????
• ?????????????????????????
• ??????????100??50??30??10??5??1??5??1?

129
(No Transcript)
130
(?)????

• ?????????,??????,????????????
• ?????????????????,?????????????S????

131
?.Protein sequencing ?????
?????
?????
????
?????
???????????
?????????
???????
???????
??N???C????
?????
132
???????

• ??????????????,???????????,??????????
• 1. ???????,????????????
• 2. ?????????????,??????????,????????????
• ??????????????????(6 N HCl,1100C,24 h)
  ??????????????????(ninhydrin)????(fluorescamine)??
  ?????????(?????????????)?

133
???????????
134

• 3. ??????N-??C-?????
• ????2,4-?????(DNFB)?????????????N-??C-???????

135
DNFB???N-??????
N-????? ??????N-??? ?-NH2?2,4-?????(EDNB)?
????(DNS)?? ?6 N HCl?????????????????????????N-???
?????????
136
?????C-??????
137

• 4. ???????(?????)?????(????)??????????????????(
  ?????)?

138
????????
????
????
????
???
139
????(trypsin)?????
140
BrCN?????
141
5. ????????? 6. ??????????,?????????????????Edma
n???,???????????,???????,??????????N-????-NH2?????
??(PITC)???????????-?(PTC??)?????????????(????????
?) ??????PTH-????
142
Edman??????????
??????
143
????????
144

• 7. ?????????????????,??????????????????

145
(No Transcript)