Quenching-resolved emission anisotropy: a steady state fluorescence method to study protein dynamics

;kmmld AND

Journal of Photochemistry and Photobiology

ELSEVIER

B:ItiGLOGY

B: Biology 27 (1995) 55--60

Quenching-resolved emission anisotropy: a steady state fluorescence method to study protein dynamics p

Zsuzsa Lakos, Agnes Szarka, Lfiszl6 Koszorfis, B61a Somogyi Department of Biophysics, Medical Unive~ity School of P~cs, P.O. Box 99, H-7601 P~cs, Hungary Received 8 March 1994; accepted 20 July 1994

Abstract Fluorescence techniques can be used to obtain information about biological objects in a non-destructive manner. O n e of

these techniques is fluorescence quenching which involves a decrease in the fluorescence emission of a biological object by externally added quenchers. Quencher molecules produce two kinds of quenching: static and dynamic. Static quenching occurs due to encounter pair formation between quencher and fluorophore molecules, while dynamic quenching requires bimolecular collisions. Unless one of the mechanisms can be neglected, steady state quenching experiments cannot provide information on the contributions of the two processes. However, time-resolved experiments are sensitive only to the dynamic process, and thus provide selective information about the relative motion of the quencher and fluorophore. Since the two quenching events are controlled by different physicochemical parameters, it is necessary to resolve them. In this paper, we describe a steady state method to resolve the static and dynamic quenching constants (rather than time-resolved techniques). Our method is based on the simultaneous determination of the fluorescence intensity and emission anisotropy data and can be regarded as the further development of quenching-resolved emission anisotropy (QREA). Since the steady state anisotropy and fluorescence lifetime are inversely related, by determining the steady state fluorescence anisotropy, changes in the fluorescence lifetime (and hence the dynamic quenching process) can be monitored (if other parameters influencing the anisotropy remain constant). We present a theoretical description of the method, computer simulations testing its accuracy and results of model experiments with pyridoxamine-phosphate-labelled lysozyme and acrylamide. By changing the external viscosity, we obtained data on the theoretical inverse relationship between the dynamic quenching constant and viscosity. The application conditions are also discussed. Keywords: Fluorescence quenching; Dynamic quenching; Fluorescence anisotropy; Steady state fluorescence

1. Introduction

Both the intrinsic fluorescence of biological macromolecules and the fluorescence emitted by different dyes attached to macromolecules are extremely sensitive to environmental parameters (such as the temperature, viscosity, ionic strength, pH, etc.). This is mainly due to the altered dynamic behaviour of the neighbouring protein matrix around the fluorophore. This feature makes fluorescence a powerful tool to obtain information on biological systems. One particular method deals with the quenching of the emitted fluorescence by external quenchers. The quenchers are molecules which, by definition, can quench the fluorescence whenever they are in contact with the excited fluorophore. The ratio of the fluorescence lifetimes in the absence (~'o) and presence (~-) of quencher molecules is described

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by the classical Stern-Volmer equation [1-3]

T

=

1 + k+ ~'o[O]

(1)

where k+ is the bimolecular rate constant characteristic of the collision rate between the fluorophore and the quencher molecules (in the case of a weak quencher, when the quenching efficiency is smaller than unity, the second term in Eq. (1) contains a corresponding factor, 3', accounting for this feature [4]). From the usual size of the applied quencher molecule and the fact that to falls into the nanosecond time regime, k÷ reports on the relative transport rate (e.g. diffusion) of the fluorophore and the quencher. However, if the quenching takes place in the interior of a protein, k÷ is related to the internal dynamics of the protein matrix.

56

Z. Lakos et al. / J. Photochem. Photobiol. B: Biol. 27 (1995) 55--60

As shown by Eq. (1), k+ is readily available from lifetime measurements carried out in the absence and presence of quencher molecules. Due to the costly instrumentation and time consumption of these experiments, they are often replaced by simpler steady state measurements. This is based on the fairly good assumption that the lifetime and the steady state fluorescence intensity are proportional to each other. In this case, the corresponding form of Eq. (1) is F0 = 1 +k+ to[Q] = 1 +Ksv[Q] F

(2)

where/70 and F are the appropriate steady state fluorescence intensities and Ksv is the Stern-Volmer constant. Eq. (2) is valid when all the fluorescence is quenchable. When a fraction of the fluorescence cannot be quenched (even at infinitely high quencher concentration), the modified form of Eq. (2) is applicable [5] Fo Fo-F

1 1 1 + a aKsv [Q]

(3)

where a is the accessible fraction of the protein fluorescence. With regard to the application of steady state measurements, a serious problem emerges in how to obtain the value of the bimolecular quenching constant, k+. In the bimolecular reaction of quencher and fluorophore molecules, quenching occurs on collision. This collision, due to the "cage" effect [6], should happen during encounter pair formation. Such an encounter complex may exist at the time of excitation. This leads to the existence of "dark" complexes, in which the excitation energy is taken away by the quencher molecule in the complex at the time of excitation. As a consequence, the steady state fluorescence intensity is decreased by both the quencher molecules in dark complexes and by those colliding with the fluorophore during the natural excited state lifetime, r0. Accordingly, steady state measurements are sensitive to both static (due to the presence of dark complexes) and dynamic (collisional) quenching, while lifetime measurements are related only to the dynamic quenching process. Therefore Eq. (2) can only be regarded as an approximation, which is valid only when the static quenching contribution is much smaller than that of dynamic quenching. The form of Eq. (2), which accounts for the presence of both static and dynamic quenching, is [2-4] Fo = (1 +Ksv[Q])(1 + V[Q]) F

ponents, the Fo/F vs. [Q] function has upward curvature, which provides the possibility to separate (but not identify) the two components by mathematical analysis of the curve [4,7,8]. In many cases, however, this curvature is absent, even in the presence of a static contribution [2], when Ksv is much larger than V (in the case of a long-lived fluorophore or a small, rapidly diffusing quencher) [4]. Even if V is not negligible, at low [Q] values the second-order term in [Q] can be disregarded in Eq. (4) [2,3]

(4)

where V is the static quenching constant with many possible interpretations (see, for example, Refs. [3,7]). The form of Eq. (4) shows that, if there is a significant contribution from both the static and dynamic eom-

F

= 1 + (Ksv + V)[Q]

(5)

Eq. (5) enables us to obtain the sum (Ksv+ 10 from the initial slopes by measuring the steady state fluorescence intensities. The appropriate form of F_,q. (3) then becomes Fo

1

Vo-V

=

-

+

1 1 (Ksv+V) [0]

(6)

In the next section, we describe a steady state fluorescence approach capable of resolving the static and dynamic quenching constants by polarized fluorescence intensity measurements at different quencher concentrations. 2.

Theory

As noted in the preceding section, the sum of Ksv and Vcan be determined by measuring the fluorescence intensities at different quencher concentrations. The fact that Ksv and V are related to different physical parameters of the system (e.g. Ksv depends on the viscosity of the medium while V is independent of it) means that it is necessary to determine their values independently. In general, static and dynamic quenching can be distinguished by their different dependence on temperature and viscosity [3], or preferably by lifetime measurements. In order to replace the costly instrumentation required by lifetime measurements, we have developed a new approach to determine the value of Ksv selectively for certain cases. The method is based on the fact that the emission anisotropy (like the dynamic quenching process) depends on the excited state lifetime, and therefore steady state measurements of the emission anisotropy at different quencher concentrations can provide information about the change in the fluorescence lifetime. When fluorophore excitation is carried out by polarized light, the anisotropy (r) of the emitted fluorescence is related to the actual lifetime of the fluorophore by the Perrin equation [9] r=~o

1(1

kT ) + ~-~h r

(7)

57

Z. Lakos et aL / J. Photochem. Photobiol. B." Biol. 27 (1995) 55--60

r~, of Eq. (7) refers to the limiting anisotropy, while ~" is the fluorescence lifetime, 77 is the viscosity of the medium, Vh is the hydrated volume of the rotating species carrying the fluorophore, k is the Boltzmann constant and T is the absolute temperature. The form of Eq. (7) shows that 1/r is linearly related to the fluorescence lifetime of the fluorophore. Con.,equently, it is related to the dynamic quenching process and is insensitive to static quenching. Accordingly, the t:mission anisotropy, by reporting exclusively on the dynamic quenching process, seems to be an appropriate parameter for determining the value of Ksv provided that other parameters in Eq. (7) remain constant on addition of the quencher. We should note here that, in cases where the rotation of the fluorophore is composed of two (or more) independent rotations, the right-hand side of Eq. (7) becomes more complicated. Computer simulations have ,&own, however, that, even in this case, the linear :-elationship between 1/r and ~-holds over a wide range of parameters determining the rotational motion of the ituorophore [10]. In the following section, we restrict our discussion :o cases where the fluorescence emission is fully quench~ble (a-- 1) (homogeneous fluorescence population). ~uch systems include small fluorophores or protein -nolecules with surface-exposed tryptophan (Trp) groups 3r external labels. When a is significantly smaller than anity, the procedure outlined below is not applicable. To proceed further, Eq. (1) can be inserted into Eq.

7) 1

- = -

1[1 roL

+

kT To ] nVh 1 + Ksv[Q]

(8)

where % is the lifetime of the fluorophore in the absence 3f the quencher. Using r, and [Q,] as reference values of r and [Q]. we can write the expression of 1/r,-1/r

A

= l+Ksv[Or] + A

(12) AKsv

A[Q]

we obtain a linear relationship between the reciprocals of A1/r and A[Q]. By forming the ratio of the slope s to the intercept i of this linear curve, we obtain s 1 + [O,] i Ksv

(13)

Eq. (13) can be used to obtain the value of the dynamic quenching constant Ksv since [Q,] is known ([Qr] can be zero or some other value).

3. Accuracy of the method The application of Eqs. (12), (13) and (14) (see later) requires a further transformation of the measured polarized intensity data leading to an increase in the error in the value of Ksv. Thus it is necessary to test the effect of accumulating experimental errors on Ksv. The accuracy of the method was tested by computer simulations: the theoretical values of Ksv and the limiting anisotropy ro were chosen as 6 M - ~ and 0.4 respectively. The calculated values of Ksv and the corresponding errors were derived from 15 independent runs, where the concentration of the quencher and the fluorescence intensity contained the error introduced by the random number generator of the computer (see Fig. 1). From the figure, we can conclude that, assuming an average experimental error (approximately 1%) and an initial anisotropy value in the range 0.05-0.3, the error in Ksv determined by the method is comparable with the error of classical quenching experiments. Eq. (11) shows that the anisotropy range covered by the measurements (A1/r) is greater when the product of Ksv[AQ] is greater (if [Qr]--O, then [AQ] = [Qmax]).

3S 150

1

1

kTro r

1

,r

r = ro~TVhL1+ K s v [ Q r ]

1

]

1

1 +Ksv[Q] = A -r

(9)

Using the notations

Ksv (go)

(10)

the resulting form of Eq. (9) is

A 1 =A Ksva[Q] r (1 + Ksv[Q~]) 2 + Ksva[Q](1 +Ksv[Qr]) Then taking the reciprocal of both sides

~,

t

T

75

and A[QI = [Q] - [Q~]

[

T

kTro ro~lV,

5o oo

I 0.4

i o Z

i 03

04

r(IQl=0)

(11)

Fig. 1. Simulated average value of Ksv and the corresponding error as a function of the initial anisotropy of the fluorophore (measured in the absence of quencher). The limiting anisotropy is ro=0.4; the pipetting error plus the error in the determination of the fluorescence intensity is 1%; (100%ffi6 M-I).

58

Z. Lakos et al. / J. Photochem. Photobiol. B: Biol. 27 (1995) 55--60

While the values of Ksv and [Qm~,] fall in a welldefined range, we examined the effect of the product Ksv[Qm~,] on the error in Ksv as a function of the initial anisotropy of the fluorophore. According to computer simulations, the error in Ksv is dominated significantly by the emission anisotropy of the fluorophore in the absence of the quencher and shows a negligible dependence on the product Ksv[Qm~] in the range 10-4).1 (data not shown).

4. Materials and methods

Pyridoxamine-phosphate (PMP) conjugated to hen egg-white lysozyme enzyme (Sigma, USA) (fluorophore), acrylamide (Sigma, USA) and potassium iodide (KI) (Merck, Germany) (quenchers) were used. The labelling of lysozyme was performed by the method of Churchich [11] applying pyridoxale-5'-phosphate (Fluka, Germany) and NaBH4 (Sigma, USA) as reducing agent. The labelling ratio was determined spectrophotometrically according to Churchich [11] and Boyer [12], and was found to be PMP:lysozyme--1.3:l. Measurements were carried out using 50 mM phosphate buffer (pH 7.0). Glycerol (Sigma, USA) was used as viscogenic agent. The relative viscosities of the glycerol solutions were taken from Ref. [13]. According to viscosity measurements with an Ostwald viscometer, applying bovine serum albumin (Reanal, Hungary) as reference protein, the presence of protein at the concentration used in the experiments has no influence on the relative viscosity of glycerol solutions compared with the values given in Ref. [13]. Fluorescence measurements were carried out at 20.0 °C on a Hitachi-Perkin-Elmer MPF 4 spectrofluorometer equipped with a thermostatically controlled cell holder and polarizers. Excitation and emission wavelengths were 325 and 390 nm, with 4 and 8 nm slits respectively. The absorbance of the protein samples was kept below 0.05 at both the excitation and emission wavelengths. Data were collected by an IBM PC/XT connected to the fluorometer through an A/D converter and were corrected for dilution on titration of the sample. The anisotropy of the fluorescence was determined

and emission light beams respectively. Results were evaluated by applying the modified Stern-Volmer plot (Eq. (6)) as well as the quenching-resolved emission anisotropy (QREA) equation (Eq. (12)). The fluorescence intensity was evaluated as F=Ivv + 2GIvH

(15)

and corrected for the dilution effect and the absorbance of the exciting light due to the changing amount of acrylamide according to Ref. [14]. The lysozyme-bound PMP fluorescence (and thus the fluorescence lifetime) was not affected by the presence of glycerol (checked by independent measurements).

5. QREA of lysozyme-bound PMP

After testing the accuracy of the method by computer simulations, it was further tested by quenching experiments with a simple system containing a solvent-exposed fluorophore (PMP) covalently attached to lysozyme and KI as ionic and acrylamide as neutral quenchers. According to experiments with KI, linear Stern-Volmer plots were obtained, indicating the presence of a single class of fluorophores with equal accessibility to the quencher (data not shown). Figs. 2 and 3 show the experimental results with acrylamide. As shown in Fig. 2, the plot of Fo/(Fo-F) vs. 1/[Q] gives a straight line. From the slope of this curve, the sum of Ksv and V can be determined (see Eq. (6)). For the PMP-labelled lysozyme-acrylamide quenching system (Ksv + V) was found to be 4.0 M-1, which is consistent with other dynamic quenching data obtained with acrylamide [15]. The value of Ksv was obtained from a different plot. According to Eq. (12), the [A(1/r)]-1 values were plotted against 1/A[Q] (see Fig. 3) and, from the parameters of the resulting curve, Ksv was determined (Eqs. (12) and (13)) (Ksv = 1.7

4-

Fo/~F 3-

as

Ivv - GIv~ I w + 2GIw-i where

G

=

IHv IHH

(14)

1

2

3

I/[Q]

where I denotes the fluorescence intensity; the first and second subscripts are related to the positions of the polarizer (H, horizontal; V, vertical) in the excitation

4

5

6

M -~

Fig. 2. Quenching of the PMP fluorescence of PMP-lysozyme conjugate by acrylamide in the presence of 30% (w/w) glycerol. Lehrer plot of the steady state quenching data.

59

Z. Lakos et aL / J. Photochem. Photobiol. B: BioL 27 (1995) 55--60 18 16 ¸ 14• 12In const 108~ 06~

4,

I/A(]/r)

3

25J

,&,~ / ~ -

.0,~i -04~

~ c

-o.6~

'01

.

0

1

.

.

2

.

3

4

I/[Q]

5

6

"'able 1 Dynamic (Ksv) and static (V) quenching constants determined by the quenching of lysozyme-bound PMP fluorescence by acrylamide ~n the presence of varying glycerol content

0 :0

i:0 •,0

0

.

01

M"1

t:ig, 3. Change in the emission anisotropy data of PMP in PMPlabelled lysozyme as a function of I/[Q] in the presence of 30% {w/w) glycerol.

Glycerol % (w/w))

-o.sl

Relative viscosity

Ksv + V (M -j )

Ksv (M - j )

V (M -j)

1 1.288 1.734 2.453

4.0 3.8 2.9 2.2

1.7 1.5 0.6 0.9

2.3 2.3 2.3 1.3

!vl- 5). From these data, the value of the static quenching constant can be obtained (V=2.3 M-S). Quenching experiments were performed with solutions of different viscosity in order to influence the dynamic quenching process selectively. Table 1 summarizes the results. The dynamic quenching constant J~sv, containing the relative transport rate of the mol<~cules, shows an inverse proportional dependence on l he medium viscosity as predicted by theory. On the other hand, the value of the static quenching constant i falls in the range suggested for the affinity of ;tcrylamide to proteins [15] and is unaffected by the ~xternal viscosity (see Fig. 4). The above data demonstrate that, by the application of this approach, the contributions of static and dynamic mechanisms can be separated and the appropriate quenching constants determined.

6. Conclusions The simultaneous determination of the steady state fluorescence intensity and anisotropy data at different quencher concentrations provides an easy-to-use method to resolve the different quenching parameters. Computer simulations and model experiments confirmed ~:heapplicability of the method. The method is applicable

.

0.2

.

03

.

04

0.5

0.6

07

08

09

In ~]rel Fig. 4. Dependence of the static and dynamic quenching constants on the relative viscosity of the medium. Double logarithmic representation of the experimental data. The slope of the fitted line is -0.93. A, ln(Ksv+V); I--I, in Ksv; *, In V.

over a wide range of experimental systems, where the quenchable fraction of the fluorescence is close to unity (a = 1) and the emission anisotropy of the fluorophore in the absence of the quencher is below 0.3. (Above this limit, the anisotropy range covered by the measurements is too narrow.) These requirements are fulfilled in a large variety of proteins containing surfaceexposed (intrinsic or extrinsic) fluorescent groups or groups fully accessible to the applied quencher. Such systems involve the family of flexible proteins containing a single tryptophan residue, e.g. glucagon, adrenocorticotropin (ACTH), apolipoprotein C-1 and others [16]. In such cases, costly time-resolved fluorescence experiments can be replaced by simpler, cost-effective steady state measurements to determine selectively the dynamic quenching constant.

Acknowledgement This work was supported by a grant from the National Scientific Research Foundation of Hungary (OTKA grant nos. 1448 and T6185).

References [1] B. Somogyi and Z,s. Lakos, Protein dynamics and fluorescence quenching, Z Photochem. Photobiol. B: Biol., 18 (1993) 3-16. [2] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, New York, 1983, Chapter 9. [3] M.R. Eftink, Fluorescence quenching: theory and applications, in J.R. Lakowicz (ed.), Topics in Fluorescence Spectroscopy, Plenum, New York and London, 1991, pp. 53-126. [4] M.R. Eftink and C.A. Ghiron, Review of fluorescence studies with proteins, Anal Biochem., 114 (1981) 199-227. [51 S.S. Lehrer, Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds

60

[6] [7]

[8]

[9]

[10]

Z. Lakos et aL / J. Photochem. Photobiol. B: BIOL 27 (1995) 55-60

and of lysozyme by iodide ion, Biochemistry, 10 (1971) 32543263. A.M. North, The Collision Theory of Chemical Reactions in Liquids, Wiley, London, 1964, p. 7. J. Keizer, Nonlinear fluorescence quenching and the origin of positive curvature in Stern-Volmer plots, J. Am. Chem. Soc., 105 (1983) 1494-1498. W.R. Laws and P.B. Contino, Fluorescence quenching studies: analysis of nonlinear Stern-Voimer data, Methods EnzymoL, 210 (1992) 448-463. F. Perrin, Mouvement brownien d'un eilipsoide (II). Rotation libre et dipolarisation des fluorescences. Transaction et diffusion de mol6cules ellipsoides, Z Phys. Radium, VII (1936) 1---44. M.R. Eftink, Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins, Biophys. J., 43 (1983) 323-334.

[11] J.E. Churchich, Energy transfer in protein pyridoxamine-5phosphate conjugates, Biochemistry, 4 (1965) 1405-1409. [12] P.D. Boyer, The Enzymes, Vol. VII, Academic Press, New York and London, 1972, p. 725. [13] R.C. Weast, CRC Handbook of Chemistry and Physics, CRC Press, Boca Raton, FL, 1986-1987, p. D-232. [14] B. Somogyi, J.A. Norman, M. Punyiczki and A. Rosenberg, Viscosity dependence of acrylamide quenching of ribonuclease T1 fluorescence. The gating mechanism, Biochim. Biophys. Acta, 1119 (1992) 81--89. [15] M.R. Eftink and C.A. Ghiron, Does the fluorescence quenching acrylamide bind to proteins? Biochim. Biophys. Acta, 916 (1987) 343-349. [16] M.R. Eftink, Fluorescence techniques for studying protein structure, in C.H. Suelter (ed.), Methods o f BiochemicalAna~sis, Vol. 35:Protein Structure Determination, Wiley, 1991, pp. 127-205.