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PAS Domain Receptor Photoactive Yellow Protein Is Converted to a Molten Globule State upon Activation*

Byoung-Chul Lee, Paula A. Croonquist, Tobin R. Sosnick, and Wouter D. HoffDagger

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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].

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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.


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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 = infinity  (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 beta -sheet in PYP and its N-terminal two alpha -helices. The side chain of Tyr-118 is surrounded by the opposite face of the central beta -sheet in PYP and alpha -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 alpha -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.


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Fig. 2.   Formation of the pB photocycle intermediate results in the exposure of hydrophobic patches as probed by ANS fluorescence. Changes in ANS fluorescence (trace 2) were measured during the recovery of the initial pG state of PYP after photoactivation, as monitored by fluorescence from the chromophore in PYP (trace 1), and described by a monoexponential decay.

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.


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Fig. 3.   Formation of the signaling state of PYP results in increased quenching of fluorescence from aromatic side chains, indicating enhanced solvent penetration. Fluorescence from PYP at pH 4.0 was determined for the initial state of PYP (filled circles), for the signaling state of PYP (open circles) by essentially quantitative photoconversion of PYP to its signaling state, and for the unfolded state of PYP (filled squares) in the presence of 4.0 M GdmCl. The Stern-Volmer constants K for the initial state, signaling state, and fully unfolded state were found to be 1.7, 3.4, and 10.3 M-1, respectively.

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.

Dagger 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.