From the Physical Biosciences Division, Lawrence
Berkeley National Laboratory, Berkeley, California 94720 and the
§ Graduate Group in Biophysics, the ¶ Department of
Plant and Microbial Biology, and the
Department of Chemistry,
University of California, Berkeley, California 94720
Received for publication, February 2, 2001
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ABSTRACT |
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The crystal structure of
BeF Two-component signal transduction systems control a variety of
cellular processes, including chemotaxis and expression of some genes
in bacteria and lower eukaryotes (1-4). Signal transduction is
mediated by phosphotransfer from a histidine kinase to a conserved aspartyl residue of a response regulator. To date, more than 300 response regulators have been identified based on homology in a domain
of ~120 residues, commonly referred to as a receiver or regulatory
domain (5). The structures of several receiver domains have been solved
(4). They all have a similar ( CheY, the response regulator of bacterial chemotaxis, has served as the
model for understanding phosphorylation-induced activation of response
regulators (7, 8). Early biochemical, genetic, and structural studies
on CheY indicate that phosphorylation induces a structural change from
an inactive to an active conformation (for a review, see Ref. 6). The
five conserved active site residues were shown to be important for
phosphorylation and/or conformational changes subsequent to
phosphorylation. Asp57 was established as the site of
phosphorylation (9) and, along with Asp12 and
Asp13, is required for magnesium binding (10-12). While
Thr87 and Lys109 were implicated in
post-phosphorylation events (10, 13-15), it is only recently that
their roles have been defined (see below). Finally, the rotameric
position of Tyr106, another conserved residue adjacent to
the active site, was thought to be correlated with the signaling state
of CheY (16). Although the importance of these residues has been noted
in numerous studies of inactive and mutant forms of CheY as well as
other response regulators (17, 18), a detailed understanding of the
mechanism of the phosphorylation-induced transformation from an
inactive to active conformation could not be reached because of the
short half-life of the aspartyl-phosphate linkage (half-life of a few seconds to hours).
We have shown through biochemical and structural studies that
BeF Recently, the crystal structures of the phosphorylated forms of two
other response regulators, Spo0Ar (21) and
FixJr (22), have been reported (superscript r denotes
receiver domain). Spo0Ar was unknowingly crystallized in
the phosphorylated state with calcium as the divalent metal rather than
magnesium, and FixJr was crystallized in the absence of a
divalent metal ion to circumvent problems associated with hydrolysis of
the phospho-aspartate. It is known that removal of magnesium from
phosphorylated CheY does not alter its enhanced affinity for FliM (23).
This suggests that, while magnesium is important in the chemistry of
phosphorylation of receiver domains, it is probably not required for
stabilizing the active conformations. Thus, the structures of
P-Spo0Ar and P-FixJr do likely represent the
phosphorylation-activated states, although the activities of these two
proteins in the conditions used for crystallization cannot be directly
assessed. Importantly, the residues homologous to Thr87 and
Tyr106 in both structures adopt similar conformations to
those seen the NMR structure of BeF We have recently determined the crystal structures of
BeF Escherichia coli--
CheY was overexpressed and purified as
described previously (20, 25) and was prepared as a solution
containing 2 mM CheY, 8 mM BeCl2,
50 mM NaF, and 4 mM MnCl2 at pH of
8.4. Crystals of the complex were obtained at room temperature using
the hanging-drop vapor diffusion method using a well solution
containing 1.8 M ammonium sulfate, 5-10% glycerol, and
100 mM Tris (pH 8.4). The crystallization droplets
contained the CheY solution mixed with an equal volume of the well
solution. Crystals appeared after 1 day and grew to ~0.7 × 0.4 × 0.4 mm after 3 days. The concentration of glycerol in the
well solution was increased (5% at each step) every 2 days to a final
concentration of 25 volume %. The glycerol was added as a
cryoprotectant to allow freezing for data acquisition. The protein
crystallized in space group P212121
with unit cell dimensions a = 53.5 Å,
b = 53.8 Å, and c = 161.3 Å with two
molecules in the asymmetric unit.
The diffraction data were collected at 100 K on the Mar345 detector at
the Stanford Synchrotron Radiation Laboratory using beamline 7-1 (wavelength of 1.08 Å). A crystal detector distance of 180 mm was used
to collect data to 2.37-Å resolution. The data set was integrated and
scaled to 2.37-Å resolution, using DENZO and SCALEPACK (26).
Cystal Structure of BeF
An unbiased (2Fo
The electron density for beryllofluoride on Asp57 was
clearly seen (10 BeF BeF-strand
4 and helix H4. The tyrosine side chain is stabilized
in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu89. This hydrogen bond
appears to stabilize the active conformation of the
4/H4 loop.
Comparison of the backbone coordinates for the active and inactive
states of CheY reveals that only modest changes occur upon activation,
except in the loops, with the largest changes occurring in the
4/H4
loop. This region is known to be conformationally flexible in inactive
CheY and is part of the surface used by activated CheY for binding its
target, FliM. The pattern of activation-induced backbone coordinate
changes is similar to that seen in FixJr. A common feature
in the active sites of BeF
and the phosphate or its
equivalent, beryllofluoride. This suggests that, in addition to the
concerted movements of Thr87 and Tyr106
(Thr-Tyr coupling), formation of the
Lys109-PO
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
/
)5 fold with an active
site comprised of five highly conserved residues, including three
aspartates, a lysine, and either threonine or serine (6).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
c) were
calculated from this partially refined MR model using SFALL of the CCP4
package (29). These phases were improved by 20 rounds of 2-fold
noncrystallographic symmetry averaging and solvent flattening at
2.4 Å using the RAVE package (30), with a mask made from the MR
solution and operators from the MR solution refined by the IMP program
using the 2Fo
Fc MR map. In
each round the map was calculated using coefficients 2Fo
Fc, with
Fc and
c calculated from the
density-modified map of the previous cycle.
Fc) map
calculated with phases and Fc from the final
symmetry-averaged, solvent-flattened map was displayed using the
graphics program O (31) and used as a guide in modeling
Tyr106, positioning Mn2+ ions, and manually
modifying the model in places where it did not fit the electron
density. Refinement was performed using CNS (28). Anisotropic
B-factor and bulk solvent corrections as well as the
cross-validation method (32) were applied throughout the refinement.
15.0 to 2.37 Å data were included in the refinement with tight
noncrystallographic symmetry restraints (300 kcal
mol
1 Å2). Water picking was
performed after the R-factor/Rfree
dropped to 24%/27% using CNS.
) in the resulting CNS Fo
Fc SIGMAA weighted map. This moiety was
modeled on both protomers and refinement continued giving a final
R-factor and Rfree of
21.0/24.0%. Geometric parameters for the structure were monitored
using PROCHECK (33) and WHAT_CHECK (34).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
5-H5 face of the second molecule. For CheY,
dimer formation in the crystal must be due to lattice packing forces
and is not biologically relevant, because in solution
BeF
View larger version (48K):
[in a new window]
Fig. 1.
Ribbon diagram of the two
BeF
Data collection and refinement statistics
The overall crystal structure of BeF/
)5 fold of receiver domains (6) (Fig. 1) and is
very similar to the NMR structure of BeF
coordinates (residues 6-125) of the
BeF
coordinates between BeF
4/H4 loop, the
5/H5 loop, and the N
terminus of helix H5. The significance of the changes in the
4/H4
loop are particularly hard to interpret, because it adopts different
conformations in the various crystal structures of inactive CheY (6).
Indeed, dynamics studies of Mg2+-bound CheY showed that
this region is flexible in solution (37), and a superposition of the
x-ray structure of BeF
4/H4 loop of the
active (x-ray) structure falls on the edge of the bundle formed by the
inactive (NMR, Mg2+-bound) structures. Rather than a
conformational change, we prefer to view the activation-induced changes
in the
4/H4 loop as a stabilization of the active conformation that
may be sampled by the inactive protein. Unfortunately, it is hard to
make similar conclusions for the
5/H5 loop, because the relaxation
data for residues in this loop could not be reliably interpreted due to complications caused by chemical exchange of Mg2+ in the
active site (37).
|
Active Site of BeF-strand
4 (along with Thr87) is displaced, and
the aromatic ring of Tyr106 becomes buried in a hydrophobic
pocket between helix H4 and
5. However, the NMR data did not define
the positions of either the BeF
(O
-Be distance 1.5 Å) in a tetrahedral
configuration. The aromatic ring of Tyr106 is seen
exclusively in the buried position, stabilized in this rotameric
conformation by a hydrogen bond that was not previously identified in
the NMR studies between the tyrosine hydroxyl group and the backbone
carbonyl oxygen of Glu89 as well as hydrophobic
interactions. The divalent cation (Mn2+) is located
adjacent to Asp57-BeF
,
Asp57 O
, backbone carbonyl oxygen of Asn59,
a fluorine atom, and two water molecules. Finally, the side chain of
Lys109 forms a salt bridge with BeF
.
|
Active Site Comparisons with P-Spo0Ar,
P-FixJr, and Phosphono-CheY--
Comparison of the
BeF, and hydrogen
bonds with Thr87 O
and the backbone amides of
Trp58, Asn59, and Ala88. The
measured distances for the common interactions in the structural models
of BeF
|
Although lacking a divalent cation, the distances for the analogous
interactions in P-FixJr are also similar to those seen for
BeF and Asp12 O
in
P-FixJr, indicating that this salt bridge is broken in the
absence of metal. It is interesting to note that, although a divalent
cation is necessary for the chemistry of phosphorylation and
dephosphorylation of CheY, removal of the metal after phosphorylation
apparently does not alter the affinity of P-CheY for FliM (23).
Similarly, the fact that P-FixJr purifies as a dimer in the
absence of metal, consistent with its activated state, suggests that
the metal is not required for inducing the active conformation of
FixJr. This may be a general feature of receiver domains.
In phosphono-CheY, except for the salt bridge between
Lys109 N and a phosphonate oxygen, the distances
measured for the analogous hydrogen bonds are outside of the acceptable
range (2.5-3.1 Å) (Table II). The absence of these interactions leads
to much smaller changes in the
4/H4 and
5/H5 loops (Fig.
2c). The modest structural differences relative to inactive
CheY appear to be consistent with the partial activity of
phosphono-CheY, which shows an 8-fold increase in affinity for N16-FliM
(38), whereas BeF
-C
bond in phosphono-cysteine is only 0.5 Å longer than the
C
-O
bond in phospho-aspartate, it is surprising that the
phosphonate analog does not better activate CheY. Since the salt bridge
formed by Lys109 N
and an active site partner
(BeF
5/H5 loop (40, 41).
Activation-induced Conformational Changes--
CheY and
FixJr are the only receiver domains that have been solved
with sufficient resolution in both active (22) and inactive (36, 42)
states to allow a detailed comparison of activation-induced structural
changes. The largest activation-induced C coordinate changes for
both proteins occur in loop regions, particularly the
4/H4 loop. In
addition, the
5/H5 loop shows significant displacement in CheY,
potentially due to the Lys109 N
-BeF
4/H4
loop is conformationally flexible according to solution NMR studies
(37). Activation results in the formation of a new hydrogen bond
between the hydroxyl of Tyr106 and the backbone carbonyl of
Glu89. This likely helps to stabilize the active
conformation of this loop. Thus, a comparison of just the active and
inactive CheY crystal structures could lead one to conclude that
activation induced dramatic conformational changes in the
4/H4 loop.
However, in light of the NMR data, the exact magnitude of this change
is hard to quantify. In FixJr the residue homologous to
Tyr106 is a phenylalanine, which cannot stabilize the
4/H4 loop through a side chain-backbone hydrogen bond. It would be
interesting to determine whether this loop in FixJr is also
conformationally flexible in the inactive state and becomes stabilized
upon activation.
Even though the loops show significant activation-induced changes,
activation of CheY and FixJr does not result in any major
structural rearrangements. Whereas some -strands and
-helices are
slightly displaced, the actual residues that define these elements of
secondary structure remain unchanged in both proteins. In both
BeF
|
The structures of Spo0Ar and NtrCr have also
been determined in both the active (21, 43) and inactive states (44,
45). It was difficult to analyze the activation-induced structural changes for Spo0Ar, because the inactive form crystallized
as a domain-swapped dimer, the biological relevance of which is
unclear. In contrast to CheY and FixJr, the low resolution
NMR structures of active and inactive NtrCr show major
structural differences, especially for residues that define helix H4. A
higher resolution structure of BeF
Conclusions--
The comparable interactions in the active sites
of BeF
Given the high sequence conservation, it is perhaps not surprising that
the structures of P-Spo0Ar, P-FixJr, and
BeF
Comparison of just crystal structures suggests that there is a coupling
between phosphorylation of CheY and FixJ with structural changes,
especially in the 4/H4 loop. The extent to which phosphorylation induces an actual conformational change versus a
stabilization of the active state from a pre-existing equilibrium
between the active and inactive conformations in solution is not clear.
Positive evidence for the idea of stabilization comes from NMR studies of inactive, constitutively active mutant forms (46), and
phosphorylated NtrC (43), which indicate that phosphorylation
stabilizes the active conformation (47). Additional NMR and x-ray
studies of active and inactive forms of response regulators will help
to clarify this issue and will help define how phosphorylation-induced conformational changes ultimately regulate the diverse processes controlled by two-component signal transduction.
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ACKNOWLEDGEMENTS |
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We thank the staff at Stanford Synchrotron Radiation Laboratory and, in particular, Drs. T. Earnest and D. Shin for providing invaluable advice on crystallographic data collection and processing. We also thank Prof. T. Yeates at UCLA for help with analysis of crystal twinning. We thank L. Huang for help with crystallization procedures and Dr. D. King for expert help with mass spectral analysis. We also thank R. Bourret for a critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Office of Energy Research, Office of Health and Environmental Research, Health Effects Research Division of the United States Department of Energy under contract number DE-AC03-76SF00098 (to D. E. W.) and through instrumentation Grant DE FG05-86ER75281 from the United States Department of Energy and National Science Foundation Grants DMB 86-09035 and BBS 87-20134 (to D. E. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1FQW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
** To whom correspondence should be addressed. Tel.: 510-486-4318; Fax: 510-486-6059; E-mail: dewemmer@lbl.gov.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M101002200
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ABBREVIATIONS |
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The abbreviation used is: MR, molecular replacement.
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REFERENCES |
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---|
1. | Nixon, B. T., Ronson, C. W., and Ausubel, F. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7850-7854[Abstract] |
2. | Ota, I. M., and Varshavsky, A. (1993) Science 262, 566-569[Medline] [Order article via Infotrieve] |
3. | Parkinson, J. S., and Kofoid, E. C. (1992) Annu. Rev. Genet. 26, 71-112[CrossRef][Medline] [Order article via Infotrieve] |
4. | Stock, A. M., Robinson, V. L., and Goudreau, P. N. (2000) Annu. Rev. Biochem. 69, 183-215[CrossRef][Medline] [Order article via Infotrieve] |
5. | Grebe, T. W., and Stock, J. B. (1999) Adv. Microb. Physiol. 41, 139-227[Medline] [Order article via Infotrieve] |
6. | Djordjevic, S., and Stock, A. M. (1998) J. Struct. Biol. 124, 189-200[CrossRef][Medline] [Order article via Infotrieve] |
7. | Stock, J. B., Stock, A. M., and Mottonen, J. M. (1990) Nature 344, 395-400[CrossRef][Medline] [Order article via Infotrieve] |
8. | Volz, K. (1993) Biochemistry 32, 11741-11753[Medline] [Order article via Infotrieve] |
9. |
Sanders, D.,
Gillece-Castro, B. L.,
Stock, A. M.,
Burlingame, A. L.,
and Koshland, D. E., Jr.
(1989)
J. Biol. Chem.
264,
21770-21778 |
10. |
Lukat, G. S.,
Lee, B. H.,
Mottonen, J. M.,
Stock, A. M.,
and Stock, J. B.
(1991)
J. Biol. Chem.
266,
8348-8354 |
11. | Lukat, G. S., Stock, A. M., and Stock, J. B. (1990) Biochemistry 29, 5436-5442[Medline] [Order article via Infotrieve] |
12. | Needham, J. V., Chen, T. Y., and Falke, J. J. (1993) Biochemistry 32, 3363-3367[Medline] [Order article via Infotrieve] |
13. |
Appleby, J. L.,
and Bourret, R. B.
(1998)
J. Bacteriol.
180,
3563-3569 |
14. |
Volz, K.,
and Matsumura, P.
(1991)
J. Biol. Chem.
266,
15511-15519 |
15. |
Zhu, X.,
Rebello, J.,
Matsumura, P.,
and Volz, K.
(1997)
J. Biol. Chem.
272,
5000-5006 |
16. | Zhu, X. Y., Amsler, C. D., Volz, K., and Matsumura, P. (1996) J. Bacteriol. 178, 4208-4215[Abstract] |
17. | Feher, V. A., and Cavanagh, J. (1999) Nature 400, 289-293[CrossRef][Medline] [Order article via Infotrieve] |
18. | Weinstein, M., Lois, A. F., Monson, E. K., Ditta, G. S., and Helinski, D. R. (1992) Mol. Microbiol. 6, 2041-2049[Medline] [Order article via Infotrieve] |
19. |
Yan, D.,
Cho, H. S.,
Hastings, C. A.,
Igo, M. M.,
Lee, S.-Y.,
Pelton, J. G.,
Stewart, V.,
Wemmer, D. E.,
and Kustu, S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14789-14794 |
20. | Cho, H. S., Lee, S.-Y., Yan, D., Pan, X., Parkinson, J. S., Kustu, S., Wemmer, D. E., and Pelton, J. G. (2000) J. Mol. Biol. 297, 543-551[CrossRef][Medline] [Order article via Infotrieve] |
21. | Lewis, R. J., Brannigan, J. A., Muchova, K., Barak, I., and Wilkinson, A. J. (1999) J. Mol. Biol. 294, 9-15[CrossRef][Medline] [Order article via Infotrieve] |
22. | Birck, C., Mourey, L., Gouet, P., Fabry, B., Schumacher, J., Rousseau, P., Kahn, D., and Samama, J.-P. (1999) Structure (Lond.) 7, 1505-1515[CrossRef][Medline] [Order article via Infotrieve] |
23. | Welch, M., Oosawa, K., Aizawa, S.-I., and Eisenbach, M. (1994) Biochemistry 33, 10470-10476[Medline] [Order article via Infotrieve] |
24. | Lee, S.-Y., Cho, H. S., Pelton, J. G., Yan, D., Henderson, R. K., King, D. S., Huang, L., Kustu, S., Berry, E. A., and Wemmer, D. E. (2001) Nature Struct. Biol. 8, 52-56[CrossRef][Medline] [Order article via Infotrieve] |
25. | Bruix, M., Pascual, J., Santoro, J., Prieto, J., Serrano, L., and Rico, M. (1993) Eur. J. Biochem. 215, 573-585[Abstract] |
26. | Otwinowski, Z. O., and Minor, W. (1997) Methods Enzymol. 276, 307-326 |
27. | Navaza, J. (1994) Acta Crystallogr. Sect. A Cryst. Struct. Commun. 50, 157-163[CrossRef] |
28. | Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve] |
29. | Bailey, S. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve] |
30. | Kleywegt, G. J., and Jones, T. A. (1999) Acta Crystallogr. D Biol. Crystallogr. 55, 941-944[CrossRef][Medline] [Order article via Infotrieve] |
31. | Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve] |
32. | Brünger, A. T. (1992) Nature 355, 472-475[CrossRef] |
33. | Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef] |
34. | Hooft, R. W. W., Vriend, G., Sander, C., and Abola, E. E. (1996) Nature 381, 272[Medline] [Order article via Infotrieve] |
35. | Kar, L., Matsumura, P., and Johnson, M. E. (1992) Biochem. J. 287, 521-531[Medline] [Order article via Infotrieve] |
36. | Stock, A. M., Martinex-Hackert, E., Rasmussen, B. F., West, A. H., Stock, J. B., Ringe, D., and Petsko, G. A. (1993) Biochemistry 32, 13375-13380[Medline] [Order article via Infotrieve] |
37. | Moy, F. J., Lowry, D. F., Matsumura, P., Dahlquist, F. W., Krywko, J. E., and Domaille, P. J. (1994) Biochemistry 33, 10731-10742[Medline] [Order article via Infotrieve] |
38. | Halkides, C. J., McEvoy, M. M., Casper, E., Matsumura, P., Volz, K., and Dahlquist, F. W. (2000) Biochemistry 39, 5280-5286[CrossRef][Medline] [Order article via Infotrieve] |
39. | McEvoy, M. M., Bren, A., Eisenbach, M., and Dahlquist, F. W. (1999) J. Mol. Biol. 289, 1423-1433[CrossRef][Medline] [Order article via Infotrieve] |
40. | Bellsolell, L., Cronet, P., Majolero, M., Serrano, L., and Coll, M. (1996) J. Mol. Biol. 257, 116-128[CrossRef][Medline] [Order article via Infotrieve] |
41. | Sola, M., Lopez-Hernandez, E., Cronet, P., Lacroix, E., Serrano, L., Coll, M., and Parraga, A. (2000) J. Mol. Biol. 303, 213-225[CrossRef][Medline] [Order article via Infotrieve] |
42. | Gouet, P., Fabry, B., Guillet, V., Birck, C., Mourey, L., Kahn, D., and Samama, J.-P. (1999) Structure (Lond.) 7, 1517-1526[CrossRef][Medline] [Order article via Infotrieve] |
43. | Kern, D., Volkman, B. F., Luginbühl, P., Nohaile, M. J., Kustu, S., and Wemmer, D. E. (1999) Nature 402, 894-898[CrossRef][Medline] [Order article via Infotrieve] |
44. | Lewis, R. J., Muchova, K., Brannigan, J. A., Barak, I., Leonard, G., and Wilkinson, A. J. (2000) J. Mol. Biol. 297, 757-770[CrossRef][Medline] [Order article via Infotrieve] |
45. | Volkman, B. F., Nohaile, M. J., Amy, N. K., Kustu, S., and Wemmer, D. E. (1995) Biochemistry 34, 1413-1424[Medline] [Order article via Infotrieve] |
46. | Nohaile, M. J., Kern, D., Wemmer, D. E., Stedman, K., and Kustu, S. (1997) J. Mol. Biol. 273, 299-316[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Volkman, B. F.,
Wemmer, D. E.,
and Kern, D.
(2001)
Science
291,
2429-2433 |