From the Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304
Received for publication, May 22, 2000, and in revised form, November 22, 2000
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ABSTRACT |
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The complex formed between the TATA-binding
protein (TBP) and the "TATA box" of eukaryotic class II promoters
is the foundation for assembly of the complex to which RNA polymerase
II is ultimately recruited. TBP binds productively to canonical and
diverse variant TATA sequences with >100-fold differences in
transcription efficiency. Co-crystals of canonical sequences and >11
variant sequences bound to various TBP molecules all have ~80°
bends. In contrast, the bend angles for TBP·TATA complexes in
solution, derived from distance distributions, are ~80° for a
canonical sequence but range from 30° to 62° for five variant
sequences (1). We show in this study that the osmolytes used to
crystallize TBP·TATA complexes induce profound increases in the DNA
bends of two transcriptionally active TBP-bound variant sequences to a
common angle of ~80° but have little effect on a transcriptionally
inactive variant. The effect of osmolyte on the TBP-induced DNA bend of
a variant TATA box sequence is also manifest in the kinetics of
association, demonstrating a functional consequence of an
osmolyte-induced structural change.
The complex formed between the TATA-binding protein
(TBP)1 and eukaryotic class
II promoters of consensus sequence TATA(a/t)A(a/t)N is the foundation
for assembly of the preinitiation complex to which RNA polymerase II is
ultimately recruited. TBP binds productively to canonical and diverse
variant TATA sequences with >100-fold differences in transcription
efficiency (2). A key feature of the solution geometry of the
adenovirus major late promoter (AdMLP) and five variant
sequences bound to Saccharomyces cerevisiae TBP has been
determined using time-resolved fluorometry coupled with Förster
resonance energy transfer (FRET) (1). The mean end-to-end distances in
these six TBP-bound sequences vary widely and correspond to TBP-induced
bends of 76° for the consensus AdMLP sequence and from 30° to 62°
for the variant sequences.
High resolution co-crystal structures of canonical TATA sequences bound
to S. cerevisiae (3), Arabidopsis thaliana (4), and human (5, 6) TBPs are extremely similar. These binary structures
are characterized by ~80°-induced bends in the DNA helical axes, in
excellent accord with the solution bend angle obtained subsequently for
AdMLP using FRET. However, in contrast to the bends determined using
FRET, the co-crystal structures of 11 TATA sequence variants (including
those examined by FRET) bound to A. thaliana TBP are all
very similar, with the DNA bent ~80° as in the strong promoters (6,
7).
Because assembly of the transcription complex proceeds by sequential
structural alterations, a clear picture of the relevant discrete
structures is fundamental to an accurate understanding of these
processes. The present study was therefore undertaken to further
investigate the relationship between the solution and co-crystal
structures of TBP·promoter complexes.
Osmolytes used as crystallizing agents for TBP·TATA complexes were
postulated to induce in the bound variant sequences severe bends
approaching those observed in the strong promoters. End-to-end distance
distributions were therefore obtained using FRET fluorometry for the
reference AdMLP and three variant sequences, free and bound to S. cerevisiae TBP, in solutions containing 0-3 M
ethylene glycol or 3.5 M glycerol. Determination of the
corresponding solution bend angles revealed little change for TBP-bound
AdMLP upon addition of osmolyte. In contrast, osmolytes induced
profound increases in the bend angles of the transcriptionally active
variant sequences, to a common angle of ~80° but little change in
the solution geometry of the transcriptionally inactive variant (T The top strands of each DNA duplex (denoted
T*14-merdpx*F) are labeled via 6-carbon linkers with
3'-fluorescein and 5'-TAMRA, constituting a FRET donor-acceptor pair.
The labeled and unlabeled DNA as well as the full-length S. cerevisiae TBP were as described previously (1, 8-11), with the
specific DNA sequences shown below in Table I. Studies were conducted
in 10 mM Tris-HCl (pH 7.4), 100 mM KCl, 2.5 mM MgCl2, 1 mM CaCl2,
and 1 mM dithiothreitol. Ethylene glycol was added to
0.5-3 M (0.5-3.8 osmolal) and glycerol to 3.5 M (5.3 osmolal) as noted. All measurements were made at 30 ± 0.05 °C.
Detailed theoretical discussions of Förster resonance energy
transfer relevant to this study have been published previously (1, 9, 11, 12 and references therein). All instrumentation, data acquisition,
and analyses and error estimates were exactly as described (1).
The Förster distance, R0, was determined
independently for free and TBP-bound AdMLP in buffer with and without 3 M ethylene glycol and for free and TBP-bound T6 in buffer
alone and with 0.5, 1, 2, and 3 M ethylene glycol. The
overlap integrals were determined as described (1). The corresponding
refractive indexes for the ethylene glycol solutions are 1.3349, 1.3388, 1.3444, and 1.3500. To ensure sufficient dye mobility in the
presence of osmolyte consistent with End-to-End Distance Distributions for Free and TBP-bound AdMLP and
Two Variant Sequences--
The TBP-induced bend in DNA bearing the
AdMLP promoter sequence and DNA bearing an A
The effect of osmolytes on the unbound duplexes was similar. The values
of
Remarkably, osmolytes affect complexes of TBP with the three DNA
sequences very differently. In the absence of osmolyte,
Similarly, the value of
Control experiments were conducted to ensure that the changes in the
value of
The overlap integrals determined for free AdMLP and T6 in 3 M ethylene glycol were identical, with
R0 = 60.9 Å. The 3'-fluorescein emission and
5'-TAMRA absorption spectra were likewise invariant for TBP-bound AdMLP
and T6, with R0 = 61.1 Å. For free T6 in 0.5, 1, and 2 M ethylene glycol, R0 = 61.1, 61.0, and 61.0 Å, respectively. The corresponding values for
TBP-bound T6 were 61.2, 61.2, and 61.1 Å. Because the overlap
integrals were nearly sequence- and osmolyte-independent, the integral
determined for free and bound T6 in 3 M ethylene glycol was
used to calculate R0 values of 60.3 and 60.5 Å for free and bound C7 in 3.5 M glycerol (
In addition, the osmolyte independence of the slow phase of the
anisotropy decays, which correspond to the rotational correlation times
for the TBP·DNA complex, suggests no osmolyte-induced aggregation of
the protein. This conclusion is further supported by
ultracentrifugation studies in 5% glycerol (14) that show no effect of
the osmolyte on TBP aggregation and in 10%
glycerol2 that show a limited
de-stabilization of TBP oligomers. Finally, the reference
AdMLP sequence serves as an effective internal control. The similarity
of P(R) for the AdMLP·TBP complex with and
without osmolyte suggests that the large changes in
P(R) observed for bound T6 and C7 do not derive
from osmolyte-induced TBP aggregation.
Solution Bend Angles for TBP-bound TATA Duplexes in the Absence and
Presence of Osmolytes--
The bend angle for TBP-bound
T*MLdpx*F obtained from the ratio of
The solution bend angles corresponding to this model for the three
TBP-bound duplexes in the absence and presence of osmolyte are shown in
Table II. The bend for the bound AdMLP
duplex is similar, ~80°, in buffer and in osmolyte. The solution
bend angles for the bound C7 and T6 variants in buffer, 52.3° and
36.9°, respectively, are much smaller than for the strong promoter
sequence, in distinct contrast to the ~80° bends observed for both
variants in their co-crystals (7). However, upon addition of osmolyte,
the solution bends for both variants increase dramatically (48 and
112%, respectively), to closely resemble the ~ 80° bend of
the consensus sequence. For the T6·TBP and C7·TBP complexes, the
ethylene glycol-induced change is sequence-independent and, for the
C7·TBP complex, the osmolyte-induced change is osmolyte
species-independent.
To induce crystallization, co-crystals are grown in solutions of
osmolytes such as ethylene glycol + glycerol + polyethylene glycol (3)
or glycerol (4, 7) at osmolalities commensurate with those used herein.
The differences in geometry observed for the TBP-bound variant TATA
sequences in buffered solutions and in co-crystals thus appear to be
attributable to the presence of the osmolytes used in crystallization.
As the bend angles for the bound variants increase in the presence of
osmolyte to approach that of the consensus sequence, Minimal Effect of Osmolytes on the TBP-bound A3
Variant--
Patigkoglou et al. (7) reported that all
attempts to crystallize TBP bound to the A3 variant were entirely
unsuccessful. Because only complexes with ~80° bends have been
crystallized, we hypothesized that osmolytes would not induce the
AdMLP-like structure in TBP-bound A3, the only sequence not known to
occur naturally of those investigated in this and the accompanying
studies. The effect of osmolytes on A Two-state Model Relating the T6 Bend Angle and Osmolyte
Concentration--
The ethylene glycol concentration dependence of
In the accompanying paper (1), a two-state model provided insight into
the close relationships among TATA sequence, solution bend angle, and
transcription activity. The relationship between the T6 bend angle and
osmolyte concentration has been considered in the same manner. In this
model, the TBP·T6 complex exists in two forms,
conformerML and conformerTI (Fig. 4A
in Ref. 1). The DNA in conformerML is bent ~80° as for
the consensus sequence, with the DNA in the transcriptionally inactive
conformerTI much less bent. The population distributions of
conformerML and conformerTI are osmolyte
concentration-dependent, with the equilibrium shifting toward
conformerML as osmolyte concentration increases.
The probability distributions, determined for bound T6 at all five
osmolyte concentrations, were analyzed globally to obtain P(R) for conformerTI,
The FRET measurements yield P(R) values for these
complexes that vary both with sequence (1) and with osmolyte
concentration. Remarkably, this two-dimensional data set is well fit in
its entirety by only two parameters corresponding to the
two-state model,
The free energy required to convert confomerTI to
conformerML is sequence-dependent due to
variations in contacts, interactions, and solvation along the minor
groove/protein interfaces, and inherent ease of deformability. The
osmolyte concentration dependence of the free energy required to
convert conformerTI
The A3 results may also be considered within the context of the
two-state model. The mole fraction of conformerML predicted to be present in a solution of TBP and A3 approaches zero at all TBP
concentrations explored, assuming the Ka measured in
buffer. Co-crystals would be unlikely from such a solution, because
crystallization has been reported only for a
conformerML-like structure. The solution and
crystallographic results thus present a coherent perspective considered
either directly or within the context of the two-state model.
Osmolyte Concentration-dependent Changes in TBP + C7
Stopped-flow Association Kinetic Curves--
The association binding
kinetics of AdMLP·TBP have been previously investigated in our
laboratory using FRET stopped-flow (10, 11). Analogous initial
association curves have been obtained for TBP binding to the C7 variant
in 0-3 M ethylene glycol (Fig. 4). In the absence of osmolyte, the TBP
association kinetics are remarkably dependent on the C7 substitution in
the canonical sequence (compare the uppermost, C7, and
lowest, AdMLP, curves in Fig. 4). In the presence of
osmolyte, however, the kinetic trace for TBP binding to C7 changes
dramatically to approach that of AdMLP. The trace for the reference
AdMLP sequence is nearly unchanged in the presence and absence of
osmolyte.4 The
sequential changes in binding as osmolyte concentration is increased
appear to correspond to the osmolyte-induced changes in the solution
conformation of the TBP·C7 complexes. A detailed analysis of the
kinetics of TBP binding the C7 variant is in progress and will be
published elsewhere. Such strong coupling between structure and
function for the TBP·TATA complex further validates the central
role of the conformation of this binary complex in the process of
transcription.
Conclusions--
This work establishes the osmolyte dependence of
the DNA bend angles in transcriptionally active variant DNA·TBP
complexes. The large body of work to date investigating the TBP·DNA
binary complex, multiprotein transcription complexes, and transcription efficiency has been done using a wide range of osmolyte species and
concentrations. The interpretation of these collective results is
complicated by recognition of the effects of osmolytes on the TBP·DNA
conformation. This complexity is particularly apparent in light of the
very small energetic differences we have found between the two putative
conformers for a given sequence, because the effects of binding of
other proteins on the TBP·DNA conformation in osmolyte is not known.
The sequence-dependent differences in association binding
suggest that TBP·DNA kinetics may also play a significant role in
ultimately determining transcription activity. We have suggested that a
stable intermediate conformer in the TBP·DNA binding pathway may be
the binary complex to which subsequent proteins bind (11).
Transcription efficiency would then depend on both the solution
conformation and the concentration time profile of the intermediate species.
Recent reviews have summarized the role of osmolytes in preferential
solute·solvent interactions with macromolecules and the associated
changes in ligand equilibria and conformational changes (15-17). We
have begun a detailed study of osmolyte effects on the structures and
populations of the intermediate species detected along the TBP·DNA
binding pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
A substitution at position 3). The conformation of the DNA within
TBP·TATA complexes appears to be conserved only in the presence of osmolytes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
for AdMLP and C7 in 3 M
ethylene glycol derived from four composite curves
(each characterizing 15 separate decays) for each case, yielding
4 × 4 matrices for both sequences. For T6 in 0.5-3 M
ethylene glycol, 2 × 2 matrices were generated. One composite
curve was obtained for each case of A3 in 3 M ethylene glycol and C7 in 3.5 M glycerol. For these latter two
conditions, the values of
were determined
from 5 × 5 matrices constructed from the five representative
curves (each comprising three separate decays).
2 = 2/3, semi cone
angles were determined as described (1) for both TAMRA and fluorescein
for free and bound AdMLP in 3 M ethylene glycol.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
C substitution at
position 7 (C7) and an A
T substitution at position 6 (T6) was
probed using FRET fluorometry. The mean end-to-end distance for each
sequence free (
for
the distance distribution in buffer, buffer plus 3 M
ethylene glycol, and buffer plus 3.5 M glycerol are listed
in Table I.
The mean 5' dye-3' dye distance, for the
probability distribution characterizing the AdMLP, C7, and T6 duplexes
free and TBP-bound at 30 °C, in the absence and presence of osmolyte
3
M osmolyte. The slight increases observed in
free for the unbound
duplexes also increased in the presence of osmolyte, from 7% to
42%, the latter for the T6 variant. The breadth of the distribution derives both from the motion of the dye linkers and from relatively slow fluctuations in the duplex. Because the linkers are invariant among these oligomers, the observed increases in
free
are believed to reflect osmolyte-dependent increases in the
deformability of the free duplexes. The determination of the relative
contributions of the linker arms and the duplex DNA to
free is the subject of active investigation.
3 M osmolyte, the values of
bound for TBP-bound AdMLP is
unchanged by addition of osmolyte, whereas
bound for
both variant sequences decreases significantly. Identical results were
obtained for C7 in ethylene glycol and in glycerol. The implications of
these changes are discussed below.
fast = 0.15 ± 0.03 ns,
slow,free = 5 ± 2 ns, and
slow,bound = 23 ± 2 ns, indistinguishable from those determined in the absence of ethylene glycol (1). These values
taken together reflect a high degree of rotational freedom in 3 M ethylene glycol for both dyes for the free and TBP-bound duplexes. Because the conformations of the bound AdMLP, T6, and C7
duplexes in osmolyte-containing solutions are very similar to each
other as well as to those in the corresponding co-crystals, these
controls were deemed sufficient to justify using
2 = 2/3 in all calculations of R0 for the duplexes
in osmolyte.
= 1.3704). These results confirm that the observed changes in the values of
, determined according to the relationship
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Fig. 1.
The model corresponding to Eq. 1 from which
bend angles were determined for TBP-bound DNA.
L2 = 20.4 Å, consistent with the structure of
B DNA and also closely approximating the distance from the mid
points of the helix between steps 1-2 and steps 7-8 in the co-crystal
structures (3, 4). L1 = L3 = ( 20.4 Å)/2. The reported bend angles correspond to "
."
with L2 defined as in Fig. 1.
(Eq. 1)
The solution bend angles corresponding to the model depicted in
Fig. 1 for TBP-bound AdMLP, C7, and T6 in the absence and presence of
osmolyte
bound decreases correspondingly. These observations are
consistent with our hypothesis that the observed inverse correlation
between
bound and the solution bend angle for TBP-bound
sequences is attributable to increased restriction of DNA helical
motion with increased complementarity of the protein-DNA interface
(1).
for free and TBP-bound
T*T6dpx*F is shown in Table
III together with the corresponding bend
angles. The successive decreases in the end-to-end distance of bound T6
as osmolyte concentration increases from 0 to 3 M
correspond to an overall 2-fold increase in the bend angle for bound
T6, with 86% of the total change occurring by 2 M. The
linear decrease in the breadth of the distribution as
increases is
94% complete by 2 M (Fig. 2)
and further confirms the thesis that the duplex is constrained as it
conforms to the protein binding site.
The dependence of on ethylene glycol
concentration for free and TBP-bound T*T6dpx*F together
with the corresponding bend angles
96% saturation of the duplex in the fluorescence
decay measurements.
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Fig. 2.
The inverse relationship between the breadths
of the distributions and the osmolyte-dependent bend angles
for TBP-bound T6. [EG] is the concentration of
ethylene glycol from 0 to 3 M (0-3.8 osmolal). To minimize
the contribution from the tethers, diff is defined as
(
diff versus the
sequence-dependent bend angles.
where i specifies osmolyte concentration and all else
was as described (1). The set of all five P(R)
values was very well described by the two-state model, with a
correlation coefficient of 0.999 for the analysis. The values obtained
for the two fitted parameters were
(Eq. 2)
TI,bound = 9.9 ± 0.1 Å. Bend angles corresponding
to the two-state model were then calculated as described (1) at each
osmolyte concentration; these angles differ from those in Table III by
only 1.6 ± 0.8°.
bound for conformerTI. The osmolyte
concentration-dependent analysis alone yields
TI,bound = 9.9 Å, and the
sequence-dependent analysis alone yields
TI,bound = 9.9 Å. Thus, only two conformations of the
TBP·DNA binary complex are sufficient to account for both
the osmolyte concentration- and sequence-dependent solution
conformations of these TBP-bound TATA sequences, as well as the
sequence dependence of transcription efficiency.
conformerML for the
TBP·T6 complex is shown in Fig. 3. For
this sequence variant, conformerTI is energetically favored
in the absence of osmolyte, whereas conformerML is more
stable in 3 M ethylene glycol. The largest energy
difference between conformerML and conformerTI, 1.32 kcal/mol, is only 0.17 kcal/mol per core base pair. The
differences in the solution and co-crystal structures thus result from
very small differences in
G0.
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Fig. 3.
G0 for
the conversion of conformerTI
conformerML for TBP·T*T6dpx*F. The
TBP-bound T6 variant in the absence of osmolyte has a bend angle
approximately half that of AdMLP. The successive increases in the
observed bend (Table III) from 0 to 3 M ethylene glycol
(EG) are attributable, within the context of the two-state
model, to small energetic changes that alter the population
distribution to favor conformerML.
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Fig. 4.
Stopped-flow association kinetic traces for
TBP binding to AdMLP and C7 at 30 °C in 0 to 3 M
ethylene glycol. AdMLP·TBP binding (heavy solid line)
was nearly unchanged in 3 M ethylene glycol.4
C7 binding to TBP in buffer (light solid line) differs
profoundly from that of the consensus sequence, but successively
approaches the AdMLP·TBP curve with increasing concentrations of
ethylene glycol: 0.5 M (light dotted line), 1 M (heavy dashed line, short dashes),
and 3 M (light dashed line, long
dashes).
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ACKNOWLEDGEMENTS |
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We thank Elizabeth Jamison for expression and purification of the TBP and Prof. Michael Brenowitz for helpful discussions and a critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a Fellowship from the Program in Mathematics and Molecular Biology at the University of California-Berkeley, which is sponsored by National Science Foundation Grant DMS-9406348 (to R. M. P.), by Wheeler (to J. W.) and McDonald (to R. M. P.) Fellowships from the College of Graduate Studies, University of Nebraska-Lincoln, and by National Institutes of Health Grants GM59346 and CA76049 (to L. J. P.).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.
To whom correspondence should be addressed: Dept. of Chemistry,
University of Nebraska-Lincoln, 525 Hamilton Hall, Lincoln, NE
68588-0304. Tel.: 402-472-3501; Fax: 402-472-2044.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M004401200
2 S. Morris and M. Brenowitz, personal communication.
3
The average value of
4 R. M. Powell, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: TBP, TATA-binding protein; AdMLP, adenovirus major late promoter; FRET, fluorescence resonance energy transfer; TAMRA, carboxytetramethylrhodamine; T*14-merdpx*F, TATA-bearing DNA duplex with 5'-TAMRA- and 3'-fluorescein-labeled top strand; 14-mer*F, corresponding duplex with 3'-fluorescein-labeled top strand.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Wu, J.,
Parkhurst, K. M.,
Powell, R. M.,
Brenowitz, M.,
and Parkhurst, L. J.
(2001)
J. Biol. Chem.
276,
14614-14622 |
2. | Wobbe, C. R., and Struhl, K. (1990) Mol. Cell. Biol. 10, 3859-3867[Medline] [Order article via Infotrieve] |
3. | Kim, Y., Geiger, J. H., Hahn, S., and Sigler, P. B. (1993) Nature 365, 512-519[Medline] [Order article via Infotrieve] |
4. | Kim, J. L., Nikolov, D. B., and Burley, S. K. (1993) Nature 365, 520-527[Medline] [Order article via Infotrieve] |
5. | Juo, Z. S., Chiu, T. K., Leiberman, P. M., Baikalov, I. B., Berk, A. J., and Dickerson, R. E. (1996) J. Mol. Biol. 261, 239-254[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Nikolov, D. B.,
Chen, H.,
Halay, E. D.,
Hoffmann, A.,
Roeder, R. G.,
and Burley, S. B.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4862-4867 |
7. |
Patikoglou, G. A.,
Kim, J. L.,
Sun, L.,
Yang, S. H.,
Kodadek, T.,
and Burley, S. K.
(1999)
Genes Dev.
13,
3217-3230 |
8. | Petri, V., Hsieh, M., and Brenowitz, M. (1995) Biochemistry 34, 9977-9984[Medline] [Order article via Infotrieve] |
9. | Parkhurst, K. M., and Parkhurst, L. J. (1995) Biochemistry 34, 293-300[Medline] [Order article via Infotrieve] |
10. | Parkhurst, K. M., Brenowitz, M., and Parkhurst, L. J. (1996) Biochemistry 35, 7459-7465[CrossRef][Medline] [Order article via Infotrieve] |
11. | Parkhurst, K. M., Richards, R. M., Brenowitz, M., and Parkhurst, L. J. (1999) J. Mol. Biol. 289, 1327-1341[CrossRef][Medline] [Order article via Infotrieve] |
12. | Parkhurst, K. M., and Parkhurst, L. J. (1995) Biochemistry 34, 285-292[Medline] [Order article via Infotrieve] |
13. | Dickerson, R. E., Goodsell, D., and Kopka, M. L. (1996) J. Mol. Biol. 256, 108-125[CrossRef][Medline] [Order article via Infotrieve] |
14. | Daugherty, M. A., Brenowitz, M., and Fried, M. G. (2000) Biochemistry 39, 4869-4880[CrossRef][Medline] [Order article via Infotrieve] |
15. | Parsegian, V. A., Rand, R. P., and Rau, D. C. (1995) Methods Enzymol. 259, 43-94[Medline] [Order article via Infotrieve] |
16. | Timasheff, S. N. (1998) Adv. Protein Chem. 51, 355-432[Medline] [Order article via Infotrieve] |
17. |
Parsegian, V. A.,
Rand, R. P.,
and Rau, D. C.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3987-3992 |