J. Phys. Chem. 1993,97, 42314238
4231
Rotational Reorientation Kinetics of Dansylated Bovine Serum Albumin Run Wang and Frank V. Bright’ Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14214 Received: December 4, 1992; In Final Form: February 3, 1993
Time-resolved fluorescence intensity and anisotropy decays of dansylated bovine serum albumin (BSA) are investigated by multifrequency phase and modulation fluorescence spectroscopy. We found that a double exponential decay law best describes the fluorescence intensity decay of covalently attached dansyl to BSA. The short lifetime component is attributed to dansyl located at the exterior surface of BSA. The longer-lived component reflects dansyl at theinterior of BSA. This result indicates that thereare twodansyl-BSA populations in the ground state. An associated model is then found to best describe the anisotropy decay kinetics of dansylated BSA. This result is consistent with the observed ground-state heterogeneity. The rotational reorientation kinetics of dansylated BSA are described by three distinct rotational correlation times. The longest is attributed to the global motion of the entire BSA molecule. The two shorter rotational correlation times are a consequence of local motions of dansyl at the exterior surface of BSA and within an internal hydrophobic site. The temperature effects on the anisotropy decay of this system were also studied and followed a simple Arrhenius rate law. The semiangle (e) for the cone of rotational reorientation associated with two local motions indicates that the surface dansyl is moderately free to rotate (e = 4 5 O ) and that temperature does not affect its reorientational freedom. In constrast, dansyl bound within the BSA matrix shows restricted motion, but this restriction wanes (17O 1 B 5 30°) with temperature.
Introduction Proteins are composed of amino acids linked by peptide bonds.[ The composition and sequence within the polypeptide chains determine the protein’s primary structure, and these chains can fold together under physiological conditions. The folding pattern, into a helix or a B structure, is often referred to as the secondary protein structure.’ Additional folding, which involves interactions between groups that are distant in the primary sequence, results in a three-dimensional (or tertiary) structure. In order to lower the system energy further, the nonpolar side chains face one another, resulting in the formation of hydrophobic sites or cavities within the protein, and polar groups are usually maintained at or on the exterior surface.’ Protein molecules are dynamic in nature? and experimental results have shown that there is considerable local motion within a protein at ordinary temperat~res.’-~Additional structural data has shown that residue or subunit displacementsplay an important role in the activity of proteins (for example, enzyme catalysis,6 hemoglobin ~ooperativity,~ and immunoglobulin action*). During the past 10 or so years, the functional significance of protein structural dynamics and internal motions in the overall biological activity has been recognized. As a result, considerableeffort has gone into characterizing the dynamical features of various protein systems. To this end, temperature-dependent X-ray diffraction, N M R relaxation, molecular dynamics simulations, and fluorescence depolarization methods have been used to address many aspects of protein dynamic~.~*~-Il Time-resolved fluorescence depolarization (anisotropy decay) studies of probes attached to proteins can provide detailed information about internal dynamics of the protein molecules.’* From the time evolution of the fluorescence anisotropy, information on the rotational mobility of the fluorophore can be extracted. In addition, if the fluorescent label is attached to a protein, it can potentially report on the overall motion of the particle and any internal rotational modes which may be present, including the local motion of the probe.I2 Time-resolved rotational reorientation studies have been used previously to demonstrate that immunoglobulin G (IgG) molecules are extremely dynamic
* Author to whom all correspondence should be sent. 0022-3654/93/2091-423 1$04.00/0
species that exhibit nanosecond segmental mobility of their F(*b’) fragments.I3J4 It was also reported that the tryptophan residue in human serum albumin (HSA) rotates freely on a subnanosecond time scale within a cone possessing a semiangle of approximately 3Oo.l5 In this paper, we report on the fluorescence intensity and anisotropy decay of dansylated bovine serum albumin using multifrequency phase and modulation techniques. This system was chosen for several reasons. First, it is in concept simpler than other protein systems. Second, it can serve as a model for the processes occurring in the new fluorescence-based biosensors being developed in our laboratory.lbIs However, prior to addressing these more complex, antibody-based systems, we study here how the protein-probe interactions affect the system dynamics. Specifically, we report on how temperature affects the anisotropy decay of dansylated BSA. Based on the form of the experimental anisotropy decay data, we have recovered information on the global motion of the entire protein and also on thelocalmotionsofthefluorescentlabelslocatedattheexterior and interior of bovine serum albumin.
Experimental Section Materials. The followingchemicalswere used: dansyl chloride, dimethylformamide (DMF), 1,4-bis(4-methyl-5-phenyl-Zoxazoly1)benzene (MeZPOPOP) (Aldrich Chemical Co.), dansylglycine, bovine serum albumin (BSA), and dialysis tubing (cellulose membrane) (Sigma Chemical Co.). Dialysis tubing was prepared following standard procedures.2 All reagents were used as received without further purification. All aqueous solutions were prepared in doubly distilled-deionizedwater. The BSA stocksolution was typically 50 MM. The stock solutions were refrigerated. Dansyl chloride was used immediately after it was dissolved in DMF. Unless otherwise noted, fresh protein solutions were used for all steady-stateand dynamic fluorescence experiments. Temperature was controlled by a Lauda RL6 temperature circulator. Preparation of Dansyl-Labeled BSA. One of the many ways to covalently label proteins is to target amine groups with sulfonyl halides such as dansyl ch10ride.I~Covalent attachment of dansyl to BSA was achieved by reacting BSA in 0.1 M phosphate buffer at pH 8.0 at room temperature with dansyl chloride dissolved in 0 1993 American Chemical Society
4232 The Journal of Physical Chemistry, Vol. 97, No, 16, 1993
a small amount of DMF. For all results reported, the initial molar concentration ratio of BSA to dansyl chloride was 1:l. After 4 h, the solutions were dialyzed exhaustively against 0.025 M phosphate buffer at p H 6.8. The purpose of dialysis was to separate the covalently labeled dansyl from any unreacted dansyl chloride and its hydrolysis product. The completeness of purification was monitored by checking for fluorescence in the solution outside the dialysis tube. Fluorescence Measurements. All steady-state fluorescence measurements were performed using a SLM 48000 M H F spectrofluorometer using a Xe arc lamp as the excitation source. All emission spectra were background subtracted and corrected for detector and monochromator transmission nonlinearities. Time-resolved anisotropy and intensity decay data were acquired in the frequency domain using a SLM 48000 M H F multifrequency phase-modulation fluorometer. An argon-ion laser (Coherent, Model Innova 90-6) operating a t 363.4 nm was used as the excitation source. A 340 f 20 nm band-pass filter (Oriel) was used to eliminate extraneous plasma discharge. Emission was observed through a 385-nm long-pass filter (Oriel). Magic angle polarization was used for all sample lifetime measurements. Me2POPOP in ethanol was used as the reference lifetime standard. Its lifetime was assigned a value of 1.45 m 2 0 Inert gas was not used to eliminate dissolved oxygen before lifetime measurements. Multifrequency phase and modulation data for lifetime and rotational correlation time measurements were acquired as described e l ~ e w h e r e . ~ lFor - ~ ~all experiments, the Pockels cell wasoperated at a repetition rateof 5 MHz. Typically, data were acquired for 60 s from 5 to 125 MHz (25 frequencies). The frequency-domain data were analyzed using a global analysis approach.24 In order to improve the precision and accuracy of the recovered kinetic parameters, multiple sets of frequency-domain data are linked together (when possible). The average experimental phase and modulation variances wered used for minimization of the xz function. In the idealized case, the best model meets simultaneously the following criteria: (1) simplest model (Le., having theminimum number of total floating parameters); (2) smallest x2 value; (3) random residual terms; (4) concordance with the steady-state experiment results; and ( 5 ) physical significance of the chosen model for a particular system. Theory
The theory of frequency-domain fluorescence spectroscopy has been described in detail e l s e ~ h e r e . ~The ~ - theoretical ~ ~ ~ ~ ~ treatments of the anisotropy decay law for heterogeneous systems were studied previously.12s26For completeness, we briefly review the key expressions. In the frequency domain, rotational reorientation kinetics are determined from frequency-dependent measurements of the differential pase angle A (4, - 011) and the polarized modulation ratio A (=mll/mi). If the time-dependent decay of the total intensity is Z ( t ) , the decay of the parallel and perpendicular components of the polarized fluorescence are given by
q t ) = I / 3 [ Z O ) ( 1 + 2r(t))l
(1)
and
where r(t) is the fluorescence anisotropy decay. For a simple isotropic rotor, r(t) decays with a single rotational correlation time, qS2
Wang and Bright takes the form of a sum of exponentials:
where j3i and ~ $ i are the fractional contribution of the total depolarization and the rotational correlation times attributed to reorientational motion i, respectively. The general expression for r(t)may involve five or more exponential terms.26 Regardless of the form of the anisotropy decay, A and A are given by the sine (Ni) and cosine (Di)transforms:
Di = Z i ( t ) cos wt dt
(6)
where Zi(t) is given by eqs 1 and 2 and w is the angular modulation frequency.
(7)
+ N L 2+ D ,
A = [ N~~~ D ‘ I 2 ] The parameters of interest, @,and+i, a r e subsequently recovered by fitting theexperimental A and A data using nonlinear regression method~.~l-~~~~~ The rotational motion of a protein is often monitored by using an extrinsic fluorophore covalently attached to the protein. However, if some form of internal (local) rotational reorientation is present, a rotational motion from the label is superimposed upon that of the entire particle. For the case of a fluorophore that is free to rotate through a limited angular range (within a cone with a semiangle e) and that is attached to a large particle that undergoes isotropic rotational diffusion, the anisotropy decay kinetics are well approximated by a double exponential decay model: 2 ~ 1
In this expression, 61 depends solely on the localized reorientation of the label when the local motion is much faster than the global motion of the whole molecule or particle (the correlation time of thelocalmotionh = l / ( l / u ~ =1/62)) and uzreflectstherotation of the entire particle (the correlation time of the global motion 4~ = U Z ) . 81 and 02 are the fractional contributions of the total anisotropy decay from the local and the global motions, respectively. The sum of j31 + j32 is equal to unity. The semiangle (e) within the cone in which the local reorientation occurs is related to the fractional contribution of local motion by15
1 - P I = cosz e ( i
+ COS e12/4
(10) If there are two fluorescing species in the ground state, the observed anisotropy decay is the sum of those two species:I2 Heref] andfz are the fractional intensity contributions of those two species, respectively. Thus, if two protein-associated species contribute to the total fluorescence, the decay of anisotropy can potentially be described by two rotational correlation times for each species (four in total). However, two of these rotational correlation times may be redundant; associated with the global motion of the entire protein. Therefore, one can expect to observe three distinct rotational correlation times from the anisotropy decay kinetics if the fluorophore is located simultaneously in two different domains. Results and Discussion
and for the more complicated anisotropic case, r(t) generally
Steady-State Fluorescence. Prior to interpretation of the dansyl fluorescence, it is important to quantify the average number of
The Journal of Physical Chemistry, Vol. 97, NO. 16, 1993 4233
Dansylated Bovine Serum Albumin
*
c
-C
. I -
1.0
0.8 0.6
C
m
p f!
-s
0.4
0.2
LL
L
0.0 400
450
500
550
600
650
700
Emission Wavelength (nm)
Figure 1. Normalized emission spectra for dansylated BSA (l), dansylglycine in 0.1 M phosphate buffer at pH 8.0 (3), and dansylglycine in 1,4-dioxane (2). The inset shows the structure of dansyl chloride.
dansyl groups attached per BSA molecule. To this end, we investigated the ultraviolet absorbance spectra of dansylglycine and dansylated BSA (0.1 M phosphate buffer a t p H 8.0). In the dansyl region, these two spectra (not shown) are effectively identical and indicate that differences in the local environment do not affect the dansyl absorbance. Thus, we can use the experimental molar absorptivity of dansyl (340 nm) and BSA (270 nm) to determine the molar ratio of dansyl to BSA. The recovered ratio is 0.6:l (dansy1:BSA). Dansyl is one of the most widely used fluorescent labels in biochemi~try.’~ This is a result of its good fluorescence yield and the fact that its spectral and temporal features are strong functions of its local environment. For example, its emission spectrum blue shifts with decreasing solvent polarity or increasing hydrophobicity.I9 Thus, emission shifts can be used as a crude measure of the local environment surrounding the probe. To obtain information on the local environment about the dansyl in the dansyl-BSA adduct, we acquired steady-state fluorescence spectra (Aex = 340 nm). Figure 1 shows the emission spectra for dansyl-labeled BSA (spectrum 1) and dansylglycine in 1,Cdioxane (spectrum 2) and 0.1 M p H 8.0 phosphate buffer (spectrum 3). These results show that the dansyl-BSA spectra are blue shifted
TABLE I: Recovered Lifetime Parameters for Dansvl-BSA modela T (“C) T I (nsY ~2 ( n d b ~
with respect to dansylglycine and are consistent with the average local environment probed by dansyl in the BSA adduct being less polar compared to dansylglycine in the bulk solvent. Time-Resolved Fluorescence. Although the steady-state fluorescence gives some insight into the local environment about the probe, it becomes problematic to use this information alone to determine if the covalently attached dansyl group(s) is (are) located in a single environment or in several environments with different physicochemical properties. In order to determine the distribution and characteristics of the local environment about the dansyl probes, one must use time-resolved fluorescence spectroscopy. Further, the heterogeneity of the dansyl ground state must be known before any accurate investigationfinterpretation of rotational reorientation kinetics of dansyl-BSA can be carried out. To recover the fluorescence decay kinetics of the dansyl-BSA adduct, we carried out a systematic series of multifrequency phase and modulation experiments. These data were then fit to various decay laws.27 Specifically, we tested models consisting of single, double, and triple exponential decay laws, unimodal continuous distributions, and a combination of discrete and distributed components. The fits to the various models are summarized in Table I. Inspection of these results indicates that the double exponential decay law best describes our experimental phase and modulation data over a broad temperature range. All other models with more floating parameters than the double exponential decay law (e.g., triple exponential decay, a distribution lifetime with a discrete component) did not significantly improve x2. Figure 2 illustrates a typical set of multifrequncy phase and modulation results for dansyl-BSA at 22 OC. In panel A, we compare our data (points) to the best single (- - -) and double (-) exponential decay law. For completenesswe also show (panel B) the corresponding residual plots. Clearly, the best fitting model is the double exponential decay law. This result is consistent with a heterogeneous dansyl ground state; the dansyl is in two discrete environments (vide infra). Before proceeding any further we questioned the uncertainty in the recovered excited-state decay parameters. In order to
~~
D
DD
DDD
LD
GD
22 30 40 50 60 22 30 40 50 60 22 30 40 50 60 22 30 40 50 60 22 30 40 50 60
17 17.4 17.4 17.1 16.2 18.8 18.4 18.5 17.9 17.2 19.2 18.5 18.5 18.0 17.2 18.5 18.6 18.5 17.9 17.2 18.6 18.3 18.3 18.1 17.0
73
(nsIb
W I (nsIc
ffld
ff2d
5
2.1 2.0 2.2 1.9 2.5 5.1 2.7 2.2 3.0 3.3 2.0 2.1 2.2 1.9 2.5 2.0 2.0 2.0 2.1 2.5
X2
42 32 29 27 20
0.8
0.71 0.75 0.75 0.76
0.80 1.4 1.4 2.1 1.6 2.0 1.9 0.006 0.04 0.04 0.001 2.4 0.24 0.24 0.47 0.44
0.67 0.74 0.75 0.76 0.80 0.85 0.86 0.86 0.87 0.89 0.72
0.76 0.76 0.76 0.82
0.1 1 0.12 0.14 0.05 0.09
1.9 1.8 1.1 3.1 0.6 2.0 1.9 1.2 3.3 0.8 2.2 1.9 1.2 3.2 0.8 2.1 1.9 1.3 3.4
a D, single exponential; DD, double exponential; DDD, triple exponential; LD, Lorentzian distribution plus a single exponential; GD, Gaussian distribution plus a single exponential. For a continuous lifetime distribution, I(r)= J ’ c ~ ( T )exp(-r/T) dt, where a ( ~=) 1/&27r exp[-0.5((~- T ‘ ) / u ) ~ ] for a Gaussian a ( r ) = 1 / exp[H/(r ~ - f ) 2 PI. f is the center lifetime, u is the standard deviation of the Gaussian, and H i s the half-width at , 7 3 are recovered lifetimes of center lifetimes f . W I is the full-width at half half-maximum (HWHM) for the Lorentzian distribution. T I , ~ 2 and maximum for a Lorentzian distribution and the standard deviation for a Gaussian distribution. CY is a preexponential factor.
+
Wang and Bright
4234 The Journal of Physical Chemistry, Vol. 97, No. 16, 1993 h
*loo
j
30
20
40
50
60
Temperature (“C)
Figure 4. Effects of temperature on the recovered lifetimes of dansylated BSA (0 and A) and dansylglycine ( 0 ) .
n
100
10
o
Frequency (MHz)
Figure 2. (A) Phase-modulationtraces for the lifetime determination of dansylated BSA. Symbols (0,0 ) are the experimentaldata. The solid line is the double exponential decay fit and the dashed line the single exponential fit. (B) Residual plot of the phase and demodulation factor. and are the phase and demodulationfactor deviations for the double exponential decay fit, respectively. A and A are the phase and demodulation factor deviationsfor the singleexponential fit, respectively. 6 ,
1
A
k /
.I-
$
I1
4
v)
0
5
10
15
20
25
0.8
1.0
Lifetime (ns)
-
0 0.0
0.2
0.4
0.6
Pro-exponential
Factor
Figure 3. x 2 error surfaces for the recovered lifetimes and preexponential factors associated with the data in Figure 2. The horizontal dashed line indicates the two standard deviation confidence level.
address this issue, we carried out a confidence interval analysis.27 In this approach,27we fix a particular decay parameter (e.g., 71) at a predetermined value and, by refitting the experimental data by adjusting all remaining parameters, calculate a new x2. This process is repeated over a range of 71 values, and x2vs 71 yields a convenient measure of the imprecision in 71. Also, because one investigates each parameter separately, any correlation between terms is compen~ated.~’ Figure 3 presents the complete confidence interval results for the multifrequency data shown in Figure 2. Similar results are found for dansylated BSA a t the other temperatures (data not shown). The dotted horizontal line is the two standard deviation
-A-A-A-AFA-A0 400.
0
455 L
A
510
L
565
f !O
Emission Wavelength (nm)
Figure 5. Decay associated emission spectra for dansylated BSA at 22
OC. (-) Normalized steady-state emission spectra, ( 0 ) 18.8-11s a m ponent, and (A) 2.1-11scomponent.
limit, and its position indicates the imprecision (measured from the points where the curves intersect the dashed line) in the decay parameters. These results demonstrate the level of precision in the recovered parameters and also provide information on our ability to resolve these decay parameters. Based on these results we conclude there are two well-resolved components that contribute to the observed fluorescence. In an effort to determine the origin of these two decay times we investigated the temperature dependence of the excited-state lifetimes. The results of these experiments are summarized in Figure 4 and show some interesting trends. For example, the recovered value for the shorter-lived component (A)is in excellent agreement with the value of dansylglycine in water (0). We also found that the lifetime of dansylglycine in water is, like the short-lived component in the dansyl-BSA adduct, temperature independent over the temperature range studied. These results are consistent with the shorter-lived component being dansyl located at the surface of BSA. The longer-lived component is thus attributed to covalently attached dansyl located within BSA in an environment that is more hydrophobic compared with the aqueous bulk solution. Decay associated emission spectra (Figure 5 ) lend additional support to this assignment. In addition, Table I shows that the preexponential factor for the longer-lived component is larger than that for the short component. Thus, the majority of the observed fluorescence is attributed to the dansyl located within the BSA. This is in agreement with our steady-state results (Figure 1). Figure 4 ( 0 )also shows that the lifetime of the longer-lived component decreases slightly with increasing temperature. This indicates that the environment of dansyl located at the BSA interior changes with temperature. This could be a manifestation of an actual denaturation process or an increase in the mobility of the dansyl label. However, previous reports on BSA have shown that denaturation is insignificant over the temperature range investigated.28 RotatioaalDiffdonKinetics. To better understand this system, we next probed the reorientational dynamics of the dansyl-labeled
Dansylated Bovine Serum Albumin
The Journal of Physical Chemistry, Vol. 97, No. 16, 1993 4235
TABLE II: Recovered Reorientation Parameters for Dansyl-BSA 6 (nsY
T ("C)
'or
x2
Model 1 22 30 40 50 60
51 39 28 21 12
0.29 linked
13
Model 2 22 30 40 50 60
52 42 34 28 23
0.71 0.64 0.61 0.56 0.49
associated with the longer lifetime T (OCI 22 30 40 50 60
0 1I*
(ns)
49 41 31 28 22
(ns)
U Z I ~
2.6 2.5 1.6 0.98 0.81
Bll'
0.91 0.89 0.85
0.336 linked
1.24
0.80 0.70
associated with the short lifetime adlinked 17228 (ns) BIZ' Model 3
0.88 0.85 0.84 0.77 0.68
49 41 31
0.74 0.60 0.49 0.13 0.04
28 22
0.43 0.31 0.30 0.50 0.40
~
ma
Y2 b
0.354 linked
1.18
~~~
a Limiting anisotropy is always linked over all temperatures. This is the global x2 for a linked analysis. The local x 2 are not shown for simplicity. Rotational correlation time for the model assuming a single rotational reorientation time. U I is the rotational correlation time for global motion, and u2 is the rotational correlation time due to local motion. Fractional contribution due to global motion. f u l l and a12are the rotational correlation times for global motion. u21 and 6 2 2 are the rotational correlation times for the local motions of labeled dansyl at the interior and exterior surface of BSA, respectively.
BSA. However, one must recall that the recovered fluorescence intensity decay kinetics have two lifetime components corresponding to surface and internal dansyl-BSA adducts. In turn, this suggests that an accurate description of the reorientational dynamics is potentially quite complicated. Therefore, an accurate description of the reorientational dynamics must explore associative and nonassociative decay of anisotropy models.12 In the nonassociative case, the recovered set of rotational correlation times describing the anisotropy decay are common to each of the lifetime components in the fluorescence intensity decay. That is, the general form of the reorientational dynamics for the dansyl located a t the exterior or interior of BSA is similar, but the actual scales are quite different. In the associative case, each excitedstate lifetime is associated with a specific set of rotational correlation times. In our case, the different rotational correlation times might be associated with dansyl at the exterior surface and within the BSA molecule. Once we identify the correct model we can begin to interpret the results with a physically significant model. We analyzed our experimental dansyl-labeled BSA data at five different temperatures using associative and nonassociative models. The recovered parameters and corresponding x2 values are summarized in Table 11. The lifetime heterogeneity of dansyllabeled BSA results from a single molecular species (dansyl) experiencing different environments. Thus, the two recovered excited-state lifetimes result from differences in the radiationless decay rate. As a result, the limiting anisotropy for the two lifetime components should be identical. Therefore, we can link2' the limiting anisotropy across all the temperature data sets. Such a linking scheme results in a much more rigorous model with less floating parameters and should result in our recovering selfconsistent results.27 From the results collected in Table 11, we can easily eliminate the model with a single correlation time based on the large x2 value. This indicates that there are a t least two reorientational motions responsible for the anisotropy decay in our dansyl-BSA system. The nonassociative model has a significantly poorer x2 (1.24) compared to the associative model (1.18). For these particular data files, with over 200 degrees of freedom, we can
-
.-5
2.4
0
i 2.2 0 0
=P
2.0
.E
1.8
-0 9
1.6
m
10 10
100
1
B
8 6 4 2
100
10 Frequency (MHz)
Figure 6. Multifrequencypolarized modulated ratio (A) and differential polarized phase angle (B) for dansylated BSA in 0.1 M phosphate at pH 8.0. Temperatures were 22 (0),30 ( O ) , 40 (V), 50 (V),and 60 ( 0 ) OC. The solid line denotes the best fit to an associated model (Table 11).
reject statistically the nonassociated model at the 95% confidence For completeness, Figure 6 shows the polarized modulation ratio (panel A) and the differential polarized phase angle (panel B) data for dansyl-BSA at five different temperatures. Clearly, the associative model (Figure 6 , solid lines) fits our experimental data (symbols) quite well over the entire temperature range studied. In addition, the limiting anisotropy recovered independently from these dynamic measurements is in excellent agreement with the steady-state result (0.353 f 0.005) for dansylglycine dissolved in glycerol at -60 "C. Therefore, we conclude that the associative model describes the anisotropy decay of dansyl-labeled BSA based on (1) the smaller x2value, (2) the agreement between
Wang and Bright
4236 The Journal of Physical Chemistry, Vol. 97, No. 16, 1993
B
1.0
t
I I I I I I I I I I I ( I I I 10
20
40
50
50
70
60
80
0
3
2
1
4
5
1.6
1.4
1.2
1 .o
t 1
0
t '
1
~
1
'
1
'
5
2
1
1
4
'
1
5
1.6
1.4
1.2
1
1.0
t 0.0
t 0.5
1 .o
1.5
2.0
0.0
0.4
0.8
1.2
1.6
Rotational Correlation Time (ns) \
1
Figure 7. x 2 error surfaces for the recovered rotational correlation times for dansylated BSA as a function of temperature. (A) The global motion of the entire BSA molecule. (B-F)The local motions for surface (- - -) and buried (-) dansyl groups at 22, 30, 40, 50, and 60 "C,respectively.
limiting anisotropy terms from completely different experiments, and (3) the physical reality (vide infra) of the model. Until quite recentlyI2it was not common to recover meaningful triple exponential decays of anisotropy or distinguish between associated and nonassociated models. Thus, before we begin to interpret our results wearecompelled toinvestigate theuncertainty in the recovered reorientational parameters. To this end, we carried out a confidence interval analysis2' (the scheme is identical to the one used for the excited-state lifetimes) on the reorien-
tational parameters for the dansyl-BSA at five temperatures. The entire set of results is shown as Figure 7. In panel A is illustrated the x2 surfaces for the longer-lived rotational correlation timeat the indicated temperatures. Panels B-Fsimilarly illustrate the two other rotational correlation times as a function of temperature. These results clearly demonstrate that we have the precision to resolve these three reorientational processes. Based on these results, we present a simple model (Figure 8) that explains the observed intensity decays and reorientational
The Journal of Physical Chemistry, Vol. 97, No. 16, 1993 4231
Dansylated Bovine Serum Albumin
TABLE III: Recovered Activation Parameters for the Reorientational Processes in Dansyl-BSA local motions involved with dansyl at global motion of exterior surface the entire BSA of BSA interior of BSA Ea (kJ/mol) 17 1 (1.8 i 0.1) A (9-I)
global m6tion Dansvl at the
elobal motion
X 1O'O
19i2 24 i 6 (1.3 i 0.1) X lo2 (3.0f 1.0) X lo"
80
A
70
Figure 8. Proposed model to explain the observed rotational dynamics of the dansyl-BSA system.
T I
AI
1
I
2 4 ,
'6
2
+6 1 3.0
3.2
3.4
Figure 9. Arrhenius plots for the reorientational dynamics of the global (0)and two local motions (within, 0 ;surface, V) of dansyl in the dansyl-
BSA system. dynamics of dansyl-labeled BSA. First, there are two excitedstate lifetimes resulting from dansyl in two discrete environments (upper and lower sections of Figure 8). Second, there are a total of three rotational correlation times. The largest correlation time (a11) is attributed to the global motion of the entire BSA molecule because the recovered correlation time for the global motion (Table 11) is comparable to that calculated for a rigid hydrated sphere with a molecular weight of 65 kDe30 Recall that BSA does not denature over the temperature range studied.28 Therefore, the global reorientation motions reported by the two different dansyl groups are, as expected, essentially the same. This is one of the reasons that we are able to successfully link the rotational correlation times for the global motions across the two lifetime components. The two faster reorientation processes (short correlation times) are attributed to the local motions of dansyl located at the two different environments (associated with T I and T ~ ) .As shown in Table 11, the local motion involved with dansyl at the exterior surface of the protein (a24 has a smaller rotational correlation time compared to that recovered for dansyl at the interior of the BSA (Q). This result is direct evidence showing that dansyl a t the exterior surface of the protein reorients more quickly compared to its counterpart buried within the BSA. Closer inspection of the results (Table 11) shows that the rotational correlation times for the global and local motions decrease with increasing temperature. Plots of the natural logarithm of the anisotropy decay rate corresponding to each reorientation process against the reciprocal of the absolute temperature were linear (Figure 9). This indicates that an Arrhenius relationship describes the rotational reorientation rates of the dansylated BSA system for the global and local motions. The corresponding activation energies and frequency factors are
20
30
50
40
Tem p e r o t u r e
60
('C)
Figure 10. Effects of temperature on the reorientational semiangle for dansyl at the surface of (A) and within (B) BSA. The error bars were determined using the uncertainty for values recovered from the x 2 error surfaces (Figure 7 ) at the 95% confidence level.
collected in Table 111. The uncertainties in the recovered parameters were obtained from the linear regression and are reported at the 95% confidence level. The frequency factor associated with the local reorientational motion of the dansyl a t the interior of BSA is significantly larger than that of the fluorophore at the exterior surface of the protein. The frequency factor due to the global motion is smaller still compared to either local motions. The activation energy associated with the interior motion is greatest followed by the surface reorientation and global motions. These results demonstrate that the energetics of rotational reorientation of the dansyl label span a significant range. In addition, they suggest that the barriers associated with reorientation within the interior of the protein are greater (e.g., restrictive) and the energetics with local surface and global motions are comparable. The amplitude (Table 11, values) of the faster local motion associated with each dansyl species can be further interpreted as the dansyl label in BSA reorienting (precessing) within a cone with a semiangle B (Figure 8).12J5 The amplitude of the local motions associated with the shorter or longer excited-state lifetime increases as the temperature increases and, using eq 10, can be used to calculate the value of the semiangle for the reorientational dynamics within the two dansyl domains. Figure 10 illustrates the effects of temperature on the semiangle associated with the surface (panel A) and interior (panel B) dansyl groups. These results show several interesting trends and merit additional discussion. First, the semiangle assigned to the surface-associated dansyl is significantly greater than that for the internal dansyl. This is not unexpected and indicates that the surface species has more freedom compared to the internal dansyl. Second, the semiangle for the surface dansyl group is unaffected by temperature. Such a result is consistent with any temperaturedependent changes in the BSA conformation not affecting the
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freedom of thesurface dansyl. Third, thesemiangle for the buried dansyl increases with temperature. This result is consistent with an increase, from the perspective of the buried dansyl label, in protein flexibility with temperature.
Conclusion The use of time-resolved fluorescence anisotropy measurements for studies of biomolecule dynamics is enjoying increased attention because of instrumental and theoretical advances.12J' Modern developments in laser technology and electronics permit subnanosecond measurements on a routine basis with considerable reliability. When a fluorescent probe is attached to a biological molecule such as a protein, the recovered anistropy decay reflects simultaneously the overall motion of the entire protein and any internal reorientational motions which may be present, including the localized motion of the probe. In turn, the reorientational dynamics of the probe are always superimposed upon that of the entire protein. In this report, we found that there are two populations of dansyl-BSA: dansyl at the exterior surface of BSA and dansyl located at an interior hydrophobic site. The recovered anisotropy decay reflects the overall motion of the BSA particle and local motion of the fluorophore within these two domains. We also found that three distinct reorientational processes are responsible for the observed anisotropy decay kinetics of dansylated BSA. The slow motion is attributed to the global reorientation of the BSA. The rotational correlation times are comparable to those predicted for a spheroid of the size of hydrated BSA. The two faster rotational correlation times (low nanosecond and subnanosecond time scales) are associated with the local motions (or wobbling) of dansyl groups within and a t the surface of BSA, respectively. The dansyl group within the BSA precesses more slowly and is restricted to a cone with a smaller semiangle. In contrast, the surface dansyl group is more free to reorient. These results, not too surprisingly, indicate that the interior of BSA is more restrictive to dansyl motion. However, temperaturedependent experiments show that the interior reorientational dynamics are a strong function of temperature and increase with temperature. The ensemble of reorientational motions are all well described by an Arrhenius expression, and all results are consistent with temperature increasing the BSA flexibility. Currently, weare investigating how protein denaturation affects the anisotropy decay kinetics of the dansylated BSA. When proteins denature they can experience a massive unfolding process. This process is expected to affect the anisotropy decay kinetics and in turn alter the observed reorientational dynamics.
Acknowledgment. This work was generously supported by the National Science Foundation (CHE 8921517).
Wang and Bright
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