Документ взят из кэша поисковой машины. Адрес оригинального документа : http://www.biochem.bio.msu.ru/assets/docs/diploma/2008/Gluhova.pdf
Дата изменения: Sun Oct 23 15:45:18 2011
Дата индексирования: Mon Oct 1 20:15:59 2012
Кодировка:

..

:

« Hsp20 Hsp27 »

: . . 5 , : ..

, 2008

............................................................................................................... 2 ................................................................................................ 4 ................................................................................................................... 5 .................................................................................................... 6
1. 2. 3. 4. 5. 6. 7. . ......................................................................6 Hsp. ............................................................8 . ............................................10 .....................................................13 . ...............................18 . ......................................20 Hsp20 Hsp27. ..........................................................21

:........................................................................................................ 24 ............................................................................................ 25
1. ....................................................................................25 1.1 1.2 Hsp20Cys ............................................................................................... 25 Hsp27 . ..................................................... 27

2. - Hsp20 Hsp27 ........................................................................................................................................28 2.1 2.2 ............... 28 ................... 29

2.3 Hsp20 Hsp27 . ............................................................................................................. 30 3. Hsp20 Hsp27 ........................30 3.1 - Hsp20 Hsp27. .................................................................................................................... 30 3.2 Hsp20 ............................................................................................ 31 4. ...................................................................................................33 4.1 4.2 4.3 1. ..... 33 SDS. ......................................................... 34 .......................................................................... 34

..................................................................................... 35
- .................................35 1.1 ............ 35 2

1.2 Hsp20 Hsp2740 1.3 Hsp20 Hsp27 ....................... 45 1.4 , «» Hsp20 Hsp27 ............................................................. 49 2. ...............................................56 2.1 2.2 ....................................................................................... 56 Hsp20 .............. 61

............................................................................................................ 70 : .................................................................................................................. 72 ............................................................................................... 73

3

G FRET HEPES Hsp Hsp20cys Hsp27_3D SDS G N,N,N',N'- () (fluorescence resonance en ergy transfer) (4-(2-)-1- (heat shock protein) Hsp20 C46S, A114C Hsp27 c S15D, S78D S82D , - ( mitogen-activated protein kinase activated protein kinase)

4

(heat shock proteins, Hsp) -, , . , . ATP (Hsp60, Hsp70 .), . ( Hsp, small Hsp, sHsp). , . Hsp -, . - , P- . . Hsp , 13 43 . 300-800 . Hsp , . . 10 , Hsp. , , . Hsp20 Hsp27 , , , . , 1% , , Hsp20 Hsp27.

5

1. .
Hsp - N , - , - , (. 1.1). 80-100 , -. (. 1.1). - , , , (van Montfort, et al., 2001). , Hsp . (Kim, et al., 1998). Tsp36 Taenia saginata. - , , , N- Hsp -

. Tsp36 , Hsp - (Stamler, et al., 2005).

N

WDPF

-

C

. 1.1. Hsp16,9 . . . Hsp16,9 . . . (van Montfort, et al., 2001) . 6

-

,

,

, 15-30 . . , Hsp - (Lindner, et al., 2000). Hsp16,9 , - , (van Montfort, et al., 2001) N- Hsp . Hsp . Hsp N- WDPF (. 1.1), . N- - - . , , (Narberhaus, 2002). , N- , Hsp . , 9 24 (Narberhaus, 2002), Hsp (Bukach, et al., 2004; Kim, et al., 2004). Hsp , . . , Hsp (Sobott, et al., 2002), , - -, , (Haley, et al., 1998; Horwitz, et al., 2004) . : Hsp16,5 Methanococcus jannaschii (Kim, et al., 1998) Hsp16,9 (van Montfort, et al., 2001). , Hsp16,9 , , 7

Hsp16,9 (Sobott, et al., 2002). 6 . . , Hsp16,9 (. 1.1). , - , . Hsp16,5 Methanococcus jannaschii Hsp16,9 23%. , , . Hsp16,9, Hsp16,5 Methanococcus jannaschii 24 (Kim, et al., 1998; van Montfort, et al., 2001). , ATP- , , , GroEL. , B- . , , - B- (Haley, et al., 1998). Hsp16,5 Methanococcus jannaschii , , N- . , Thr33, , , (Kim, et al., 1998).

2. Hsp.
, , . Hsp - . , - , B- - 560 , , , (Horwitz, et al., 2004). Hsp Bradyrhizobium japonicum 400 500 (Studer and Narberhaus, 2000). 8

, Hsp (Ehrnsperger, et al., 1999; van de Klundert, et al., 1998). , Hsp25 , Hsp27 , , . Hsp25 . , , (Dudich, et al., 1995; Ehrnsperger, et al., 1999). Hsp20 (43 ) 470 , (van de Klundert, et al., 1998). , Hsp (Bova, et al., 1997; Friedrich, et al., 2004; Sun, et al., 1998). , Hsp (Sobott, et al., 2002; van Montfort, et al., 2001). , . , (Bova, et al., 1997), (Bova, et al., 2002; Friedrich, et al., 2004). , - , , 37°, Hsp16,5 Methanococcus jannaschii ( , 94°) 50° , 72° (Bova, et al., 2002). Hsp16,5 : 30 300 , 5 20% (Bova, et al., 2002). FRET (fluorescence resonance energy transfer), . , . , , , . pH, Ca2+

9

- (Bova, et al., 1997). Hsp . , Hsp25 75° 1,3 , . , (Ehrnsperger, et al., 1999). Hsp27 30 40° , 10° (Lelj-Garolla and Mauk, 2006). Hsp26, Saccharomyces cerevisiae, 24 , , (Haslbeck, et al., 1999). , Hsp . N- (Lambert, et al., 1999; Rogalla, et al., 1999) ( 4 « »). , , . , . - , Hsp. , , , , .

3. .
, , . Hsp , (Lindner, et al., 1997). - ( , pH .). , - (Lee, et al., 1997). (Narberhaus, 2002). 10

Hsp (. 1.2). , (Hsp60 Hsp70) ATP- .

. 1.2. (, et al., 2003). , ATP . , , , Hsp25 , , , . , , . , Hsp ( 62°). Hsp27 , (85°) (Pivovarova, et al., 2005). Hsp (Ehrnsperger, et al., 1997). , , ( .) - , ATP (Biswas and Das, 2007).

11

, Hsp (Ehrnsperger, et al., 1997; Lee, et al., 1997; Lelj-Garolla and Mauk, 2006). Hsp , , , Hsp70 - ATP. , (Friedrich, et al., 2004). . , (2,6 ) B (3 ), - , . (40 ) (Bova, et al., 1997). Hsp . , . Hsp . , Hsp25 Hsp25, Hsp27 (Pivovarova, et al., 2007; Stromer, et al., 2003). . A-, B, 4 37° . 59°, B- , 37°, A- (van Boekel, et al., 1999). - Hsp. , . Hsp , . , Hsp - , .

12

4.
, . Hsp . Hsp . , Hsp20 (Beall, et al., 1999; Beall, et al., 1997), B Hsp27 (Lee, et al., 2005) MAP- (Kato, et al., 1998; Larsen, et al., 1997). . , (Lambert, et al., 1999; Rogalla, et al., 1999). . (Brophy, et al., 1999a; Lambert, et al., 1999; Rogalla, et al., 1999; Theriault, et al., 2004). (Ahmad, et al., 2008). , . Hsp . , . , B- , , Hsp27 (Ito, et al., 1997; Lambert, et al., 1999; Shi, et al., 2008). , Hsp . in vitro , , ,

13

. , Hsp20 (S16D), (Bukach, et al., 2004). Hsp27 . , (Rogalla, et al., 1999). Hsp27 3D (Ser15,78,82D) F-. (Pivovarova, et al., 2005). 3D B- (S19,45,59D) (Ahmad, et al., 2008). , in vivo Hsp27, . . , Hsp27 - Ser15 Ser90. , , (Theriault, et al., 2004). , . , , Hsp27 3 (S15,78,82D) , S15D S78D,S82D(Rogalla, et al., 1999).

Hsp20
N

Ser16
, G

120 137

C

Hsp27
Ser15 Ser78 Ser82

N
MAPKAP MAPKAP MAPKAP, Akt (B), D,

C

. 1.3. Hsp20 Hsp27 . 14

Hsp20 Hsp27, . Hsp20 . G Hsp20 Ser16 ( Hsp20 , , ), ( Hsp20 ), Ser16, (Beall, et al., 1999; Beall, et al., 1997; Brophy, et al., 1999a). Hsp20 , , 120-137 (.1.3) (Beall, et al., 1999). , Hsp20

. G . (Beall, et al., 1997; Brophy, et al., 1999a). , Hsp20 , , ( G) Hsp20 (Brophy, et al., 1999a; Woodrum, et al., 1999). Hsp27 MAPKAP 2 . Hsp27 Ser15, Ser78 Ser82 (Larsen, et al., 1997; Rogalla, et al., 1999). MAPKAP 2 p38 , . , (Knock, et al., 2005; Larsen, et al., 1997). (Shi, et al., al., 1997) Hsp27 2008).

Hsp27 MAPKAP 2 - (Larsen, et

15

Hsp27 , Akt ( B), D (.1.3) (Doppler, et al., 2005; Meier, et al., 2001; Nakajima, et al., 2005; Rane, et al., 2003; Tanabe, et al., 2008). Hsp27 p38 MAP-/Akt (Brophy, et al., 1998; Nakajima, et al., 2005). Hsp25, Hsp27 (Zoubeidi, et al., 2007). Hsp25 , , (Lee, et al., 2005). , Hsp20 Hsp27 . . , Hsp20 , Hsp27, , . , , Hsp20 Hsp27. , Hsp27, Ser15, 78 82, (, ) , . Hsp20 (McLemore, et al., 2005). , , p38 , Hsp27 MAPKAP2 , Hsp20, , (Knoepp, et al., 2000). Hsp27 , Ser78 Ser82. Hsp20, (Fuchs, et al., 2000). Hsp20 Hsp27

16

, - . Hsp20 Hsp27. Hsp25 Hsp20 . , Hsp25 , , , Hsp27 (Benndorf, et al., 1994; Ibitayo, et al., 1999; Wieske, et al., 2001). , Hsp20 (Brophy, et al., 1999b). , - Hsp (Bukach, et al., 2005; Panasenko, et al., 2003). Hsp20 14-3-3. , 14-3-3 , (Birkenfeld, et al., 2003). Hsp20 14-3-3. Hsp20 , 14-3-3 (Chernik, et al., 2007). , 14-3-3, , . , Hsp20 Hsp27 , , . , Hsp in vivo, , , . in vitro . , Hsp27 Hsp20 . Hsp27, MAPKAP 2 , Hsp20,

17

Hsp27 Hsp20. Hsp . , Hsp20 Hsp27 , . Hsp , , - , .

5.

.
, , , . Hsp. , Bradyrhizobium japonicum 10 Hsp, (Studer and Narberhaus, 2000). , (Studer and Narberhaus, 2000). , . , , Hsp (Ganea, 2001). Hsp -, . , - - - B- . , , Hsp. Hsp Bradyrhizobium japonicum Hsp, 18

, , (Studer and Narberhaus, 2000). Hsp , (Fontaine, et al., 2005; Sugiyama, et al., 2000; Sun, et al., 2004). , , Hsp18,1 Hsp16,5 IbpB E. coli Hsp Bradyrhizobium japonicum (Sobott, et al., 2002; Studer and Narberhaus, 2000).

. 1.4. Hsp (, 2006). 10 , Hsp, (. 1.4). -, HspB9 HspB10 ( , ), (MKBP, HspB3 HspB7), Hsp , (Taylor and Benjamin, 2005). . HspB3, MKBP, Hsp27, Hsp20 B-. , Hsp: Hsp20 Hsp27 B- MKBP HspB3. (Sugiyama, et al., 2000). Hsp.

19

6. .
Hsp . , (Sun, et al., 1998). Hsp. , - B-. - B-, 3:1 (Sun and Liang, 1998). Hsp . , Hsp18,1 Hsp16,9 , (Sobott, et al., 2002). -, . , - B . , - pH, . B-, , - (van Boekel, et al., 1999). , . - , 3:1 ( ) (Sun and Liang, 1998). Hsp27 -, Hsp, . 65° Hsp27 , - . , . , Hsp27 -

20

(Fu and Liang, 2003). Hsp27 (Zantema, et al., 1992). . . , Hsp27 - , . -. , , , (Zantema, et al., 1992). , . - B- (Datta and Rao, 2000). . , Hsp Bradyrhizobium japonicum (Studer and Narberhaus, 2000). , , Hsp, . - , . , , Hsp .

7. Hsp20 Hsp27.
1994 . Hsp20 . Hsp20 Hsp27 -, 7 (Kato, et al., 1994). Hsp20 Hsp27 - (Sugiyama, et al., 2000; Sun, et al., 2006). - , Hsp20 Hsp27 , (Sugiyama, et al., 2000). 5 « », Hsp20 Hsp27 21

, ( 1.1) (Kato, et al., 1994; Kato, et al., 1992). Hsp20 Hsp27. 1.1. Hsp20 Hsp27 (Kato, et al., 1994; Kato, et al., 1992). Hsp20, / 12800±2260 ( ) 2050±320 () 281±43 ( ) 1190±180 417±82 191±24 1790±360 24,6±1,6 43,5±7,8 6,4±1,0 3,5±0,3 14,1±4 3,1±0,2 Hsp27, / 2290±320 1710 1170±330 1210±560 971±81 595±303 135±37 246±70 123±70 138 171±17

, Hsp20 Hsp27. - . , ... .. , Hsp20 Hsp27 , - Hsp20 Hsp27 (Bukach, et al., 2004). . , Hsp27 , Hsp20 . , (Fuchs, et al., 2000). , , , ,

22

, . , , Hsp20 Hsp27. . - Hsp20 Hsp27 .

23

:
:
-

Hsp20 Hsp27 .

:
1. 2. 3. 4. 5. Hsp20 Hsp27. Hsp20 Hsp27 . Hsp20 Hsp27 . - Hsp20 Hsp27 -. Hsp20 Hsp27wt , (S15,78,82D).

24

1.
... .. - , , ... .. Hsp20wt.

1.1

Hsp20Cys
Hsp20Cys E. coli. E.coli BL21(DE3), pET23b,

Hsp20Cys, 25 LB, 0,1 / 30° 15-16 . 2 LB, 0,1 / 8-9 37°, 15 30°. (15 5000 g), 20 (50 -HCl, 8,0, 0,1 NaCl, 1 , 14 , 0,5 ) -20°. Hsp20Cys. Hsp20Cys , Hsp20 (Bukach, et al., 2004) . E. coli c 1 , 10 (50 -HCl, 8,0, 0,1 NaCl, 1 , 14 , 0,5 ), 100 (50 /) 30 4°. 35 MgCl2 (2,5 M) 50 I (5 /) 15 . , 20 22000g. , 30 , . , , 30% 1 4°.

25

(20 22000g) , 32 (20 -, 7,6, 10 NaCl, 14 , 0,1 , 0,1 ), 2 . (1 100000g). .

Hsp20Cys. Q-Sepharose HiTrap (V=5 ). , 4 (20 ) (20 -, pH 7,6, 10 NaCl, 14 , 0,1 , 0,1 ). NaCl (00,35 ) . . , Hsp20Cys . , , 0,2 , 8,0. Phenyl-Sepharose Hi-Trap (5 ). 4 (20 -, 8,0, 14 ), 0,2 (NH4)2SO4, . (NH4)2SO4 0,20,01 ; (NH4)2SO4 0,010 , 10 . , , Hsp20Cys, SDS-. , ,

. 20°. 320 1 . coli.

26

1.2

Hsp27 .
Hsp27 ,

(, 2006) . Hsp27. E. coli c 2 , 5 (50 -HCl, 8,0, 0,1 NaCl, 1 , 14 , 0,5 ), 200 (50 /) 30 . 32 MgCl2 (2,5 M) 100 I (5 /) 15 . , 25 23700g. , 30 , . 30% 1 . , 10 (20 -, 7,6, 10 NaCl, 14 , 0,1 , 0,1 ), . (1 100000g). 30% 50%, 1 4°. 10 . (1 100000g). . Hsp27. Q-Sepharose Hi Trap (V=5 ). , 4 (20 ) 1 (20 -, pH 8,0, 10 NaCl, 14 , 0,1 , 0,1 ). NaCl (00,35 ) 1. . ,

27

Hsp27,

(

«»)

Q-Sepharose . - Superdex 200 HR 26/60. , 0,15 NaCl. Hsp27 . , Hsp27, , . 20°.

2.

-

Hsp20 Hsp27
2.1
. , Hsp20 Hsp27 . , Hsp20 (105 ) Hsp27 (105 ) (20 -, pH 7,6, 10 NaCl, 0,1 , 0,1 , 14 ), 15 4° 15 , 30 , 1 3 37° 45°. (0,25 HCl, 6,8, 20% ) (« », 4.1). Hsp20 Hsp27 , Hsp20 (2 /) Hsp27 (2 /) , 37° 30 1 . 4° 15 . (0,25 -HCl, 6,8, 20% ) . , 37° 1 30 .

28

-. -. 100 , Hsp20 (100 ) Hsp27 (100 ), 1 3 37° 1 45°. 10 (200 -, 7,6, 100 NaCl, 140 , 1 , 1 ) Superdex 200 HR10/30, , 150 NaCl. 100 . , . Image Quant, Hsp27/Hsp20 .

2.2
Hsp20 Hsp27 -. . (V=100 ), Hsp20 Hsp27, ( 30 150 ), 3 37°. , 10 (200 -, 7,6, 100 NaCl, 140 , 1 , 1 ) Superdex 200 HR 10/30, , 150 NaCl. 100 . , Hsp20 Hsp27 3 37° ( 150 ). , , , . , . Image Quant, Hsp27/Hsp20 .

29

Hsp20, Hsp27 . Hsp20 Hsp27 (44 ) 1 45°, . , Hsp20 (88 ) Hsp27 (88 ) 50 , pH 7,5, 150 NaCl, 2 . 20 44 ( Hsp20 Hsp27). Beckman E, 0,6°, 8000 / ( 7,5 ). 292 . SEDFIT.

2.3 Hsp20 Hsp27 .
Hsp20 Hsp27 , , 210 , Hsp20/Hsp27 5/1, 3/1, 2/1, 1/1, 1/2, 1/3, 1/5. 3 37°, . , Hsp20 Hsp27 , - (« », 2.1 2.2).

3.

Hsp20

Hsp27
3.1 - Hsp20 Hsp27.
- . - Hsp20 Hsp27 . -. 30

, . (Farahbakhsh, et al., 1995) . (7,2 /), 2,5% , (300 ) , 0,24 /. 10 37° 20 . (50 , 7,5, 50 NaCl,) 37°. Hsp20 Hsp27 1,5 12 , 0,75 . Hsp20 Hsp27 1 45° 3 37°. Hsp20 Hsp27, 3 37° 1 45°. 340 Ultraspec 3100 pro. - . 5 (30 HEPES, 1 MgCl2, pH 7,3). F- (1 /), 58,5° ( ). 340 Cary Eclipse (Varian). . Hsp20, Hsp27 1,5 , 0,75 58,5 °. . .. .. . .. .

3.2 Hsp20

Hsp20 Hsp27 . Hsp20 () Hsp20 (110

31

) (20 -, 7,6, 10 NaCl, 14 , 0,1 , 0,1 ) ATP (0,4 ), ( ) (2*10-3 /), ( 400 150 ) -32P ATP (25 HEPES-NaOH, 7,5, 25 Na-, 20 MgCl2, 2 ), 30° 30 , 1 2 . Hsp20 Hsp20 Hsp20 (110 ) Hsp27 (110 ) 1 37° 1 45° . , . ATP . ( 4.1) ( 4.2). . Hsp20 Hsp27 Ser (Hsp27_3D) . Hsp20 Hsp27_3D ( 110 ) 45° . , , . Hsp20. , Hsp20 (50 ) Hsp27 (0 - 200 ), 1 45° Hsp20 Hsp27 (1/0; 2/1; 1/1; 1/2 1/4). , ATP ( 200 ), -32P ATP . 90 30°, (20 ) Whatmann 3MM. 3 20 10% , 5 Na2HPO4 5 Na2P2O7, 3 96% . ( POPOP PPO 32

) RackBeta 1214 (LKB) . Hsp20 1 Hsp20 1 ATP . Hsp20 Hsp27wt Hsp27_3D, Hsp27wt 1 45°. Hsp27 Hsp20 . .. .

4.

4.1
(6 15%) . 2.1. 2.1. 30% , 0,8% , , , 10 % , , 2 4% 0,27 0,5 (0,5 -HCl, 6,8) 1,23 10 2 4 6% 0,8 1 (1,5 -HCl, 8,8) 2,2 10 2 4 15% 2 1 (1,5 -HCl, 8,8) 1 10 1

(0,25 -HCl, 6,8, 40% ). : (0,025 -HCl, 8,6), (0,192 -, 8,6). 10 / 3 . 10%

33

, 0,2% R-250, 10% 30% . . - (440 ), (66 128 ) (43 ).

4.2

SDS.
: 15% ,

0,4% , ј (1,5 -HCl, 8,8, 0,4% SDS), 0,05% . 0,1%. : 6% , 0,16% , ј (0,5 HCl, 6,8, 0,4% SDS), 0,05% . 0,1%. 1/3 (0,25 -HCl, 6,8, 8% SDS, 40% , 0,004% , 5% ). : - 0,192 -, 8,6, 0,1% SDS; 0,025 -HCl, 8,6. 10/ 20 / . 10% , 0,2% R-250, 10% 30% . .

4.3

.

, 2.2.

2.2. . 280 0,556 1,779 0,955

Hsp20 Hsp27

Swiss-Prot: 14558 Swiss-Prot: 04792 Swiss-Prot: 01317 34

1. -

1.1

, . , (Bova, et al., 1997). Hsp , .

- (Sun, et al., 1998), . Hsp20 Hsp27 . , (Kato, et al., 1994; Kato, et al., 1992). , 37°. , Hsp27 , (Sugiyama, et al., 2000), , . , (37°) (45°). Hsp20 Hsp27 -. , (. 3.1), 4° 15 . , Hsp20 Hsp27 (. 3.1, 1).

35

200

150

3, 4 D
280
100

2
50

0 8 10 12 14 16

1
18

,
. 3.1. Hsp20 Hsp27. . . Hsp20 (105 ) Hsp27 (105 ) 15 4є (1), 15 (2), 30 (3) , 1 (4) 3 (5) 45° 15 (6), 30 (7), 1 (8) 3 (9) 37°. Hsp20, Hsp27, - . . - Superdex 200 HR 10/30. , , 150 NaCl, 100 Hsp20 (100 ) Hsp27 (100 ), 4° (1), 1 37° (2), 3 37° (3) 1 45° (4). . 36

37°, , Hsp20 Hsp27 170 260 . 3 (. 3.1, 6-9). 37° , Hsp20 Hsp27. 45°C, Hsp20 Hsp27 15 (. 3.1, 2-5). , , Hsp20 Hsp27, , , 60°, ( ). , , (. 3.1). Hsp20 Hsp27, 4°, 730 50 , Hsp27 Hsp20 (. 3.1, 1). 1 37°C 3 630, 57 120 , , , Hsp27 Hsp20, . Hsp27 (630 kDa) (734 kDa), Hsp20 (50 57 kDa). , - - . C , , (. 3.1), , Hsp27 , , Hsp27, Hsp20. , Hsp20 . , , 37°C Hsp20 Hsp27

37

, , . Hsp20 Hsp27, 3 37°, , 400 140 (. 3.1, 3). , 1 45°C (. 3.1, 4). , , , , . , , 37° 45° Hsp20 Hsp27. 4° , , , Hsp20 Hsp27. Hsp20 Hsp27 37° , (30 1 ), 4° (15 ) , 37° (. 3.2).

. 3.2. Hsp20 Hsp27 4. . Hsp20 Hsp27 ( 2 / ), 15 4° (1), 30 37° (2), 30 37° 4° 15 (3), (4), 1 37° 4° 15 (5), Hsp20 (6) Hsp27 (7). 38

, 4° (. 3.2, 2 3; 4 5). , , . , , 4°, Hsp20 Hsp27 3 37° 15 45°, . . , Hsp (Bova, et al., 1997; Friedrich, et al., 2004; Sun, et al., 1998). (Bova, et al., 1997). , Hsp. Hsp20 Hsp27 , - (Sun, et al., 1998). 45°, , , . Hsp . (, Hsp25 ), , (, Hsp26 ) (Ehrnsperger, et al., 1999; Haslbeck, et al., 1999). Hsp20 Hsp27 4° 45° - . (. 3.1, 2-5 9), - (. 3.1, 3 4) , . , , , , Hsp20 Hsp27 39

, . .

1.2

Hsp20 Hsp27
« », , (Ehrnsperger, et al., 1999; van de Klundert, et al., 1998). , Hsp Hsp20 Hsp27 . , , . Hsp20 Hsp27 (150, 100, 50 30 ) 3 37° . - (. 3.3, ). , Hsp20 Hsp27 150 , , , ( 3.3, ). , (30 ), , (. 3.3, 1), 5 , (. 3.3, 4). , , , Hsp20 Hsp27, . , , .

40

. 3.3. Hsp20 Hsp27. Superdex 200, , 150 NaCl, Hsp20 Hsp27, 3 37°. , 150 ( ) ( ). 1 200 , 150 ; 2 100 , 150 ; 3 100 , 100 ; 4 100 , 50 ; 5 100 , 30 ; 6 Hsp20 Hsp27 200 , 25 . - . 41

, , . , , , , , . , (. 3.3). , , , . 30 (. 3.3, 2) 6 (. 3.3, 5), 420 340 , 175 105 . , , , . , (- ) , , . - , . , , , , . , , , «» , . , , , Hsp20 Hsp27 , 42

, . Hsp, , Hsp25 Hsp20 (Ehrnsperger, et al., 1999; van de Klundert, et al., 1998). , Hsp25 , , , (Dudich, et al., 1995; Ehrnsperger, et al., 1999). , Hsp20 Hsp27 . . , , , -. , , . . SEDFIT, (s) (Schuck, 2000). S , . :

, M , f , - , (, ). . (f/f0), f , f0 , , . s, f/f0 :

43

, M , f /f0 , , (, ), - , Na . . 3.4 (s) Hsp20, Hsp27 .
0.4

2

0.3

3 c(s)
0.2

1

0.1

0.0

4

8

12

16

20

24

, s
. 3.4. Hsp20, Hsp27 . (s). 1 Hsp20 (40 ); 2 - Hsp27 (40 ); 3 - Hsp20 (20 ) Hsp27 (20 ) Hsp20 Hsp27 1 45°C. Hsp20 3,7s 6,2s, , , (. 3.4, 1). Hsp27 , 15s, , , (. 3.4, 2). Hsp20 Hsp27, , , 11s (. 3.4, 3). , , Hsp20, Hsp27

44

, , , - . , (s) SEDFIT f/f0 . Hsp, , , f/f0 . , . - . , , , , Hsp20 Hsp27 . 3 37°, 45°. Hsp20 Hsp27 , . , Hsp20 Hsp27 ( 1 1) .

1.3

Hsp20 Hsp27

, , Hsp20, - Hsp27. - . Hsp20 (Bukach, et al., 2004), Hsp27 , 700 (. 1.1 « »).

45

, . Superdex 200 10 30 Hsp20 Hsp27, (. 3.5 , ). 10 Hsp20 Hsp27 1 37°, , (. 3.5). Hsp27/Hsp20, Hsp20 Hsp27. . 3.5-, ( ) Hsp27/Hsp20 ( ). , Hsp27/Hsp20 , Hsp27, , Hsp20 (. 3.5, . . 3.3). , . Hsp27 Hsp20 ( 42-46), . ( 3 37°), , , , Hsp27 Hsp20 (. 3.5 ). , . , Hsp27 (. 3.5 , 32-36), Hsp27, Hsp20 (. 3.5 , 46-50). , Hsp27 Hsp20 , . , Hsp27/Hsp20 .

46

80 70 60

28 30 32 34 36 38 40 42 44 46 48 50 52 54 56

400

80

28 30 32 34 3 6 3 8 4 0 4 2 44 46 48 50 52 5 4 5 6

20 15 4 3 2 1

Hsp27/Hsp20

350 4 3 2 1 0 18

60

40 30 20 10 0 10 12 14 16

D280, mAU

D280, mAU

50

50 40 30 20 10 0 10 12 14 16

0 18

,

,

250 200

28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 5 4 3 2 1 0 18

Hsp27/Hsp20

D280, mAU

150 100 50 0

10

12

14

16

,

. 3.5. Hsp27/Hsp20 . -. Superdex 200 HR 10/30 Hsp20 Hsp27 1 (), 3 () 37° 100 ( ), 100 , 3 37° 150 , 200 (). Hsp27/Hsp20, SDS . . . , 3 37°, Hsp20 Hsp27 (210 ), Hsp20/Hsp27. 1 5/1, 2 3/1, 3 2/1, 4 1/1, 5 1/2, 6 1/3, 7 1/5, 8 - Hsp20 Hsp27 1/1, Hsp20, Hsp27, - .

Hsp27/Hsp20

70

47

« », (Sobott, et al., 2002). , , Hsp20/Hsp27 1. . (210 ), Hsp27 Hsp20. 3 37° ( 3.5). Hsp20 Hsp27. , Hsp27 Hsp20 (. 3.5, 4). , Hsp. Hsp20 , Hsp20 (. 3.5, 1-3). Hsp27 (. 3.5, 5-7). , , Hsp27/Hsp20 . Hsp , . - B-, , 3:1 (Sun and Liang, 1998). . Hsp18,1 Hsp16,9 , (Sobott, et al., 2002). , in vivo, . , Hsp20 Hsp27 , . , Hsp20 Hsp27 ( 1.1),

48

, Hsp20 Hsp27.

1.4

, «»

Hsp20 Hsp27
Hsp20 Hsp27, , , «» . , , , , Hsp20 Hsp27 . Hsp20 Cys46, N- , Hsp27 - Cys137, - (.3.6). ,

Hsp20wt
N

Cys46

C
Cys137

Hsp27
N

C
Cys46Ser Ala114Cys

Hsp20cys
N

C

. 3.6. Hsp20wt, Hsp27 Hsp20cys . , , Hsp20 Hsp27, , . « », Hsp , « » , , . , ,

49

.

,

, , -. , B- - , N- (Sreelakshmi, et al., 2004). , , Hsp20 Hsp27, . Hsp20 Hsp27 , «» . Hsp20 Hsp27 , «» . Hsp20cys (C46S, A114C) «» , Cys137 Hsp27 (.3.6). , Hsp20cys, Hsp27 Hsp20wt . Hsp20 Hsp27 , Hsp27 Hsp20wt Hsp20cys, , . . (. 3.7) (. 3.7). (. 3.7, «+»). , «» Hsp20 ( C46S, A114C) Hsp27 (. 3.7, «+», 4 5). Hsp20wt , «» (. 3.7, «-», 1), . , (. 3.7, «-», 1). , Hsp20 , . , -,

50

. 3.7. Hsp20 Hsp27 , . , Hsp20wt (1), Hsp20cys (2), Hsp27 (3) Hsp27 Hsp20wt (4) Hsp20cys (5) (+), (-). , . . . .

. 3.8. Hsp20 Hsp27. , Hsp20wt (1), Hsp20cys (2) Hsp27 (3) , 1 45° (+) (-). (-) (+) Hsp27 Hsp20wt (4) Hsp20cys (5). () (), . 51

, . Hsp27 «» 10 40%. Hsp20cys, , Cys137 Hsp27 (. 3.7, «-», 2 3). , Hsp20cys, Hsp27 , (. 3.7, 2, 3 5). Hsp20wt Hsp27, Hsp20wt, , . , , , (. 3.7, «-», 4). Hsp27 Hsp20wt . , . , , «» . Hsp20, Hsp27 , Hsp20wt, Hsp20cys , . - «» Hsp27, , . , Hsp27 , Hsp20 Hsp27. Hsp20cys Hsp27 , «»

(. 3.5, 2, 3 5). , Hsp20cys Hsp27 . , , . , , , . 52

1h 45° . Hsp20wt Hsp27 , ( ). , , Hsp20wt, Hsp27 - . (.3.8). Hsp20wt, Hsp20cys Hsp27 , , . , 1 45°, , . , , . (. 3.8) (. 3.8). , Hsp20 , , Hsp20cys Hsp27 (. 3.8, «-», 2 3). 1 45° Hsp20 Hsp27 (. 3.8, «-», 4 5) Hsp20, . , Hsp20wt, Cys46. , Hsp27 Hsp20wt Hsp20cys, Hsp27 , Hsp20, , . , Hsp20wt Hsp20cys , , , , - Hsp20.

53

, , Hsp20 Hsp27 , Cys46 Hsp20 , Hsp20 . , , , Cys137 Hsp27 ( Cys114 Hsp20) , Hsp27 Hsp20. Hsp20 Hsp27 , , . Hsp20 Cys46 N- . Hsp16.9 Triticum aestivum, , N Hsp (van Montfort, et al., 2001). , Hsp27 (Cys137) - , , ( ) «» Hsp27 , Hsp27. ( ). , Hsp20 , , (.3.9). Hsp27

. 3.9 - Hsp20 Hsp27. , . , Hsp20, , . , Hsp20cys, , 54

Hsp27, . , , , , . , , Hsp (Sobott, et al., 2002; van Montfort, et al., 2001). Hsp20 Hsp20 Hsp27 (« », 1.3), , , 2 .. ( 100 ), 4 , , Hsp20, Hsp27. , , , «» Hsp20 , Hsp20 Hsp27. , Hsp20 . , Hsp27 . , , , Cys137 Hsp27, Hsp20. Hsp20wt, Cys137 Hsp27, Hsp20Cys .

55

2.
2.1

,

, Hsp. « », . (van Boekel, et al., 1999). - (Ganea, 2001). - . - , , , Hsp. Hsp20 Hsp27, . 1 45° 3 37°. , . , , , , 340 (. 3.10, 1). Hsp20 ( 2) Hsp27 ( 3) 1,5 - . Hsp , ( ). . 3.10, Hsp20 Hsp27, , (. 3.10, 2 3). 3 37° 1 45° ( ).

56

, %

100 80 60 40 20 0

1 2 3 4,5,6

0

5

10

15

20

25

30

,
. 3.10. Hsp20, Hsp27 -. (1), Hsp20 (2), Hsp27 (3), Hsp20 Hsp27 (4) (5 6). 3 37° (5) 1 45° (6). 0,24 /, Hsp20 Hsp27 - 1,5 . Hsp20 Hsp27 1,5 ( 0,75 ). D340 , . Hsp20 Hsp27 ( ) . ,

, Hsp20 Hsp27 3 37° 1 45° (. 3.10, 5 6). , Hsp20 Hsp27 (. 3.10, 4, 5 6). . -, , , , , . Hsp20 Hsp27 . 57

( Hsp20 Hsp27) . Hsp . . Hsp20 Hsp27 , , Hsp. .. , - , , , . , Hsp . , . Hsp20, Hsp27 F-. , . , . Hsp . , (Lambert, et al., 1999; Panasenko, et al., 2003), -. (. 3.11). (1°/) , , 340 (. 3.11, 1). Hsp20, Hsp27 ( 1,5 ), 3 37°, (. 3.11, 2, 3 4),. , Hsp20 Hsp27, , 58

, .

600 500 400 300 200 100 0 20

1 2 3,4

1 2 4 3

30

40

50

60

70

80

90 0

10

20

, °

30 ,

40

50

. 3.11. Hsp20, Hsp27 F- -. . F 1 /. . F- 58,5°. 1 , 2 - 4 Hsp20 (2), Hsp27 (3) (4). 3 37°. 0,2 /, Hsp20 Hsp27 - 1,5 () 0,75 (). Hsp20 Hsp27 1,5 () 0,75 ().

. . . 3.11 , Hsp , 58-59°. , 58,5° 1 . 340 (. 3.11). 58,5° Hsp, 1,5 ( Hsp20 Hsp27), ( ), , 0,75 Hsp20 Hsp27, . , Hsp20, Hsp27

59

. . , Hsp20, Hsp27 -, Hsp20 Hsp27, . Hsp20 Hsp27 -, Hsp20 Hsp27 , . . . - B- , Hsp Bradyrhizobium japonicum (Datta and Rao, 2000; Studer and Narberhaus, 2000). - Hsp, . , , . , 1 45 ° . ., Hsp27 75° (Ehrnsperger, et al., 1999). Hsp , . . Hsp , .

60

2.2

Hsp20

« », Hsp20 (Beall, et al., 1997; Brophy, et al., 1999a; Woodrum, et al., 1999). Hsp20 () Ser16 (Beall, et al., 1999). Hsp20, Hsp27. Hsp27 cAMP- , (Ser15,78 82) MAPKAP 2 , (Brophy, et al., 1998; Knoepp, et al., 2000). Hsp20 Hsp27 , . Hsp27 Hsp20 cAMP- ( , ). , Hsp20 Hsp27wt, (. 3.12 ). Hsp20 , -32P ATP. (. 3.12) (. 3.12). , . , Hsp20 (. 3.12, 1 2), , . , Hsp20, , , , . . , . , , Hsp20. 61

, %
60 40

1 2 3 4 5

20

0

0

40

80

120

160

,
. 3.12. Hsp20 Hsp27wt . , Hsp20wt (1), Hsp20 Hsp27wt (2), (3) (4) Hsp20 Hsp27wt (4), 30 - ATP . () (). . ( ) (- ). . Hsp20 . 1 Hsp20, 2-5 Hsp20 Hsp27. Hsp20/Hsp27wt 2/1 (2), 1/1 (3), 1/2 (4), 1/4 (5).

62

( ). , , , . , Hsp20 . Hsp27wt Hsp20 (. 3.12 , 2). Hsp20 (. 3.12, 2). , Hsp20 Hsp27 , Hsp20 (. ). Hsp20 , , Hsp27 Hsp20 (. 3.12, 4). Hsp20 (Hsp20 Hsp27 1 37°) , Hsp20, Hsp27 (. 3.12, 3). Hsp20 Hsp20 Hsp27 (. 3.12). , Hsp20 , . . , Hsp20 , Hsp20. , , Hsp20 Hsp27 . , . , , Hsp20, . Hsp20 , , , , . Hsp20 Hsp27 ( 1.1, . 22). 63

. , Hsp27 , Hsp20/Hsp27 . Hsp20 Hsp27, Hsp20 Hsp27 ( Hsp20 50 , Hsp27 0 200 ). Hsp20 , (. 3.12.). Hsp20, , , , , 100%, (. 3.12, 1). , -32P ATP Hsp20. , Hsp20, Hsp27 Hsp27 (. 3.12, 2-5). Hsp20 Hsp27 Hsp20 (. 3.12, 5). , , Hsp20 . , Hsp27, Hsp20 Hsp27 , Hsp20 , . , , Hsp20/Hsp27. , , , , Hsp27 Hsp20, , (. 3.5). , Hsp20 Hsp27, . , Hsp27, , Hsp20 Hsp27. Hsp20 , Hsp20 Hsp27. 64

Hsp27, Hsp20 . , , Hsp27 Hsp20 . , Hsp20 Hsp27_3D. Hsp27, (Ser15,78 82) MAPKAP 2 (Rogalla, et al., 1999). , Hsp27 ( Hsp27 . Hsp20, Hsp27_3D (. 3.13). Hsp20 - -32P ATP. , (. 3.13 ). Hsp20 Hsp27_3D Hsp27 (. 3.12, 4 . 3.13, 2). Hsp27, , , . . 3.13 , Hsp20 Hsp27_3D . Hsp20, , Hsp27_3D (. 3.13). , Hsp27_3D , Hsp20. , Hsp20 , , Hsp20, Hsp27wt . Hsp27_3D ( ), Hsp20 , . , Hsp20 Hsp27_3D . Hsp20 , . 3D ) Hsp20, ,

65

, %
60 50 40 30 20 10 0

1,2,3, 4,5

0

20

40

60

,
. 3.13. Hsp20 Hsp27_3D . , Hsp20 (1) Hsp20 Hsp27_3D (2), 30 - ATP . () (). . ( ) (- ). . Hsp20 . 1 Hsp20, 2 5 Hsp20 Hsp27_3D. Hsp20/Hsp27_3D 2/1 (2), 1/1 (3), 1/2 (4), 1/4 (5).

66

., , Hsp27wt Hsp20, Hsp27, MAPKAP 2 , (Fuchs, et al., 2000). , . , Hsp27 (Hsp27_3D), , MAPKAP 2 . , Hsp27. , Hsp27 , ,

. , Hsp27 Hsp27wt (Hsp27_3D). . Hsp20 Hsp27wt, . . 3.14 Hsp20 Hsp27wt, Hsp27_3D (. 3.14).

, %

70 60 50 40 30 20 10 0 0 20 40 60 80

3 4 1 2

100

,
. 3.14. Hsp20 Hsp27wt, Hsp27_3D . Hsp20 (1), Hsp20 Hsp27wt (2), Hsp27_3D (3) Hsp27wt Hsp27_3D (4). Hsp20/Hsp27 1/2.

67

Hsp20 : Hsp27wt, Hsp27_3D , Hsp20. , . , , . , Hsp27_3D MAPKAP 2 Hsp27wt. , . , Hsp27 MAPKAP 2 . Hsp27 Hsp20. , Hsp27 ,

(Lambert, et al., 1999; Rogalla, et al., 1999). , Hsp20 Hsp27_3D , Hsp27wt (Bukach, et al., 2004). Hsp20 Hsp27 ( Hsp27wt Hsp27_3D) Hsp27wt Hsp27_3D ( Hsp27). , Hsp27 Hsp20. , Hsp20 , Hsp20 . , Hsp27wt Hsp27_3D . , Hsp20 Hsp27wt Hsp27_3D, , Hsp20, Hsp27wt Hsp27_3D. Hsp20 . , , Hsp27 ., , . Hsp20 Hap27 , 37° , in vivo . , , , 68

. , , Hsp20 Hsp27. , ., , (Fuchs, et al., 2000).

69

, , , Hsp20 Hsp27, , , . , , (Bova, et al., 1997; Sun, et al., 1998). 4° , Hsp, . , (Ehrnsperger, et al., 1999; van de Klundert, et al., 1998). Hsp20 Hsp27 , . Hsp20/Hsp27 , , « » . , Hsp20 Hsp27 - , . Hsp20/Hsp27 , , . , - (Sreelakshmi, et al., 2004). Hsp20 Hsp27, , , Hsp20 Hsp27 . , Hsp20 , Cys46. N- Hsp20,

70

Hsp, , Hsp20 , . , Cys138 Hsp27, , , , . Hsp27 , , Hsp27, , Hsp27 Hsp20. , , Hsp20 Hsp27 . Hsp20 Hsp27, , Hsp20 Hsp27 . Hsp20 Hsp27 , -, . , , , Hsp20 Hsp27. , -. , Hsp20 Hsp27 . , , (Beall, et al., 1997; Brophy, et al., 1999a; Brophy, et al., 1998; Knoepp, et al., 2000). , Hsp20 Hsp27wt Hsp20 . , Hsp20 Hsp27_3D . , Hsp20 , , , .

71

:
1. Hsp20 Hsp27 , - , Hsp20 Hsp27 1. 2. 37° 45° Hsp20 Hsp27, 4° . 3. Hsp20 Hsp27 , «» . Hsp27 . 4. Hsp20 Hsp27 -. , . F-, - Hsp20 Hsp27 . 5. Hsp27 Hsp20

, Hsp27 , (S15,78,82D), .

72

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