Abstract
In this paper, we have described a stopped-flow apparatus that is capable of measuring infrared kinetics in the amide I′ region of a protein's vibrational spectrum. The dead time of this setup, determined by the reducing reaction of 2,6-Dichlorophenolindophenol by l-ascorbic acid, is between 6 to 15 ms, depending on the flow rate. Therefore, this stopped-flow IR method provides a means of measuring infrared kinetics in a time window that is not easily accessible to other mixing-based IR techniques. Using this apparatus, we have studied the alkaline transition of cytrochrome c and have found that this conformational event proceeds in a biphasic manner. The characteristic time constants of these two phases were determined to be 68 ± 20 ms and 624 ± 37 ms, respectively.
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References
1.
Roder
H.
Maki
K.
Cheng
H.
and
Shastry
M. C. R
, Methods 34 , 15 (2004 ).
2.
Elove
G. A.
Chaffotte
A. F.
Roder
H.
and
Goldberg
M. E
, Biochemistry 31 , 6876 (1992 ).
3.
Elove
G. A.
Bhuyan
A. K.
and
Roder
H
, Biochemistry 33 , 6925 (1994 ).
4.
Kuwajima
K.
Garvey
E. P.
Finn
B. E.
Matthews
C. R.
and
Sugai
S
, Biochemistry 30 , 7693 (1991 ).
5.
White
A. J.
Drabble
K.
and
Wharton
C. W
, Biochem. J. 306 , 843 (1995 ).
6.
Dunn
B. C.
and
Eyring
E. M
, Appl. Spectrosc. 53 , 292 (1999 ).
7.
Troullier
A.
Reinstadler
D.
Dupont
Y.
Naumann
D.
and
Forge
V
, Nature Struct. Biol. 7 , 78 (2000 ).
8.
Hinsmann
P.
Haberkorn
M.
Frank
J.
Svasek
P.
Harasek
M.
and
Lendl
B
, Appl. Spectrosc. 55 , 241 (2001 ).
9.
Dunn
B. C.
Marda
J. R.
and
Eyring
E. M
, Appl. Spectrosc. 56 , 751 (2002 ).
10.
Masuch
R.
and
Moss
D. A
, Appl. Spectrosc. 57 , 1407 (2003 ).
11.
Fabian
H.
and
Naumann
D
, Methods 34 , 28 (2004 ).
12.
Uno
T.
Nishimura
Y.
and
Tsuboi
M
, Biochemistry 23 , 6802 (1984 ).
13.
Segel
D. J.
Bachmann
A.
Hofrichter
J.
Hodgson
K.O.
Doniach
S.
and
Kiefhaber
T
, J. Mol. Biol. 288 , 489 (1999 ).
14.
Funahashi
S.
Ishihara
K.
Aizawa
S.
Sugata
T.
Ishii
M.
Inada
Y.
and
Tanaka
M
, Rev. Sci. Instrum. 64 , 130 (1993 ).
15.
Hoeltzli
S. D.
and
Frieden
C
, Biochemistry 37 , 387 (1998 ).
16.
Roder
H.
Maki
K.
and
Cheng
H
, Chem. Rev. 106 , 1836 (2006 ).
17.
Callender
R.
and
Dyer
R. B
, Chem. Rev. 106 , 3031 (2006 ).
18.
Marinkovic
N. S.
Adzic
A. R.
Sullivan
M.
Kovacs
K.
Miller
L. M.
Rousseau
D. L.
Yeh
S. R.
and
Chance
M. R
, Rev. Sci. Instrum. 71 , 4057 (2000 ).
19.
Kauffmann
E.
Darnton
N. C.
Austin
R. H.
Batt
C.
and
Gerwert
K
, Proc. Natl. Acad. Sci. U.S.A. 98 , 6646 (2001 ).
20.
Marinkovic
N. S.
Huang
R.
Bromberg
P.
Sullivan
M.
Toomey
J.
Miller
L. M.
Sperber
E.
Moshe
S.
Jones
K. W.
Chouparova
E.
Lappi
S.
Franzen
S.
and
Chance
M. R
, J. Synchrotron Radiat. 9 , 189 (2002 ).
21.
Kotting
C.
and
Gerwert
K
, Chem. Phys. Chem. 6 , 881 (2005 ).
22.
Tonomura
B.
Nakatani
H.
Ohnishi
M.
Yamaguchiito
J.
and
Hiromi
K
, Anal. Biochem. 84 , 370 (1978 ).
23.
Theorell
H.
and
Åkesson
A
, J. Am. Chem. Soc. 63 , 1804 (1941 ).
24.
Morishima
I.
Ogawa
S.
Yonezawa
T.
and
Iizuka
T
, Biochim. Biophys. Acta 495 , 287 (1977 ).
25.
Rosell
F. I.
Ferrer
J. C.
and
Mauk
A. G
, J. Am. Chem. Soc. 120 , 11234 (1998 ).
26.
Ferrer
J. C.
Guillemette
J. G.
Bogumil
R.
Inglis
S. C.
Smith
M.
and
Mauk
A. G
, J. Am. Chem. Soc. 115 , 7507 (1993 ).
27.
Assfalg
M.
Bertini
I.
Dolfi
A.
Turano
P.
Mauk
A. G.
Rosell
F. I.
and
Gray
H. B
, J. Am. Chem. Soc. 125 , 2913 (2003 ).
28.
Lu
J.
Fang
J. L.
Zhu
D. X.
and
Tang
W. X
, Spectrosc. Lett. 30 , 1369 (1997 ).
29.
Dong
A.
Huang
P.
and
Caughey
W. S
, Biochemistry 29 , 3303 (1990 ).
30.
Davis
L. A.
Schejter
A.
and
Hess
G. P
, J. Biol. Chem. 249 , 2624 (1974 ).
31.
Hasumi
H.
, Biochim. Biophys. Acta 626 , 265 (1980 ).
32.
Hoang
L.
Maity
H.
Krishna
M. M. G.
Lin
Y.
and
Englander
S. W
, J. Mol. Biol. 331 , 37 (2003 ).
33.
Huang
C. Y.
Getahun
Z.
Wang
T.
DeGrado
W. F.
and
Gai
F
, J. Am. Chem. Soc. 123 , 12111 (2001 ).
34.
Berners-Price
S. J.
Bertini
I.
Gray
H. B.
Spyroulias
G. A.
and
Turano
P
, J. Inorg. Biochem. 98 , 814 (2004 ).
35.
Williams
S.
Causgrove
T. P.
Gilmanshin
R.
Fang
K. S.
Callender
R. H.
Woodruff
W. H.
and
Dyer
R. B
, Biochemistry 35 , 691 (1996 ).
36.
Huang
C. Y.
Getahun
Z.
Zhu
Y. J.
Klemke
J. W.
DeGrado
W. F.
and
Gai
F
, Proc. Natl. Acad. Sci. U.S.A. 99 , 2788 (2002 ).
37.
Bykov
S.
Lednev
I.
Ianoul
A.
Mikhonin
A.
Munro
C.
and
Asher
S. A
, Appl. Spectrosc. 59 , 1541 (2005 ).
38.
Balakrishnan
G.
Hu
Y.
and
Spiro
T. G
, Appl. Spectrosc. 60 , 347 (2006 ).
39.
Weida
M. J.
Arnone
D.
and
Day
T
, Laser Focus World 42 , 109 (2006 ).
40.
Zanni
M. T.
and
Hochstrasser
R. M
, Curr. Opin. Struct. Biol. 11 , 516 (2001 ).
41.
Mukherjee
P.
Kass
I.
Arkin
I.
and
Zanni
M. T
, Proc. Natl. Acad. Sci. U.S.A. 103 , 3528 (2006 ).