From the Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Received for publication, February 26, 2001, and in revised form, April 20, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biological signaling generally involves the
activation of a receptor protein by an external stimulus followed by
protein-protein interactions between the activated receptor and its
downstream signal transducer. The current paradigm for the relay of
signals along a signal transduction chain is that it occurs by highly specific interactions between fully folded proteins. However, recent
results indicate that many regulatory proteins are intrinsically unstructured, providing a serious challenge to this paradigm and to the
nature of structure-function relationships in signaling. Here we study
the structural changes that occur upon activation of the blue light
receptor photoactive yellow protein (PYP). Activation greatly reduces
the tertiary structure of PYP but leaves the level secondary structure
largely unperturbed. In addition, activated PYP exposes previously
buried hydrophobic patches and allows significant solvent penetration
into the core of the protein. These traits are the distinguishing
hallmarks of molten globule states, which have been intensively studied
for their role in protein folding. Our results show that
receptor activation by light converts PYP to a molten globule and
indicate stimulus-induced unfolding to a partially unstructured molten
globule as a novel theme in signaling.
PYP1 displays
rhodopsin-like photochemistry (1, 2) based on the trans to
cis photoisomerization (3-6) of its unique p-coumaric acid chromophore (7, 8). PYP is a prototypical PAS domain (9) involved in photosensory processes in purple bacteria (10, 11). PAS domains are a ubiquitous signaling module
involved in regulation, sensing, the circadian rhythm, and a number of
human diseases, which were first identified in the proteins Per, Arnt,
and Sim (12). Photoexcitation of PYP triggers a series of processes
that result in the formation of a long-lived blue-shifted photocycle
intermediates (1, 2). This intermediate is considered to be the
functionally active signaling state of PYP (1, 4, 6, 10). The
three-dimensional structure of PYP has been determined at very high
resolution (5, 13, 14), providing a unique opportunity to study
photosensory signaling at the atomic level. Previous results have
indicated a link between formation of the signaling state and protein
unfolding in PYP. This partial unfolding was detected by: (i) the
non-Arrhenius temperature dependence of the photocycle kinetics (15,
16); (ii) the loss of amide-proton NMR HSQC cross-peaks (17); (iii) the
solvent exposure of amide backbone sites (16); and (iv) the exchange
broadening of NMR HSQC signals, particularly in the N-terminal 28 residues of PYP (18). Here we examine the hypothesis that the PYP
signaling state is a molten globule state. To this end, we determine to
what extent the PYP signaling state possesses the following set of
specific properties widely used as the operational definition of a
molten globule state (19-21): (i) a large decrease in tertiary
structure but only slight reduction in secondary structure as probed by
circular dichroism (CD) spectroscopy; (ii) exposure of hydrophobic
patches as revealed by 8-anilinonaphthalene-1-sulfonate (ANS)
fluorescence; and (iii) penetration of water into the core of the
protein as shown by quenching of the intrinsic fluorescence of aromatic
amino acids.
Purification of PYP--
Pure PYP was obtained as described
previously (4) from the PYP overproduction strain Escherichia
coli M15/pHisp using Ni2+-affinity and size exclusion chromatography.
Circular Dichroism Spectroscopy--
CD spectra and
time-resolved CD traces at selected wavelengths were recorded on a
Jasco J-715 spectropolarimeter. The CD spectrum of the activated state
of PYP was obtained by time-resolved CD spectroscopy during the
spontaneous decay of the signaling state of PYP to its initial state
after 20 s actinic illumination with blue light (400-500 nm) at
pH 4.0. The data were collected at various wavelengths upon the closure
of an optical shutter. A second optical shutter was used to protect the
photomultiplier tube from scattered actinic light. The signals at 446 nm showed that the conditions used resulted in 90% photoaccumulation
of the signaling state. This value was used to calculate the CD spectra of the pure signaling state.
ANS Fluorescence Spectroscopy--
PYP (2 µM) at
pH 4.0 in the presence of 100 µM ANS was exposed to
actinic light for 10 s to photoaccumulate the signaling state.
Upon switching off the light, changes in ANS fluorescence were probed
by fluorescence excitation at 310 nm and detection at 510 nm using a
Perkin Elmer LS50B fluorimeter. The contribution of intrinsic
fluorescence from PYP under these conditions was found to be small and
could be accurately corrected by using fluorescence data obtained under
the same conditions but in the absence of ANS. The recovery of the
initial state of PYP from its signaling state was detected by
fluorescence excitation at 440 nm and detection at 490 nm.
Quenching of Fluorescence by Acrylamide--
The
intensity of fluorescence emission from aromatic side chains upon
excitation at 295 nm was detected at 340 nm as a function of acrylamide
concentration using a Perkin Elmer LS50B fluorimeter. Quenching of the
fluorescence intensity F as a function of acrylamide concentration
[Q] was quantified using the Stern-Volmer constant K with the
equation F0/FQ = 1 + K × [Q].
CD spectroscopy provides information about protein secondary
structure through the far-UV signals caused by peptide bonds as well as
protein tertiary structure by near-UV signals originating from aromatic
side chains in the asymmetric chiral environment provided by the folded
protein. The CD spectrum of the initial state of PYP exhibits a minimum
at 222 nm and a shoulder at 206 nm (Fig.
1A), consistent with its
secondary structure (1) as determined by x-ray crystallography (13).
The effect of photoactivation of PYP on its CD spectrum was determined
by illuminating the protein at pH 4.0, resulting in essentially
quantitative conversion of PYP to its signaling state. Because
FTIR difference spectroscopy has shown that the structural
changes that occur during formation of the signaling state at pH 3.5 and 7.0 are very similar (16), these conditions provide reliable
information on structural changes during PYP activation under native
conditions. Light-induced conversion of PYP to its activated state
results in a 19% reduction of the CD signal at 222 nm (Fig. 1,
A and C). The recovery of the CD signal at 222 nm
occurs with the same kinetics as the decay of the signaling state of
PYP to its initial state as probed by absorbance of the
p-coumaric acid chromophore.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (37K):
[in a new window]
Fig. 1.
Light-induced conversion of PYP to its
activated state greatly reduces tertiary structure, while leaving
secondary structure largely unperturbed. The CD spectra of the
initial state of PYP (filled circles) and of its activated,
blue-shifted state (open circles) were measured in the
far-UV region (A), documenting secondary structure, and in
the near-UV/vis region (B), revealing tertiary structure.
For comparison, the CD spectrum of fully denatured PYP in the presence
of 4.0 M GdmCl is also depicted (dashed lines).
Signals above 300 nm originate from the p-coumaric acid
(pCA) chromophore in PYP. Note that the CD signal at 446 nm for the
trans pCA in the native state is positive but that the
cis pCA in the blue-shifted state at 355 nm has a negative
CD signal. The recovery of the initial state of PYP after
photoexcitation was probed at a range of wavelengths and described as a
monoexponential decay. The kinetic traces for the signals at 222 (C) and 275 nm (D), fit as a monoexponential
decay, are depicted. The resulting values for the signaling state at
t = 0 (filled circles) and for the recovered
initial state at t = (open circles) were
plotted in A and B, together with the steady
state CD spectrum of the initial state of PYP in the dark. The
photocycle kinetics as monitored by the recovery of absorbance at 446 nm are identical to the kinetics to the CD signals.
Changes in tertiary structure upon signaling state formation were
probed using the strong near-UV CD signal of PYP at 270 nm. PYP
contains five Tyr residues and a single Trp side chain. Of these 6 groups, Tyr-76, Tyr-94, and Tyr-98 are largely exposed to solvent,
whereas Tyr-42, Tyr-118, and Trp-119 are fully buried. Therefore the
latter three residues are expected to be largely responsible for the CD
signal at 275 nm. The side chain of Trp-119 is packed between the
central antiparallel -sheet in PYP and its N-terminal two
-helices. The side chain of Tyr-118 is surrounded by the opposite
face of the central
-sheet in PYP and
-helix 5. Finally, the side
chain of Tyr-42 is part of the active site of PYP, hydrogen bonded to
the p-coumaric acid chromophore, and more than 8 Å removed
from both Tyr-118 and Trp-119. Thus, these three buried aromatic side
chains probe the tertiary structure in three distinct regions of
PYP.
Upon light activation, the CD signal at 270 nm is reduced by 66% and
becomes featureless, indicating a strong reduction in the tertiary
structure in PYP in the signaling state (Fig. 1, B and
D). The recovery of tertiary structure after photoexcitation occurs with the kinetics of the photocycle transition from the signaling state to the initial state of PYP. A loss of tertiary structure around the side chain of Trp-119 is in line with recent NMR
data, which indicate a reduction in structure in the two N-terminal -helices upon signaling state formation (18). The peak position of
the near-UV CD signal at 270 nm demonstrates that moreover the
environment of Tyr side chains becomes disordered upon photoactivation of PYP. Our results show that the signaling state of PYP maintains most
of its secondary structure, whereas its tertiary structure is greatly
diminished, providing strong support (19, 20) for the proposal that the
signaling state of PYP is a molten globule.
The exposure of hydrophobic patches upon formation of the signaling
state of PYP was investigated using the fluorescent probe ANS. This
compound binds specifically to clusters of hydrophobic groups, which
are highly indicative of molten globule states (19, 20). The binding of
ANS to such clusters results in an increase in its fluorescence quantum
yield. For all protein investigated in this respect, conversion to the
molten globule state, but not the fully unfolded state, results in an
increase in ANS fluorescence (18, 19). In the case of PYP the formation
of the signaling state leads to a significant (12%) increase in ANS
fluorescence (Fig. 2). Recovery of the
initial level of ANS fluorescence occurs with kinetics identical to
that of the PYP photocycle transition from the signaling state back to
the initial state of PYP. Thus, the ANS binding properties of the PYP
signaling state are those expected for a molten globule state.
|
A third characteristic of molten globule states is a significant
increase in hydrodynamic radius as a result of penetration of water
into the folded core of the protein. To directly investigate such
solvent penetration upon formation of the signaling state of PYP, the
quenching of intrinsic fluorescence from aromatic side chains by
acrylamide was investigated. The level of fluorescence quenching in the
initial state of PYP was found to be small (Fig. 3). Conversion of PYP to its signaling
state resulted in a large increase (2-fold) in fluorescence quenching,
demonstrating increased interaction of the buried aromatic side chains
with the solvent. Conversion of PYP to its fully denatured state by the
addition of GdmCl results in maximal exposure of aromatic amino acids
and increases the effectiveness of fluorescence quenching by acrylamide 6-fold. Comparison of the fluorescence quenching in the initial state
and native state of PYP with that in fully unfolded state indicates a
20% increase in solvent penetration upon signaling state
formation.
|
Because the signaling state of PYP exhibits all three properties
specific to the molten globule states of a large number of proteins, we
conclude that activation of PYP by light converts this receptor protein
into a molten globule. This provides the first example of
stimulus-induced partial unfolding of a receptor protein to a molten
globule state. Thus, the molten globule state is not only important for
understanding protein folding but also is of direct relevance to the
field of signal transduction. The following considerations (4, 22)
explain how light activation converts PYP to a molten globule state.
Absorbance of a photon by PYP results in the trans to
cis isomerization of the ionized p-coumaric acid
chromophore in PYP (3-6). This event triggers an intramolecular proton
transfer step from the protonated side chain of active site residue
Glu-46 to the PYP chromophore (4, 22). Both Glu-46 and the chromophore
in PYP are buried residues with anomalously shifted pK values. Changes
in the protonation state of such residues at pH extremes contribute
significantly to the acid and base denaturation of proteins in general
(23). In many cases, the acid-denatured state of a protein is in fact a
molten globule (24). Because light absorbance also causes a change in
the protonation state of Glu-46 and the chromophore, this is predicated
to trigger structural changes analogous to acid/base-induced
denaturation and therefore can result in the formation of a molten
globule. The stimulus-induced transient unfolding process in PYP can
provide this receptor protein with the conformational plasticity needed
for signaling state formation. Because PYP serves as a prototype for
the PAS domain family (9), it is expected that transient partial
protein unfolding is also involved in other proteins containing this
ubiquitous signaling module. The same mechanism may also function in
unrelated signaling systems. In view of the significant fraction of
regulatory proteins inferred to be unstructured (25), the mechanism of
transient partial protein unfolding from a fully folded state to a
molten globule state reported here may well be widely used in signal transduction. In this proposal the folding status of a regulatory protein depends on its signaling status. Changes in the input into the
signal transduction chain affect the folding state of the protein and
can thus result in signal relay.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Philippe Cluzel for many stimulating discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the American Cancer Society and the Cancer Research Foundation (to W. D. H.).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. Tel.: 773-834-3098;
Fax: 773-702-0439; E-mail: whoff@midway.uchicago.edu.
Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.C100106200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PYP, photoactive yellow protein; GdmCl, guanidinium chloride; pCA, p-coumaric acid; ANS, 8- anilinonaphthalene-1-sulfonate; PAS, Per-Arnt-Sim; FTIR, Fourier transform infrared; CD, circular dichroism.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Meyer, T. E., Yakali, E., Cusanovich, M. A., and Tollin, G. (1987) Biochemistry 26, 418-423[Medline] [Order article via Infotrieve] |
2. | Hoff, W. D., Van Stokkum, I. H. M., Van Ramesdonk, H. J., Van Brederode, M. E., Brouwer, A. M., Fitch, J. C., Meyer, T. E., Van Grondelle, R., and Hellingwerf, K. J. (1994) Biophys. J. 67, 1691-1705[Abstract] |
3. | Kort, R., Vonk, H., Xu, X., Hoff, W. D., Crielaard, W., and Hellingwerf, K. J. (1996) FEBS Lett. 382, 73-78[CrossRef][Medline] [Order article via Infotrieve] |
4. | Xie, A., Hoff, W. D., Kroon, A. R., and Hellingwerf, K. J. (1996) Biochemistry 35, 14671-14678[CrossRef][Medline] [Order article via Infotrieve] |
5. | Genick, U. K., Soltis, S. M., Kuhn, P., Canestrelli, I. L., and Getzoff, E. D. (1998) Nature 392, 206-209[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Perman, B.,
Srajer, V.,
Ren, Z.,
Teng, T.,
Pradervand, C.,
Ursby, T.,
Schotte, F.,
Wulff, M.,
Kort, R.,
Hellingwerf, K. J.,
and Moffat, K.
(1998)
Science
279,
1946-1950 |
7. | Hoff, W. D., Düx, P., Hård, K., Devreese, B., Nugteren-Roodzant, I. M., Crielaard, W., Boelens, R., Van Beeumen, J., and Hellingwerf, K. J. (1994) Biochemistry 33, 13959-13962[Medline] [Order article via Infotrieve] |
8. | Baca, M., Borgstahl, G. E. O, Boissinot, M., Burke, P. M., Williams, D. R., Slater, K. A., and Getzoff, E. D. (1994) Biochemistry 33, 14369-14377[Medline] [Order article via Infotrieve] |
9. |
Pellequer, J.-L.,
Wagner-Smith, K. A.,
Kay, S. A.,
and Getzoff, E. D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5884-5890 |
10. | Sprenger, W. W., Hoff, W. D., Armitage, J. P., and Hellingwerf, K. J. (1993) J. Bacteriol. 175, 3096-3104[Abstract] |
11. |
Jiang, Z.,
Swem, L. R.,
Rushing, B. G.,
Devanathan, S.,
Tollin, G.,
and Bauer, C. E.
(1999)
Science
285,
406-409 |
12. |
Taylor, B. L.,
and Zhulin, I. B.
(1999)
Microbiol. Mol. Biol. Rev.
63,
479-506 |
13. | Borgstahl, G. E. O., Williams, D. R., and Getzoff, E. D. (1995) Biochemistry 34, 6278-6287[Medline] [Order article via Infotrieve] |
14. | Düx, P., Rubinstenn, G., Vuister, G. W., Boelens, R., Mulder, A. A., Hård, K., Hoff, W. D., Kroon, A. R., Crielaard, W., Hellingwerf, K. J., and Kaptein, R. (1998) Biochemistry 37, 12689-12699[CrossRef][Medline] [Order article via Infotrieve] |
15. | Van Brederode, M. E., Hoff, W. D., Van Stokkum, I. H. M., Groot, M. L., and Hellingwerf, K. J. (1996) Biophys. J. 71, 365-380[Abstract] |
16. | Hoff, W. D, Xie, A., Van Stokkum, I. H. M., Tang, X.-J., Gural, J., Kroon, A. R., and Hellingwerf, K. J. (1999) Biochemistry 38, 1009-1017[CrossRef][Medline] [Order article via Infotrieve] |
17. | Rubinstenn, G, Vuister, G. W., Mulder, F. A. A., Düx, P. E., Boelens, R., Hellingwerf, K. J., and Kaptein, R. (1998) Nat. Struct. Biol. 5, 568-570[CrossRef][Medline] [Order article via Infotrieve] |
18. | Craven, C. J., Derix, N. M., Hendriks, J., Boelens, R., Hellingwerf, K. J., and Kaptein, R. (2000) Biochemistry 39, 14392-14399[CrossRef][Medline] [Order article via Infotrieve] |
19. | Ptitsyn, O. B. (1992) in Protein Folding (Creighton, T. E., ed) , pp. 243-300, W. H. Freeman and Co., New York |
20. | Arai, M., and Kuwajima, K. (2000) Adv. Prot. Chem. 53, 209-282[Medline] [Order article via Infotrieve] |
21. | France, R. M., and Grossman, S. H. (2000) Biochem. Biophys. Res. Commun. 269, 709-712[CrossRef][Medline] [Order article via Infotrieve] |
22. | Xie, A., Kelemen, L., Hendriks, J., White, B. J., Hellingwerf, K. J., and Hoff, W. D. (2001) Biochemistry 40, 1510-1517[CrossRef][Medline] [Order article via Infotrieve] |
23. | Honig, B., and Yang, A.-S. (1994) Adv. Prot. Chem. 46, 27-58 |
24. | Fink, A. L., Calciano, L. J., Goto, Y., Kurotsu, T., and Palleros, D. R. (1994) Biochemistry 33, 12504-12511[Medline] [Order article via Infotrieve] |
25. | Wright, P. E., and Dyson, H. J. (1999) J. Mol. Biol. 293, 321-331[CrossRef][Medline] [Order article via Infotrieve] |