ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 271, No. 1, May 15, pp. 200-205,1989

Fluorescent Derivative of Cysteine-I 0 Reveals Thyroxine-Dependent Conformational Modifications in Human Serum Prealbumin’

GUSTAV0 GONZALEZ

Instituto de Q&mica, Facultad de Ciencias Bhicas y Matemhticas, Universidad Catdlica de Valparako, Casilla 4059, Valparako, Chile

Received October 11,1988, and in revised form January 17,1989

Fluorescence studies on the N-(iodoacetyl)-N’-(5-sulfo-l-naphtyl)ethylenediamine- labeled cysteine-10 residue of human prealbumin were carried out to detect conforma- tional changes induced by the binding of the ligand thyroxine to the two structurally identical binding sites. A red shift of the spectrum was observed and the total change was confined to the first ligand. This was interpreted as resulting from a conformational change which increases the exposure of the fluorescent probe moiety. Thyroxine also alters the effect of the collisional quencher, acrylamide, confirming the greater exposure of the probe. This modification in structure is associated with changes in relaxation time which indicate that when thyroxine is bound there is an increase in the rotational freedom of the segment or domain of prealbumin which contains the fluorescent probe. 8 1989 Academic Press, Inc.

Prealbumin (PA)2 is one of three plasma proteins responsible for the transport of the hormone thyroxine in man (1). PA is a very stable tetramer (2,3) whose structure and number of binding sites have been de- termined. X-ray crystallography (4,5) and amino acid sequencing (6, 7) have estab- lished that PA consists of four identical subunits and that two identical binding sites for thyroxine are located in a central channel formed by the association of the four monomers.

Two moles of hormone are bound per mole of protein with binding constants that are 2 orders of magnitude different for each mole (8, 9). The binding behavior of the two hormone molecules has been inter- preted in terms of two identical sites with negative cooperativity (8). Subunit pro-

1 This work was supported by Direccibn General de Investigacibn, Universidad Catblica de Valparaiso.

‘Abbreviations used: PA, prealbumin; 1,5- IAEDANS, N-(iodoacetyl)-N’-(5-sulfo-l-naphtyl)- ethylenediamine; AEDANS, N-acetyl-N’-(5-sulfo-l- naphtyl)ethylenediamine.

teins may develop cooperativity through a conformational change on binding a ligand on one subunit, altering the interactions between subunits and consequently the binding affinity for subsequent ligands. PA is exceptionally resistant to denaturation; it is totally resistant to urea; and at neu- tral pH, guanidinium hydrochloride con- centrations greater than 6 M are required to denature the molecule (10, 11). The in- ternal organization of the molecule does not appear to change between pH 3.5 and pH 12 nor is it dissociated into subunits by strong acid, alkali, or 0.1% sodium dodecyl sulfate (3). This extreme stability makes it rather difficult to expect conformational changes in the protein; however, the study reported here reveals that structural al- terations in PA are produced upon binding of thyroxine and are confined to the bind- ing of the first ligand.

PA has only one cysteine (Cys-10) per identical subunit and its location is near the entrance of the central channel of the molecule. Labeling with the fluorescent probe N-acetyl-hr’-(5-sulfo-l-naphtyl)-

0003-9861/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

200

CONFORMATIONAL CHANGES IN PREALBUMIN 201

ethylenediamine (AEDANS), provided a unique site for examining the conforma- tional changes produced by the binding of the thyroxine to PA.

EXPERIMENTAL PROCEDURES

Materials

N-(Iodoacetyl)-N’-(5-sulfo-l-naphtyl)ethylenedia- mine (1,5-IAEDANS) and L-thyroxine were obtained from Sigma. Chemicals not specifically mentioned were of reagent grade. Acrylamide was recrystallized from ethyl acetate. Human serum prealbumin, gener- ously provided by Drs. Heide and Haupt, Behringw erke, A.G., Marburg/Lahn, German Federal Repub- lic, was obtained through the Molecular Biophysics Laboratory, Oxford University, England. It was fur- ther purified by DEAE-Sepharose anion-exchange chromatography and Sephadex G-100 gel filtration (12). A single band was observed by sodium dodecyl sulfate-gel electrophoresis at pH 8.9.

Methods

Preparation of the jluwescent congugate. PA was incubated with 1,5-IAEDANS for 4 h at room temper- ature in the dark. The reaction buffer was 20 rTIM sodium phosphate, pH 6.5. The molar ratio of added 1,5-IAEDANS to protein was approximately 2:l. The derivatized protein was then freed from low-molecu- lar-weight reactants by filtration through a column of Sephadex G-25. The circular dichroism of PA and AEDANS-PA was measured using a Cary 60 spectro- polarimeter equipped with a Model 6001 CD attach- ment.

Fluorescence. Fluorescence spectra and intensity were obtained with a Spex Fluorolog photon counting spectrofluorometer at 20°C; excitation and emission wavelengths for AEDANS were 340 and 480 nm, re- spectively. Fluorescent measurements were per- formed at right angle and front-face illuminations (a facility provided by the Spex fluorometer) to check for possible inner filter effects. The only difference found between the two orientations was a decreased sensi- tivity with the front-face arrangement.

Polarization of $uurescence. The Spex Fluorolog spectrofluorometer was modified to measure polar- ization of fluorescence with the insertion of polariz- ers. The excitation and emission wavelengths were set for 340 and 480 nm, respectively. Polarization (P) is expressed as (V - GH)/( V + GH) where V and H are the vertically and horizontally polarized intensi- ties, respectively, and G is equal to the ratio V/Hwith the polarizer oriented horizontally. The polarization data were analyzed by the Perrin equation,

l/P - l/3 = (l/PO ~ l/3)(1 + 37/p)

where T is the lifetime and p the harmonic mean re- laxation time.

Fluorescence lifetime measurements. Lifetime de- terminations were done with a cross-correlation flu- orometer (SLM Instruments, Urbana, IL).

Acrylamide quenching aaalysis. Acrylamide fluo- rescence quenching data were analyzed according to the Stern-Volmer equation,

where r0 and 7 are the lifetimes of the Auorophore in the absence and presence of quencher (Q); K,, is the dynamic quenching constant.

RESULTS

AEDANS-PA Churacterixation

The number of moles of conjugated AE- DANS (per mole of subunit) was 0.4, as de- termined by the absorbance at 337 nm and using 6100 as the molar extinction coeffi- cient (13). That AEDANS was uniquely bound to the thiol moiety of PA was ascer- tained using Ellman’s reagent (14). Amino acid analysis indicated the presence of car- boxymethyl-Cys as the sole alteration in the amino acid composition. The CD mea- surements showed that in no case did the presence of the fluorescent probe appear to affect the circular dichroism spectra of PA in the near- and far-ultraviolet spectral re- gions. PA derivatization with AEDANS did not alter its binding behavior toward thyroxine as measured by quenching of tryptophan fluorescence (15).

A EDA NS Fluorescence

The interpretation of alterations in the fluorescence spectra of AEDANS-PA due to thyroxine binding can be facilitated by knowledge of the three-dimensional struc- ture of PA. The molecular model shows that the derivatized cysteinyl residue (Cys-10) is located close to the central channel of the tetramer (5). This position is ideal for detecting conformational changes induced by thyroxine at the bind- ing site (Fig. 1).

The fluorescence intensity of AEDANS- PA is strongly diminished and the emis- sion peak is red-shifted to 480 nm from 462

202 GUSTAV0 GONZALEZ

FIG. 1. A schematic drawing of the four identical monomers of prealbumin associated to form the bind- ing channel. The hatched areas show the location of the cysteine residue in each subunit. The DE loop is also shown. Drawn from the X-ray data of Blake et UL (4). Adapted from Somack et al. (16).

nm when thyroxine is bound to the protein (Fig. 2). This effect does not arise from di- rect interaction of thyroxine with the AE- DANS moiety since addition of thyroxine to the model compound AEDANS-Cys showed no effect on the fluorophore emis- sion.

The emission maximum shifts continu- ously to the red as the extent of site satura- tion increases, while the fluorescence in- tensity decreases (Figs. 3 and 4). These effects are observed only until near satura- tion of the first site is reached. No further

AEDANS -PA

01

450 500 550

WAVELENGTH (nm)

FIG. 2. Effect of thyroxine (T4) on the fluorescence emission spectrum of AEDANS-PA. AEDANS-PA (15.0 pM) in 0.05 M Tris, 0.1 M NaCI, at pH 8.6, and 20°C. AEDANS-PA-T,, mole ratio = 1.70. The excita- tion wavelength was 340 nm.

MOLE RATIO T,/PA 0.5 1.0 1.5 20

I I I I

375 1125 188 26 3 [TOTAL Ti.1 Mx106

FIG. 3. Fluorometric titration of AEDANS-PA with thyroxine. The ordinate is the fluorescence in- tensity at the emission maximum obtained at differ- ent mole ratios. The lower abscissa is the total thy- roxine added to the AEDANS-PA solution. The upper abscissa is the number of moles of thyroxine bound per mole of AEDANS-PA. The excitation wavelength was 340 nm. AEDANS-PA solutions were as in Fig. 2.

effect is observed with saturation of the second site. The two affinity constants (Kl = 3.2 X lo8 M-‘; K2 = 5.0 X lo6 M-‘) obtained by fluorescence procedures (9) were used to calculate the number of thyroxines bound per mole of PA.

The size of the observed effects reflects a rearrangement of the protein, since the shift extent corresponds to that observed when l,&IAEDANS dissolved in ethanol (LX = 460 nm) is compared to 1,5-IAE- DANS dissolved in 80% ethanol in water (&3X = 485 nm) (13), suggesting a change in the polarity environment of AEDANS when thyroxine is bound to PA. The ob- served decrease in AEDANS-PA fluores- cence intensity also reflects the same de- scribed phenomenon; however, its magni- tude is much larger than expected for just a polarity effect (13). Probably a quenching effect by neighboring groups is also in- volved.

Acrylamide Quenching The relative accessibility of the AE-

DANS group to the solvent can also be as-

CONFORMATIONAL CHANGES IN PREALBUMIN 203

MOLE RATIO T&/PA

05 10 1.5 20

I I I I I

375 11 25 188 263

[TOTAL TLl MxlO'

FIG. 4. Fluorometric titration of AEDANS-PA with thyroxine. The ordinate is the wavelength shift from 462 (emission maximum in the absence of thy- roxine) to the value obtained at different mole ratios. The lower abscissa is the total thyroxine added to the AEDANS-PA solutions. The upper abscissa is the number of moles of thyroxine bound per mole of AE- DANS-PA. The excitation wavelength was 340 nm. AEDANS-PA solutions were as in Fig. 2.

certained by measuring the ability of the polar nonionic quencher acrylamide to quench its fluorescence intensity. The ad- dition of acrylamide decreased the lifetime of the AEDANS attached to PA in accor- dance with the Stern-Volmer law, as shown in Fig. 5. The form exhibited in the plot is linear, indicating a simple colli- sional mechanism for the quenching pro- cess. The quenching constant, KS,, is the slope of the acrylamide quenching plot, and its value increases from 12 X 10e7 to 20 X 1O-7 M-’ s-l when thyroxine is bound, indicating that binding of the hormone makes AEDANS more easily quenched and therefore is more in contact with the polar solvent molecules. This result is con- sistent with the previous finding that thy- roxine promotes a change in the polarity environment of AEDANS. Quenching by acrylamide cannot be interpreted as a di- rect resonance energy transfer phenome- non since the acrylamide absorption spec- trum does not display Fiirster overlap with the fluorescence emission spectrum of AE- DANS-PA.

Polarization of Fluorescence The rotational diffusion coefficient, i.e.,

the relaxation time of PA, was evaluated from the dependence of the polarization of AEDANS fluorescence on the viscosity of the solution. A lifetime of 15 ns was found at 20°C for the AEDANS group of labeled PA, and this value did not change signifi- cantly when thyroxine was bound.

The increase in the AEDANS polariza- tion of PA, and of PA with bound thyrox- ine with addition of sucrose is plotted in Fig. 6 according to the Perrin equation. The binding of thyroxine resulted in a de- crease in polarization. Relaxation times of 126 and 69 ns at 20°C were calculated from the data in Fig. 6 for PA and bound thyrox- ine PA, respectively. The smaller relax- ation time of the latter suggests that bind- ing of thyroxine results either in a de- crease in the asymmetry of an equivalent ellipsoid of revolution, i.e., a decrease in the axial ratio of the PA molecule, or in an increase in the rotational freedom of the segment or domain of PA which contains the AEDANS chromophore.

I I I I

0 0.1 02 03

[ACRYLAMIDEI,M

FIG. 5. Stern-Volmer plots of quenching by acryl- amide of the fluorescence of AEDANS-PA (0) and AEDANS-PA-thyroxine (TJ. mole ratio = 1.70 (A). AEDANS-PA solutions were as in Fig. 2. Acrylamide was added from a concentrated stock solution and the fluorescence intensity was corrected for dilution and the inner filter effect.

204 GUSTAV0 GONZALEZ

I I I

44 0 1 2 3

T/q&

FIG. 6. Perrin plots of the polarization of the AE- DANS fluorescence of AEDANS-PA (0) and AE- DANS-PA-thyroxine (TJ, mole ratio = 1.70 (a). AE- DANS-PA solutions were as in Fig. 2; T = 20°C. The wavelengths of excitation and emission were 340 and 480 nm, respectively. The lifetimes in sucrose solu- tions were obtained from the change in fluorescence with sucrose, assuming proportionality. The fluores- cence of AEDANS-PA decreased by 14% between 0 and 45% sucrose while that of AEDANS-PA-thyrox- ine decreased by 5%.

DISCUSSION

PA is a tetrahedral tetramer of struc- turally identical subunits (2, 3, 17). Each subunit is composed of 4-stranded p- sheets. A dimer is formed by hydrogen bonds between two subunits. The associa- tion of two dimers forms a 16-stranded p- cylinder 55 A long (4, 5). The channel formed by the two dimers has a diameter of about 8 A, except at its center where a local constriction is found. The two thyrox- ine binding sites are located in the cylin- drical channel. Since the two sites are re- lated by twofold symmetry they are iden- tical.

The only amino acid residue in PA which was found to carry covalently bound AE- DANS was Cys-10. According to the three- dimensional structure of PA this residue is located close to the cylindrical channel, specifically, if the dimer is considered, at

the start of strands A and A’ in sheet I (4). The tetramer is assembled by opposing the two sheets I of both dimers positioning cysteine near the entrance of the binding channel.

The changes in the AEDANS fluores- cence spectrum produced by the binding of thyroxine on the first site of PA reveal that a conformational change takes place with binding. The red-shift in the spectrum in- dicates a rearrangement of the AEDANS moiety, either by its greater exposure to the solvent or by new interactions with more polar residues. Absorption studies of PA aromatic chromophores led Irace and Edelhoch (18) to propose that thyroxine binding produces a minor conformational change which involves an increase in the polarity of their environments.

The quenching studies of AEDANS emission by acrylamide also support the interpretation of a conformational change leading to a greater exposure of AEDANS to the solvent. The polarization of fluores- cence clearly shows changes with thyrox- ine binding which can be interpreted in terms of a modification in PA molecular di- mensions or in the degree of exposure of AEDANS to the solvent.

The various methods employed in this study support the evidence that thyroxine binding produces structural alterations in PA that necessarily result in an increase in the exposure of the AEDANS probe which is located in the outer part of the thyroxine binding sites. It is also evident that these alterations are produced upon the binding of thyroxine to the first site, giving rise to the difference in binding con- stants between the two sites. These inter- pretations can be correlated with the re- sults of Blake and Oatley (19), who, on the basis of X-ray studies of PA crystals, de- tected large displacements in the outer part of the thyroxine binding site which are apparently obligate to give the ligand access to the inner part of the binding site. These displacements might increase the environment polarity of the AEDANS moiety through its greater exposure to the solvent and/or through its approximation to the 11-residue DE loop, joining the monomer D and E strands, which contains several polar side chains (Fig. 1) (4,5).

CONFORMATIONAL CHANGES IN PREALBUMIN 205

ACKNOWLEDGMENTS

I thank Dr. Alejandro Paladini, Jr., for the fluores- cence lifetime measurements and Dr. Adelaide Fal- joni-Alario for making available the CD spectropo- larimeter. I also thank Dr. Colin Blake for the gift of human serum prealbumin.

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