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Дата индексирования: Mon Oct 1 20:16:14 2012
Кодировка:

. . . . ..

5

-, Arg91Gly

. .. : , ..

2008

2

............................................................3

...........................................................................4
1. ...................................4 2. .............................................................5 3. ....................6 .................................................................6 .............................................9.... 4. ........................................ 14 5. ).................................15 .........................................................................16 F-...............................................16 F- ...............................19 ..................................................................20

..................................................................
.................................................... F- ............................................................ .................. - ............................................. SDS............................ ................................................................... -........................................... ().................................. F . .................................. .......................................................................... F-..................................................... ....................................................

........................................................
- (-NmTm)................................................. - R91G............................................................................ - R91G F-................................................................. -NmTm WT -NmTm R91G F- .............................................................................................. -NmTm WT -NmTm R91G F-...................................................................... - (-SkTm).................................................... , -SkTm WT -SkTm R91G................................................................................ -SkTm WT -SkTm R91G................................................................................ - R91G F-............................................................. -SkTm WT -SkTm R91G F- ... -SkTm WT -SkTm R91G F-........................................................................... .................................................................................. ........................................................................................ .....................................................

3

ATP DTT Hepes 2-[4-(2-)-1-] F- - - R91G Arg91 S1 1 SDS TPCK -L-- m -Tm WT -m -Tm R91G -m R91G -NmTm -m -SmTm -m -SkTm -m LMW m Tm HMW m Tm

4

.

,

[, 2004]. ( 50 /), ,

() () . , , . , , [, 2000]. , () , . (" ") , ("") [, 2000].

1. . . F-, , G-.

: , G-, "-", "-" F-. , Mg2+ [Dos Remedios et al., 2003]. , , , ( , ), - . ( 1 , S1)

5 [Holmes and Geeves, 2000; ., 1995]. , , S1, S1 . -.

2. . , . "" , S1 . , Ca2+ , , [Squire and Morris, 1998; Perry, 2001]. Ca2
+

, ( ) , . « ». , , .

blocked

closed +Ca2+ -Ca2+

open

Tm
S1 power stroke

. 1. , ("blocked", "closed" "open") [McKillop & Geeves, 1993]. . . (S1, ) . . .

6 , [McKillop and Geeves, 1993]. «blocked», «closed» «open» (. 1). "blocked" S1 . Ca2+, . "closed" ( Ca2+) «» ( , DP·Pi), "open" «» «» ( ADP ). , .

3. .

. - , 100%- - . , (, , , ) [Perry, 2001]. 4 , 20 : TPM1 (), TPM2 (), TPM3 () TPM4 (). , : 1) (HMW), 281 284 ; 2) (LMW), 247 . HMW, LMW. TPM ( HMW) ( LMW) [Gunning et al., 2005]. , .

7 (TPM1 TPM2) - - [Perry, 2001]. , (3-4):1 -. , : , . in vivo 90% ( ). , -, - , [Nilsson & Taijsharghi, 2008]. - - -, , , . , , 39 (~14%), - (Cys-190), - (Cys-36 Cys-190). , , - -, , 31% . - [Perry, 2001] . -. . 2 2 (): () LMW HMW () [Libri et al., 1990; Swiss-Prot]. 3, 4, 5, 7 8 . 1 2 , 1 (HMW). , 1', , [Libri et al., 1990]. - (LMW). , : 6a/6b 9a/9d. 6a/9d , 6b/9a [Gunning et al., 2005;Perry, 2001]. , -: -Tm (LMW -Tm), -Tm (-SkTm)

8 HMW- ,

(-SmTm/-NmTm) [Pittinger et al., 1995]. , -Tm, Swiss-Prot, .. . -Tm, «» «», 6b/9d 6a/9a [Wang et al., 1992]. , , , , .

. 2. - (TPM2) [Libri et al., 1990; Swiss-Prot]. 1, 6 9 -Tm. . .

9 . 1946 . [Bailey, 1948] , . , , ~90% . . ,

, [Astbury et al., 1948]. , . , , , . - , , , , , «». , . , , 33 (284 ) -. coiled-coil " ", , , [Brown et al., 2005]. () ( ). (bcdefg). , a d, , e g . d ( ) (. 3). E 1953 . . coiled-coil, «knobs in holes» («

10 »), (a d) , [Brenden & Tooze, 1999]. e g: i- (g) i'+5 ( , ). , [Kohn et al., 1997], (i/(i+3) i/(i+4)). , - , [Brown et al., 2005]. 284 , 40 .

. 3. , coiled-coil- [Kwok & Hodges, 2004]. a-g . . .

, . . [Kwok & Hodges, 2004] « »,

coiled-coil. , , a d , (Val, Leu, Ile, Phe, Tyr, Met) ( 14 ). , , , [Kwok & Hodges, 2004]. , -

(~74%). ,

11 , . 2001 . [Brown et al. 2001] N- - (1-81), (Lys15-Ala18-Ala22-Ala25-Lys29-Ala32-Ser36). , . -, ~1,2 е; , 2 е , , . , .

( 120150), 137 (d-) , coiled-coil , , - . , (Arg133) [Sumida et al., 2007]. , Asp137 . , . [Sumida et al., 2007]. " " 8-9 C- N- [Perry, 2001]. N- C- ( 1948 [Astbury et al., 1948]). , , (406 ± 1.4 е [Phillips et al., 1986]) (423 е). , , N- [Perry, 2001]. E.coli N- «» Ala-Ser, , , [Monteiro et al., 1994]. F- [Kremneva et al., 2004].

12 , .

7 ( , I C 1:1:1) [Squire and Morris, 1998; Perry, 2001]. , . , . 1974 . , ,

. , - . (- -) , - [Stewart and McLachlan, 1975]. , : 10,5 е, [Perry, 2001]. 2005 . ( coiled-coil ) 2,3 е [Brown et al., 2005]. . 12 N- coiled-coil . 4 (169-172), . , , , . , 169172 . , , 169172.

13 (I172T, D175N, E180G, E180V, L185R) , 167-184 N- 5- . , . .

-

,

, , [Brown et al., 2005]. ,

-. ,

( ) . (LMW) (, ) . ADF/ , [Gunning et al., 2005; Cooper, 2002] Arp2/3 [Blanchoin et al., 2001]. (HMW) . : (-SkTm) - , (-SmTm/-NmTm) ( ) .

. . - , . , 6b, 6a, , , [Maytum et al., 2004] () [Kremneva et al., 2006]. , , 9a F-, () 9d [Pittenger et al., 1995]. , , 9a 9d - [Coulton et al., 2008]. ,

14 1, 6 9. 4. .

, .. , . [Donner et al., 2002], [Robinson et al., 2007] (FHC, Familial Hypertrophy Cardiomyopathy) (DCM, Dilated

Cardiomyopahy) [Seidman and Seidman, 2001; Fatkin and Graham, 2002]. [Donner et al., 2002]. () - [Robinson et a l ., 2007] .

, , . FHC , () . , .

, FHC , ; , . DCM , , , . , , - (FHC [Golitsina et al., 1997; Kremneva et al., 2004] DCM [Mirza et al., 2007]), - ( [Donner et al., 2002], [Robinson et al.,2007]), T, I C (FHC, DCM [Fatkin and Graham, 2002; Mirza et al., 2005]) , , C- [Fatkin and Graham, 2002]. , , .

15 , , . , . , ( ), . - TP ,

in vitro motility. , . , . [Mirza et al., 2005] ATP . , , . , [Hitchcock-DeGregori et al., 2006], [Golitsina et al., 1997], () F- [Kremneva et al., 2004; 2006]. . [Mirza et al., 2007], -, DCM E40K E54K, F-.

5. (). , , . .

16 , , Potekhin, 1986]. ,

[Privalov and

, , [Privalov and Dragan, 2007]. ( ) , [, 2004].

. ( - [Orlov et al., 1998; Levitsky et al., 2000]) . ,

[Williams and Svenson, 1981]. ~43є ~54є. SH, ,

Cys-190 [Kremneva et al., 2004]. , -

SH-, , N- , C- Cys-190 [Williams and Svenson, 1981]. , , , , [, 2005] .

F-. [Kremneva et al., 2004; ., 2003] [Levitsky et al., 2000] F- , , ,

17 F-

(. 4). F-, ,

(~80є) [, 2004]. , F- (. 4), , (. 4) [, 2004].

()

1- 2-
-Tm

-Tm

+ F-

100

. . (%)

80 60 40 20 0

20

30

40

50

60

70

80

90

( C)

o

. 4. [, 2004]. . - (-Tm) F-, . -Tm, F-. , . : 30 -Tm, 46 F-, 70 20 Hepes-KOH, pH 7,3, 2 MgCl2 1 -. 10 . 1 /. . -TmF-, , , (). 100% -TmF- 25 F-, , ( 65). -TmF-.

18 , ,

F-. , , F-, .. F- . , . F- : , Tm - ; F-. , Td ,
iss

F-

(Wegner, 1979; Kremneva et al., 2004). Te - , TA
T

, F ; Tdiss. , , F- Tm T , T , T
diss

[Kremneva et

al., 2004;, 2004; , 2005].
m diss.

, F- T
diss,

,

. , , , F- . [Levitsky et al., 2000; Kremneva et al., 2006], Tm 45є , T
diss.

, , , T
m

, Tm.

TM2. , ..

19 3,6є , [, 2005] .

F- . , 100є TnT TnI [ ., 2003]. TnC , . B 1980 . , TnC , - N- TnC [Tsalkova and Privalov, 1980]. TnC , , 2+ [ ., 2003].

30 25

(µW)

FA FA FA Tn Tn

+ Tm + Tm + Tn(Ca) + Tm + Tn(EGTA) (Ca) (EGTA)

20 15 10 5 0 30 35 40 45 50
o

55

60

65

( C)
. 5. F- - (Tm) (Tn). Ca2+ (0,5 mM CaCl2) Ca2+ (1 mM EGTA). F , . Tm 10 µ M, 10 µ M, F- 46 µ M, 60 µ M. 30 m Hepes, pH 7,3, 100 mM KCl, 1 mM MgCl2. 1 /. [, , , ].

20 , , Tn Tm. F- Tm-Tn, Tm; Ca2+ (.. "blocked") [ ., 2003]. , (. 5). ( ~3,5є), . (. 5) , F- Tm, [ ., 2003; , 2005]. . , , , . -, FHC- D175N E180G [Kremneva et al., 2004] DCM- E40K E54K [Mirza et al., 2007], [Sakura et al., 2006], -

, . , (, Tm, T
diss,

),

( ) F-. , E180G T
diss,

, , F- [Kremneva et al., 2004].

21 F-. Arg91Gly , HMW--. , , F- [Robinson et al., 2007]. F- . , - . - , , - , -. - [Orlov et al., 1998; Levitsky et al., 2000], - . - , .

, , - F- Arg91Gly (R91G). : 1. R91G / - . 2. ( ) F- R91G.

22

.
: Hepes, ATP, , , "Sigma Chemical Co." (); , SDS, MgCl
2

"Serva" (), -

"ICN" (). , , ... .. -Skm -Smm/-Nmm R91G - (University of Oxford, UK).

. . (1 ) 20 G (2 -HCl, 0.2 , 0.2 CaCl2, 0.5 mM -) 45 . 28000 / Ti-60 ( "L" "Beckman" ()). , , KCl MgCl2 50 2 , . , . 2 KCl 0,6 ( 0,55 , , 0,05 KCl). . Ti75 29000 / 3 . G . ( ~3,5 ) G F-. G, 0,1 NaN3. F- Ti-75 40000 / . , , 1
%

290 , 6,3 -1. G- 3

; G- F-, G- MgCl2 2 .

23 F- . F- , F- (1,43 ) , 1,33 .

. m, 20 M -ME 60 50, 10 70. -

1 mM -ME SH- Cys-190 [Kremneva et al., 2004]. - -ME 50-60є.

- . , . [Ishii & Lehrer, 1990]. ~2 / , 5 , 20 mM DTT, 1 mM MgCl2, 30 mM Hepes, pH 7,0, 60 40 . DTT 500 NAP-5 (Pharmacia),

DTT, 1 . (1,0-1,2 /), . 5- - (Molecular probes),

, . DTT. DTT NAP-5 (Pharmacia), DTT. (2 ) , 30 mM Hepes, pH 7,3, 100 mM KCl, 1 mM MgCl2. 3
44

,

=

2,2.104.-1.-1. . 90%.

24 SDS. MiniProtean 3 ell (Bio-Rad) [Laemmli, 1970] 16%- 200 V. 0,75 .

. . Cary-100 (Varian),

0,27 (SwissProt P07951). . , 1 10 (1 ) [ , 1989].

-. , -L-- (TPCK-, Worthington biochemical corporation). , 30 mM Hepes, pH 7,3, 100 mM KCl, 1 mM MgCl2. (0,6 /) - (0,5 / 1 mM HCl) 300/1. 25, . 2- (0,125 M -HCl, pH 6,8, 4% SDS, 9M , 0,004% , 5% ME). SDS .

(). . -4

( , . --) 0.47 . 1 10 100є; 2 2 .5 .

W 25 .

25 . (« ») [Kremneva et al., 2006]. : , , ,

, , . (. 6), , . , , , . , , . . , ; , , . , , - [Kremneva et al., 2006]. , . [Freire & Biltonen, 1978] «Origin 1.16» (MicroCal). , , . , ( ), . , , , , (Hcal),

, (Tm).

26

10

( )

-SkTm WT

0

, µW

-10

-20

-30

-40

10

20

30

40

50

60

,

. 6. (« ») (-Skm). . .

F . mF-, , 90

[Levitsky et al., 2000; Kremneva et al., 2004; 2006]. 350 Cary Eclipse (Varian, Australia), ,

( , , . . 1 ) . F- , mF-. m, F-. mF- F-, m

27 . , mF- m

. , , «Origin 7.0» (MicroCal) . , , Tdiss, . . , 50 %. . () Chirascan Circular Dichroism Spectrometer (Applied Photophysics) 20 ( 5) 2 . 222 nm (
222

)

5 60 1/, . . , . , Tm 0.1 /, Hepes- 10 - (pH 7.3), 100 mM NaCl, 1 mM MgCl2 1 mM -ME. F-. - 20. F- (10 µM) c Tm (05 µM) (30 mM Hepes, pH 7,3, 100 mM KCl, 1 mM MgCl2, 1 mM -ME) 100 µl. , Tm 100000 g 30 (Beckman TL100A). , 100 µl (. « »), 3 SDS- 16%- . ( Epson 1640SU) MatLab.

. - ( , Beckman), , , on-line. ,

28 An-F Ti , 52000 /. 280 . 1 / -. (30 mM Hepes, pH 7,3, 100 mM KCl, 1 mM MgCl2); . , SEDFIT.

... .. ... .. .

29

- (-NmTm).
(HMW) - (-NmTm),

(-NmTm w.t.) -NmTm, R91G (-NmTm R91G). - -Tm (. 2), (-NmTm). -NmTm

(-SkTm), SH- (Cys-36 Cys-190), SH- (Cys-36), .

- R91G. -Tm R91G (. « »). , .. ,

. c. 7. , , Tm, cal, , 1. - (-NmTm WT) 3 (. 7): 1, 2 3 28,7, 38,1 42,8 є, 17%, 57% 26% cal, . , ~15 1. , -NmTm w.t. . ,

30 , .. .

50 40

A
2 3

-NmTm WT

Cp, kJ mol K

-1 -1

30 20 10

1
0 50 40

-NmTm R91G

Cp, kJ mol K

-1 -1

30

3
20

1+2
10 0

20

30

40
o

50

60

, C

. 7. -NmTm w.t. () -NmTm R91G () , . 30 . : 30 mM Hepes, 100 mM KCl, 1 mM MgCl2, pH 7,3. 1 /. . . , 1, 2. 3.

31
1. , -NmTm w.t. -NmTm R91G (. 7).

-NmTm -NmTm w.t. 1 2 3 -NmTm R91G 1+2 3

Tm (oC) 2 8 ,7 3 8 ,1 4 2 ,8

H

cal

(kJ/mol) 120 380 175

2 7 ,8 4 3 ,8

255 290

R91G -NmTm (. 7 1), ~27,8. 27,8 43,8є; , , 47% 53% cal. , R91G -NmTm, , ( 1+2 . 7). , R91G N- -NmTm, , 1 2 -NmTm
w.t., ( 2

1, 1+2), N- , 3 C- -NmTm. 91- g- (. « »). , . R91G. , , , [, , 2002], -. , , -NmTm R91G , -NmTm w.t. ,

32 -4 10. , - . (45). . 8 -NmTm WT -NmTm R91G (. 8) (. 8).

-NmTm WT -NmTm R91G
100

A

, F(%)

80 60 40 20 0 1

0 -1

dF/dT

-2 -3 -4 -5 -6

10

20

30

40
o

50

60

, C

. 8. -NmTm w.t. ( ) -NmTm R91G ( ), 222 (). 1 /, 0,1 /. : 10 - (pH 7.3), 100 mM NaCl, 1 mM MgCl2 1 mM -ME. () , ().

33 -NmTm WT . 8 37,7, 2 -NmTm WT (. 7). - -NmTm R91G , 26 (.8). -NmTm R91G, 91- . , R91G -NmTm

, . , , NmTm WT, -NmTm R91G , , .. 5. , , (. 8), (. 7). , coiled-coil « » [Dragan, Privalov, 2005]. coiled-coil, a d- . , , , , , ( ) . , , , coiled-coil. , , . , «» («pretransitions»), (~15% ) . , , , , -

, , ( ). , ,

- . , , , , ,

34 -NmTm. ( a d) , , 100% . , . [Kwok & Hodges, 2004], «» , -,

. R91G , , , . , , ? , (Cys-36) -NmTm. , coiled-coil, Cys-36 (.. -) , . (. 9), 1 / =485 ( ), , .. . -NmTm WT -NmTm R91G . 10. ,

( ). c. 11 -NmTm WT -NmTm R91G ( . 10). , , , , , . , , : 2 -NmTm WT (. 7), 1+2, -NmTm R91G (. 7), N-

35 -NmTm, Cys-36 .

200

, ..

150

100

50

0 350 400 450 500 550 600

, nm

. 9. , ys-36 -NmTm WT. 340 . 1 . 10 .

IAP- -NmTm WT, T1/2= 36.8 C IAP- -NmTm R91G, T1/2= 27.5 C
, ..
80 70 60 50 40 30 20 10
o

o

10

20

30

40
o

50

, C
. 10. 485 , ys-36 -NmTm WT ( ) -NmTm R91G ( ). 340 . 1 . : 30 mM Hepes, 100 mM KCl, 1 mM MgCl2, pH 7,3. 1 /. 10 .

36 ,

- Cys-36. , ( . 11), , .

50 40

-NmTm WT

Cp, kJ*mol *K

-1 -1

30 20 10 0 50 40

-NmTm R91G

Cp, kJ*mol *K

-1 -1

30 20 10 0 20 30 40 50
o

60

, C
. 11. -NmTm WT -NmTm R91G ( ) ( . 10). , , , .

- (SkTm). , [Williams & Svenson, 1981; Kremneva et al., 2004]. - Cys-190,

37 - , , - ( ). , ,

, . , , , , . , , - , , ( -NmTm WT, -NmTm R91G), -. , , , - [Anfinsen & Scheraga, 1975]. , «» . , -NmTm WT 25

/ 1/1000 1/100 ( 510 ) 20 17,5 (. 12). (20 ), 10- /Tm, 1/300. -NmTm WT - (-SkTm), [Ueno, 1984] . [Sumida et al., 2007]. , , . -NmTm WT, , 20 17,5 (. 12), -SkTm ( ) 17 15 (. 12A).

38

. 12. -SkTm () -NmTm () /, 1/300. 16%- . () 25.

, -SkTm NmTm WT 85%- . [Sumida et al., 2007], -SkTm Arg133.

Asp137, d- , , coiled-coil, - . - , , ,

, . . -, -SkTm, -NmTm WT , -NmTm WT . -NmTm WT , , -SkTm. , -NmTm WT SkTm, ,

-NmTm WT (, - ), . , ,

39 -NmTm WT ( , ) . , . , . R91G -NmTm (. 13). 25, -NmTm R91G (. 7). , . (~ 10- /-NmTm = 1/100) -NmTm R91G (. 13A) 14 . , (. 13) / ( 1/100) ( 90 ). , R 91G

-NmTm. , - .

. 13. -NmTm R91G () -NmTm () /, 1/300. 16%- . () 25.

40 , : R91G -NmTm,

N- .

- R91G F-. -NmTm WT -NmTm R91G F- . 14 , F- NmTm WT -NmTm R91G. , - (. 14). (

= 38є)

diss

(37,2єC),

.. -TmF-, (. 15, ). F- [remneva et al., 2004; , 2004]. . ( 37,2єC), . , ,
diss,

. , , , ( ) N- NmTm WT,
diss.

-NmTm R91G - F- (. 14). , F- , ; ( 1 1,5) (. 14). , (. 15), ,

41 ( .
diss

= 30,3єC), ..

- F- + F-
80

A
60
-1

-NmTm WT

Cp, kJ mol K

-1

40

20

0 80

60

-NmTm R91G

Cp, kJ mol K

-1

-1

40

20

0

20

30

40

, C
. 14. -NmTm w.t. () -NmTm R91G () F-, ( ). 65є, F-, . . , 90є 5 є, .. F-. 10 . : 46 F-, 70 , 30 mM Hepes, 100 mM KCl, 1 mM MgCl2 1 mM -ME, pH 7,3. 1 /.

o

50

42

-NmTm WT (Tdiss = 37,2 C) -NmTm R91G (Tdiss = 30,3 C)
o

o

, %

100

80

60

40

20

0

20

25

30

35

40
o

45

50

, C

. 15. -NmTm w.t. -NmTm R91G F-, . . 100% 25є TmF- F-, . , . 14. 1 /. Tdiss F-, .. , 50%.

-NmTm WT -NmTm R91G F. . 15 F- -NmTm WT -NmTm R91G. , .. . - -NmTm R91G F- (T (T
diss diss

= 30,3є),

= 37,2є) (. 15). ,

, , . , -NmTm WT, -, R91G, F-

43 . - . , F- 100000120000 g. , , (, ), . (.. , ) , . -NmTm w.t. () -NmTm R91G () . 16. , -NmTm w.t. -NmTm R91G , . [Robinson et al., 2007] .

1.6 1.4

-NmTm WT -NmTm R91G

Tm, M

1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Tm, M

. 16. -NmTm w.t. -NmTm R91G F-. F- (10 µM) c Tm (05 µM) (30 mM Hepes, 100 mM KCl, 1 mM MgCl2, 1 mM -ME, pH 7.3) 100 µl. , Tm, 100000 g 30 . (. « »). SDS-. Tm F- Tm, F- , Tm, .

- Tm F , Tdiss (, , , .. , F-

44 ) Tm/, [Levitsky et al., 2000]. , Tdiss, R91G -NmTm (. 15), -NmTm F- (. 16). ( -NmTm w.t. -NmTm R91G) F- , . -NmTm R91G , NmTm w.t. (. 15), (Tdiss = 30,3є), 1+2 -NmTm R91G (Tm = 27,8є) (. 7, . 1), ( 11.5) , N- -NmTm R91G (. 14). , R91G / -Tm, . N- -NmTm, , , , -NmTm F- , , .

- (-SkTm).
, , R91G () -. [Robinson et al., 2005] , , R 91G -,

, ( ) . . [Robinson et al., 2005] , -NmTm R91G , -NmTm WT, - ATP 10-9 10-5 . , , ,

- . - (-NmTm).

- (-SkTm, . . 2) R91G, .

45

, -SkTm WT -SkTm R91G. ,

-SkTm -SkTm R91G . -, -SkTm (Cys-36 Cys-190), . -, . , (30 mM Hepes, pH 7,3, 100 mM KCl, 1 mM MgCl2) , ( 360 1520 % 280 ). , , -SkTm -NmTm, . -SkTm WT, -SkTm R91G (. 17), , , NmTm.

. 17. (16%), (-NmTm WT, -SkTm WT -SkTm R91G) SDS- () 6 (). . .

(620 % 1017 %). -SkTm WT

46 -SkTm R91G 6 (. 17). , ( ) , - ( -NmTm, -SkTm WT -SkTm R91G) (. 17). , , 2% SDS -ME, 6 . . : ( . 18) . , -SkTm (, ) [Kremneva et al., 2004]. SkTm . -SkTm WT ( . 18) , -ME 2 . . 18. , , ( . 18), ( . 18) , -SkTm WT, ( ). , , , (,

). - -SkTm WT,

(. 18). , [ ., 1998] , pH ( 7,3) . , , -SkTm -

, ( , , ), ,

47 (. 18). , (, -SkTm), (Cys-190), .

15 10

-SkTm WT
1 2 (+ 2 -) 3

, µW

5 0 -5 -10 -15 20 30 40

50

60

,

. 18. , -SkTm WT, (). ( ) , -ME 2 . . 30 . : 30 mM Hepes, 100 mM KCl, 1 mM MgCl2, pH 7,3. 1 /. . .

-SkTm (. 17), -ME, . , , -SkTm WT -SkTm R91G NmTm -SkTm,

.

(

48 ) . 19. 2 S 4 S 2,3 S 2,8 S. , -SkTm WT -SkTm R91G (. 19). , , -ME . , -SkTm WT -SkTm R91G SDS- (. 17) .

1.25

1.00

-SkTm -NmTm WT -SkTm WT -NmTm R91G -SkTm R91G

0.75

c(s)
0.50 0.25 0.00

0

2 4 6 , S

8

. 19. . c(s), 52000 /. 1 /. : 30 mM Hepes, 100 mM KCl, 1 mM MgCl2, pH 7,3.

-SkTm WT -SkTm R91G -ME. ,

49

-SkTm WT -SkTm R91G. , -ME , , , . SH- -SkTm WT DTT, , -SkTm WT, DTT, , -SkTm WT -ME. -SkTm WT, , . , -ME -SkTm, . -SkTm, -ME.

-SkTm WT -SkTm R91G. -SkTm WT -SkTm R91G . -SkTm WT -SkTm R91G . 20. , -NmTm WT NmTm R91G, ,

( ). -SkTm WT 2 ( 1 2 . 20), 34% 67% cal, ( 2). , -SkTm WT (. 20), , -NmTm WT (, «1» . 7A). , -NmTm WT (. 7). , 2 SkTm WT 2 -NmTm WT (. 7), N .

50

60 50

A

-SkTm WT

Cp, kJ mol K

40 30 20 10

-1

-1

2 1

0 60 50
-1

B

-SkTm R91G

Cp, KJ mol K

40 30

-1

2
20 10 0

1

10

20

30

40
o

50

60

, C
. 20. -SkTm w.t. () -SkTm R91G () , . 30 . : 30 mM Hepes, 100 mM KCl, 1 mM MgCl2, pH 7,3. 1 /. . . , 1 2. 2. , -SkTm w.t. -SkTm R91G (. 20).

-SkTm -SkTm w.t. 1 2 -SkTm R91G 1 2

Tm (oC) 2 9 ,1 3 7 ,5 2 9 ,3 3 5 ,4

H

cal

(kJ/mol) 215 420 315 200

51

-SkTm R91G 2 , 1 2, 61% 39% cal, (. 20, 2). , 2 -SkTm WT -SkTm R91G (37,5 35,4, ), -SkTm R91G , SkTm WT (. 20, 2). 1 -SkTm R91G 29,3, -NmTm R91G ( 1+2 27,8 . 7). , SkTm WT (. 20). -SkTm WT 2 , N- - . N- NmTm WT ( 2 . 7A) -SkTm WT ( 2 . 20) (38,1 37,5, ). , -SkTm WT -NmTm WT Ser-188, - (- 6 9, . . 2 « »). , , - -SkTm WT ( 2 37.5 . 20) , -NmTm WT ( 3 42,8 .7). - -SkTm WT N- 2, -Tm, 37,5 . , - , 6, , 6b, - [Maytum et al., 2004; Kremneva et al., 2006]. 1 -SkTm WT, , ,

52 -SkTm WT ( 130188), .

-SkTm WT -SkTm R91G
100

A

, F(%)

80

60

40

20

0 1 0 -1 -2 -3 -4 -5 -6

d F /d T

10

20

30

40
o

50

60

, C

. 21. -SkTm w.t. ( ) -SkTm R91G ( ), 222 (). 1 /, 0,1 /. : 10 - (pH 7.3), 100 mM NaCl, 1 mM MgCl2 1 mM -ME. () , ().

R91G -SkTm (. 20). -NmTm R 91G

53 N- (. 7). , -SkTm R91G. -, R91G N- . ( 1 . 20 29,3) , N- -NmTm R91G (. 7). 2 (. 20, 2). , 2 -SkTm WT N- - . -SkTm WT -SkTm R91G , -NmTm, ( , .. 5) (. 21). (. 21) , R91G -SkTm.

- R91G F-. -SkTm WT -SkTm R91G F- . 22 , F- SkTm WT -SkTm R91G. , - (. 22). (

= 37.9є)

-SkTm WT, 1,7 , ( 2 ). , -SkTm WT
diss

(37,5єC), .. -TmF-,

(. 23, ). : « ?». , , , ,

54 . , -SkTm .

+ F- - F-
200

A

-SkTm WT

Cp, kJ mol K

-1

-1

150

100

50

0 200

-SkTm R91G

-1

Cp, kJ mol K

150

-1

100

50

0

10

20

30

40

50
o

60

, C

. 22. -SkmTm w.t. () -SkmTm R91G () F-, ( ). 65є, F-, . . , 90є 5 є, .. F-. 10 . : 46 F-, 70 , 30 mM Hepes, 100 mM KCl, 1 mM MgCl2 1 mM -ME, pH 7,3. 1 /.

55

, 1,7 , , , (. « ») . , -NmTm, -SkTm. , «» . F- - [Kremneva et al., 2006]. -, R91G (. 22). -SkTm, ( 1,4 ) . , F- -SkTm R91G 3644 (. 22). - , ( 2 . 20). -SkTm R91G (
diss

= 34, . 23) ,

(. « »), .

-SkTm WT -SkTm R91G F (. 23) , -SkTm, R91G, ((
diss

= 34), (

diss

= 37,5).

, (

= 37.9є SkTm WT 33,8 diss

SkTm R91G). , , (3,5

4,1

) -SkTm , -NmTm,
diss

-NmTm WT -NmTm R91G

7 (. 15). ,

56

. , , -Tm R91G , () -Tm.

-SkTm WT (Tdiss=37.5 C) -SkTm R91G (Tdiss=34.0 C)
, %
100
o

o

80

60

40

20

0

20

25

30

35

40

45
o

50

55

, C

. 23. -SkTm w.t. -SkTm R91G F-, . . 100% 25є TmF- F-, . , . 22. 1 /. Tdiss F-, .. , 50%.

57

, , . , R91G . , , Arg-91 -NmTm, -SkTm. N , . , , 5 ( -) . , (R91G) -, , , ,
N- . , R91G

. . -.

(-NmTm -SkTm) . , , (1020 ). , - . , -SkTm WT (. 20) 37,5C, -SkTm , , 43 [Kremneva et al., 2004]. , , - ( R91G!) . , . , . - , , ,

58

-SkTm WT, -SkTm R91G 6. , -SkTm . - -NmTm -SkTm. , -SkTm , -NmTm. , N- - . -NmTm -SkTm 188 , - ( 188
284). , -SkTm

-NmTm , 6b
(. 2). [Kremneva et al., 2006],

, 6a, -NmTm. . -SkTm , -NmTm. , -SkTm. , -NmTm R91G, -SkTm R91G. , - (. . 2) , , -.

59

1.

,
R91G -,

N- .

2.

, -
F-.

3.

,

- ,

.

- , -.

60

.. (1998) - . , 5, 2-16. .. (2000) . , 6(8), 24-32. .., .., .., .. (2003) . , 68, 976-984. , .., , .., ., ., , .. (1998). . . . 63, . 3., 422-429. .. (2005) F-. . , . .. , , 2005. .. (2004) . , 69, 1447-1462. .. (2004) - (). . , 44: 133-170. .., .., .. (1995). . " ", 1: 249-293, ( . .., ..), , . , .., , .. « ». 2- . «», , 2002.
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