Structural Investigation of Tet Repressor Loop 154–167: A Time-Resolved Fluorescence Study of Three Single Trp Mutants

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 346, No. 2, October 15, pp. 230–240, 1997 Article No. BB970290

Structural Investigation of Tet Repressor Loop 154–167: A Time-Resolved Fluorescence Study of Three Single Trp Mutants Patrizia Alberti,*,† Elisa Bombarda,*,† Martin Kintrup,‡ Wolfgang Hillen,‡ Hans Lami,† Etienne Pie´mont,† Silvia M. Doglia,* and Marie Chabbert†,1 *Dipartimento di Fisica, Universita` degli Studi di Milano, Via Celoria 16, 20133 Milano, Italy; ‡Lehrstuhl fu¨r Mikrobiologie, Institut fu¨r Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander Universita¨t Erlangen-Nu¨rnberg, Staudtstasse 5, 91058 Erlangen, Germany; and †Laboratoire de Biophysique, Faculte´ de Pharmacie de l’Universite´ Louis Pasteur de Strasbourg, CNRS URA 491, BP 24, 67401 Illkirch, France

Received April 4, 1997, and in revised form July 7, 1997

We have studied the time-resolved fluorescence of three engineered Tet repressor (TetR) mutants bearing a single Trp residue at positions 162, 163, and 165 in the C-terminal part of the loop joining helices 8 and 9. Detailed analysis indicates that, at 207, the fluorescence decay of each Trp can be described as the sum of three exponential components with lifetimes in the 1-, 3-, and 6-ns range. Emission wavelength and temperature dependence studies are consistent with a model in which these components are due to the existence of three classes of Trp residues non-interconverting on the nanosecond timescale. Within the framework of the rotamer model, the weak temperature dependence of the lifetimes strongly suggests that the secondary structure of the loop, at least in the 162– 165 range, is not altered with temperature. The equilibrium between the rotamers is characterized by an enthalpy–entropy compensation effect which strongly suggests the involvement of background structural regions of TetR in the thermodynamics of the process. The very high DH7 and TDS7 observed (up to 18 kcal/ mol) should reflect the temperature-dependent conformational change of a large part of the protein which would alter the rotamer distribution of the Trp residues. Taken together, our results are consistent with the existence of (at least) two conformations of the loop and suggest a model for loop motion. q 1997 Academic Press

Key Words: TetR; Trp; time-resolved fluorescence; loop.

1

To whom correspondence should be addressed.

Class B Tet repressor (TetR)2 controls the transcription of the Tn10-encoded tet genes conferring resistance to tetracycline in gram-negative bacteria. TetR responds to the antibiotic tetracycline which acts as an inducer (1). In the absence of tetracycline, TetR binds to the two tet01 and tet02 operators and prevents transcription of the tetA and tetR genes, encoding for the resistance protein TetA and for TetR. Upon tetracycline binding, TetR undergoes conformational changes and dissociates from the corresponding operators, allowing the synthesis of TetA, an efflux protein which pumps tetracycline out of the bacteria, and its own synthesis. The crystal structure of the homologous class D TetR complexed to 7-chlorotetracycline has re˚ resolution (2). TetR occurs cently been resolved at 2.1 A as a homodimer. Each monomer, composed of 207 residues, folds into 10 a-helices with connecting turns and loops. The DNA binding domain, which has an a-helix– turn– a-helix structure, is formed by the N-terminal three a-helices. The core of the protein is formed by ahelices 5 to 10. It is responsible for dimerization and contains the inducer binding pocket. All parts of the polypeptide chain could be fitted without problems except for the segment ranging from residue 156 to residue 164 (2). This segment, which is located on the surface of the protein, is part of the loop joining helices 8 and 9 (Fig. 1a). It is indirectly involved in inducer binding, since it is connected to a-helix 9, which forms the hydrophobic pocket for the D ring of tetracycline (2). The loop located from residue 154 to residue 167 is one 2 Abbreviations used: TetR, Tet repressor; b-ME, b-mercaptoethanol; HEPPS, hydroxyethylpiperazine propanesulfonic acid; MEM, maximum entropy method.

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the loop. We show here that, independently of the Trp position (162, 163, or 165), the fluorescence decay at 207C can be described as the sum of three exponential components. The temperature dependence of the decay parameters has been thoroughly studied and is discussed in relation to the loop structure, within the framework of the rotamer model. MATERIALS AND METHODS

FIG. 1. (a) Backbone structure of class D TetR dimer complexed with tetracycline, with ribbon drawing of a-helices 8 and 9. Drawn with INSIGHT. (b) Sequence of the class B TetR loop joining ahelices 8 and 9. Positions where Trp residues were introduced by molecular engineering are indicated by arrows.

of the least conserved regions and the only part of the polypeptide chain where insertions and deletions are found among the seven tetracycline resistance determinants present in gram-negative bacteria (classes A–E, G, and H) (2, 3). It might be essential for the positioning of a-helix 9 and the correct recognition of the drug. A recent mutagenesis study (4) has shown that the loop fragment located from residues 161 to 166 is important for inducibility by tetracycline. Deletions in this fragment have an induction-deficient TetRS phenotype, whereas substitutions do not or only slightly affect inducibility, suggesting that the length of the loop is important for the structural transition between Tc-bound and operator-bound conformations. To further investigate the structure of this fragment, we have investigated the time-resolved fluorescence of three single Trp mutants of TetRB whose Trp residues are located at positions 162, 163, and 165 (Fig. 1b). These mutations do not alter tetracycline binding and inducibility (Kintrup et al., to be published elsewhere). These Trp can be used as fluorescent probes to study the structure and dynamics of the C-terminal part of

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Materials. All the chemicals were of reagent grade or better. Ultrapure water (Milli Q instrument from Millipore Corp.) was used throughout the experiments. The experiments were carried out in 100 mM NaCl, 5 mM MgCl2 , 10 mM b-mercaptoethanol (b-ME), and either 10 mM Tris–HCl, pH 8.0, or 10 mM HEPPS, pH 8.0. We did not observe any significant difference between the two buffers at 207C. The HEPPS buffer was preferred to Tris for temperature studies because of the much weaker temperature dependence of its pKa (DpKa /7C Å 00.007). The engineered single Trp mutants of class B Tet repressor used in this study, Y43F75W162 TetR, Y43F75W163 TetR, and Y43F75W165 TetR, were purified as described elsewhere (5). The proteins were stored at 0187C in solutions containing 10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 10 mM b-ME, and 50% (v/v) glycerol. Spectroscopic methods. The concentrations were determined spectrophotometrically using a Cary 4 spectrophotometer. Steadystate fluorescence spectra were recorded on a MPF66 spectrofluorometer (Perkin–Elmer). Quantum yields were measured relative to that of the tryptophan zwitterion at pH 7.0 (0.14) (6). Time-resolved fluorescence measurements were carried out with the pulse fluorometry technique. The excitation source was a modelocked Ti-Sa laser (Spectra-Physics) pumped by an Ar/ laser. The 890-nm light pulses of 1 ps duration were converted to 296.6 nm through a BBO frequency tripler. At this wavelength, there is selective excitation of Trp, without contribution from the Tyr residues. The repetition rate of the excitation pulses was 4 MHz. The fluorescence emission at right angle to the excitation beam was detected by a microchannel plate photomultiplier (Hamamatsu R3809U), after going through a polarizator set at 54.77 from the excitation polarization. The emission wavelength was selected by a 16-nm bandwidth monochromator (Jobin-Yvon H10). The instrument response function was recorded with a polished aluminum reflector. Its full width at half-maximum was 40 ps. The calibration of the multichannel analyzer was 25.5 ps/channel. The total number of channels used was 1024. The typical total number of counts was 8 1 105. Each measurement was repeated n times (typically 5) on the same sample in order to estimate experimental errors. The protein concentrations ranged from 4 to 8 mM. Decay data analysis as sum of exponential terms. The fluorescence decay data were analyzed as the sum of discrete exponential terms: I(t) Å Saie0t/ti

[1a]

Sai Å 1

[1b]

with

by using a nonlinear least-squares reconvolution procedure based on the Marquardt algorithm. The minimal number of exponential components necessary to fit the decay was determined by increasing the number of exponentials progressively until the lowest possible x2 was reached and the weighted residuals appeared randomized. The weighting factors used in the x2 test were determined from the

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Poisson law. Experimental weighting factors (7, 8) were not used in order to compare these results with those obtained with the MEM (see below). Rigorous error analysis was carried out as described by Beechem (9). Analysis of the set of n decays was carried out with a fixed lifetime. The resulting x2 value was plotted as a function of the fixed lifetime to give the x2 error surface. The x2 increase corresponding to a given confidence interval was determined by F statistics (10). Maximum entropy method. Data analyses were performed using the maximum entropy library MEMSYS2 software (MEDC Ltd., Cambridge, UK). In the maximum entropy method, the fluorescence decay law is assumed to be the sum of a continuous distribution of positive exponential components:

I(t) Å

*

`

A(t)e0t/tdt.

[2]

0

The Skilling–Jaynes entropy function is defined as

SÅ

*

`

[A(t) 0 m(t) 0 A(t)log(A(t)/m(t))]dt,

[3]

0

where m(t) is the initial guess, which must be set to a flat distribution in log t space when one has no a priori knowledge about the distribution A(t) (11). In the present work, 150 lifetime values equally spaced in logarithmic scale and ranging typically from 0.001 to 15 ns were allowed for the distribution A(t). The distribution A(t) which minimizes the x2 value and maximizes the entropy function is searched, starting from the flat model m(t). The spectrum A(t) is divided in as many peaks as can be clearly separated by two successive well-defined minima. The average position tk of the peak k is the barycenter

tk Å Si Aiti /Si A i

[4]

calculated over all values of i included in the peak. The amplitude ak of the peak k is the ratio of the peak surface SiAi over the total surface of the spectrum (in log t scale). The average lifetime is defined as t Å Skaktk . The uncertainties given by the ME program are calculated from the covariance matrix as described by Brochon (12).

RESULTS

1. Fluorescence Decay Analysis The tryptophan fluorescence decay data at 207C were analyzed as sums of discrete exponential components and as sums of nonparametrized distributions of exponentials using the maximum entropy method. The interest of the MEM is that there is no a priori parametrization of the lifetime distributions. Its main inconvenience is the drastic effect of the signal/noise ratio in determining the distribution width (13). The width of the distributions recovered with the MEM can thus be considered the maximum limit of the true distribution width. The fluorescence decay of Trp-162 was not adequately described by a biexponential function. Addition of a third component in the fitting law led to a marked

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decrease in the x2 value from 3.1 to 1.5 (Table I). The weighted residuals appeared random (Fig. 2a). The x2 error surfaces (Fig. 4b) were determined for each decay component to carry out a rigorous error analysis on the recovered lifetimes. They indicated that the lifetimes were recovered with a good precision at the 67% confidence level (Table III). The lifetime distribution obtained with the maximum entropy method (Fig. 4a) indicated the existence of three narrow lifetime distributions centered around 6.1, 2.5, and 0.7 ns. The average lifetimes of the peaks and their amplitudes (Table II) were fully consistent with the discrete lifetime values and the corresponding preexponential terms obtained with the Marquardt algorithm, corroborating the triexponential decay law. The analysis of the fluorescence decay of Trp-163 and Trp-165 as sum of exponentials did not give satisfying results. Two-component analysis did not allow a correct fit of the decay data. Addition of a third component in the fitting procedure led to a decrease in the x2 value of about 0.4, but the weighted residuals showed an upward deviation, indicating that, in both cases, the recovered long lifetime was too short to adequately fit the decay tail (Fig. 2). The fourth-component analysis did not converge. Triexponential analysis of a set of decays on the same sample indicated two different behaviors: for Trp-163, decay analyses converged to very reproducible data with three lifetimes ranging from 0.6 to 4.4 ns, whereas for Trp-165, there was splitting of either the long or the short component of the biexponential analysis into two neighbor components, leading to data with larger uncertainties (Table I). The maximum entropy method, however, allowed an adequate fit of decay data as shown by random distribution of weighted residuals (Fig. 3). In both cases, the lifetime distribution obtained with the MEM indicated the existence of three decay components with lifetimes in the 1-, 3-, and 6-ns range (Figs. 4c and 4e; Table II). The x2 error surfaces obtained from triexponential analysis with a fixed lifetime (Figs. 4d and 4f) corroborated the maximum entropy analysis. The long lifetimes recovered from the x2 error surfaces were in the 6-ns range, in agreement with the maximum entropy analysis (Table III). The precision on these lifetimes, however, was very low. No maximum limit could be set up at the 90% confidence level. Figure 4 qualitatively shows the flattening of the x2 error surface of the long component when the corresponding amplitude decreases. This correlation explains the difficulty in analyzing decay data with the Marquardt algorithm when the amplitude of a component is about 0.10 or less. In this case, the maximum entropy method works out much better. Determination of the x2 error surfaces, however, allows poor fits to be resolved and may be of great help when the MEM is not available (Table III).

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FLUORESCENCE STUDY OF TetR LOOP 154–167 TABLE I

Fluorescence Decay Analysis as Sum of Exponentials Trp 162 163 165

t1 (ns) 2 3 2 3 2 3

exp exp exp exp exp exp

4.69 6.45 3.99 4.41 3.64 4.26

{ { { { { {

0.04 0.39 0.01 0.15 0.02 0.45

t2 (ns)

a1 0.409 0.184 0.535 0.402 0.442 0.235

{ { { { { {

0.004 0.029 0.004 0.048 0.007 0.070

0.85 2.66 1.03 1.82 1.07 2.01

{ { { { { {

0.03 0.26 0.02 0.31 0.01 0.68

a2 0.591 0.347 0.465 0.374 0.558 0.372

{ { { { { {

0.004 0.014 0.004 0.025 0.007 0.058

t3 (ns)

a3

0.66 { 0.03

0.469 { 0.017

0.63 { 0.10

0.224 { 0.065

0.87 { 0.08

0.393 { 0.104

x2 3.06 1.49 2.11 1.66 2.07 1.76

Note. Measurements were carried out in 10 mM HEPPS, pH 8.0, 100 mM NaCl, 5 mM MgCl2 , 10 mM b-ME, at 207C. Data represent the averages ({standard deviation) of five decays on the same sample.

2. Wavelength Dependence of the Decay The wavelength dependence of the fluorescence decay of each Trp has been studied using the analysis as sums of discrete exponential components. For Trp-162, all the lifetimes in the fitting procedure were free. For Trp-163 and Trp-165, because of the difficulty to recover the long component, the fluorescence decay analyses were carried out with the longest lifetime fixed at the value obtained at 350 nm with the MEM. For wavelengths greater or equal to 340 nm, all the decays

could be fit by a triexponential law. For wavelengths £330 nm, an additional component in the picosecond range was required for a satisfying fit of the data. Its weight markedly increased at 330 nm. In this case, this ultrashort component could be related to Raman scattering. At 315 and 320 nm, it could be due either to residual Raman or Rayleigh light scattering or to the existence of ultrafast relaxation processes. This component, whose weight was less than 1%, was not taken into account for data presented here. The main three lifetimes did not display significant dependence on the emission wavelength (Fig. 5). The absence of significant wavelength dependence for the shortest two components of Trp-163 and Trp-165 justifies the a priori choice of performing the decay analysis with a fixed long component. The spectrum associated with each component of the decay could be calculated from (14) Fi(l) Å F(l)tiai/(Sjtjaj).

FIG. 2. Weighted residuals for the best triexponential fit of a typical fluorescence decay of Trp-162 (a), Trp-163 (b), and Trp-165 (c). Measurements were carried out in 10 mM HEPPS, pH 8.0, 100 mM NaCl, 5 mM MgCl2 , 10 mM b-ME. The temperature was 207C.

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[5]

The decay-associated spectra of Trp-163 and Trp-165 (Figs. 5d and 5f) were very similar. In both cases, the weight of the middle-lived component was preponderant (about 65%), whereas the long component contributed only to 15–20% of the total fluorescence intensity. In the case of Trp-162, these two components had a similar weight (about 40%) (Fig. 5b). For the three Trp studied, there was not a significant difference in the emission maxima of the decay-associated spectra which were similar to that of the corresponding steady-state spectrum (347, 345, and 348 nm for Trp-162, Trp-163, and Trp-165, respectively). The absence of emission wavelength dependence of the decay strongly suggests that the three mathematically resolved decay components have a physical meaning and can be attributed to three classes of Trp residues. Under the assumption that there is no interconversion between these classes on the nanosecond timescale, the recovered lifetimes correspond to the intrinsic lifetimes of the classes. Under the supplemen-

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ALBERTI ET AL. TABLE II

Fluorescence Decay Analysis with the Maximum Entropy Method Trp

t1 (ns)

a1

Dt1 ({)

t2 (ns)

a2

Dt2 ({)

t3 (ns)

a3

Dt3 ({)

t (ns)

x2

162 163 165

6.12 6.83 6.23

0.208 0.078 0.059

0.56 0.58 0.62

2.52 3.18 2.93

0.334 0.578 0.487

0.32 0.28 0.35

0.69 0.92 1.00

0.458 0.344 0.455

0.06 0.03 0.05

2.43 2.68 2.24

1.39 1.42 1.49

Note. Measurements were carried out in 10 mM HEPPS, pH 8.0, 100 mM NaCl, 5 mM MgCl2 , 10 mM b-ME, at 207C. Analyses using the MEM were carried out on an individual decay. The corresponding lifetime distributions are shown in Fig. 4. It was checked that the result was independent of the individual decay considered. The lifetime ti corresponds to the average position of peak i and ai to its amplitude as defined under Materials and Methods. Dti ({) represents the width of peak i and t the average lifetime.

tary assumptions that the classes have the same extinction coefficient and radiative decay rate, the amplitudes of the decay components calculated from

* F (l)t

Ci Å (

01 i

i

dl)/(Sj

* F (l)t j

01 j

dl),

[6]

correspond to the relative concentrations of the corresponding classes. For each Trp, the relative concentration of the long component class was low (from 5% for Trp-165 to 17% for Trp-162), whereas the shortest two components had similar concentrations.

3. Temperature Dependence of the Decay The fluorescence decay of each Trp residue as a function of the temperature was analyzed with the MEM. The excitation wavelength was 350 nm. The uppermost temperature used for this study was 357C because the denaturation temperature of the mutants was 427C (not shown). Figure 6 shows the lifetime distributions obtained at 10 and 357C with the MEM for the three Trp. Temperature increase induced a marked change in component populations without reorganization of the lifetime distributions. This indicates that the fluorescence decay components were in slow exchange on the nanosecond timescale and that the equilibrium between them was temperature dependent. The long component of Trp-163 and Trp-165 decays disappeared at 35 and 307C, respectively. Analysis of the fluorescence decays for a set of temperatures ranging from 10 to 357C (Fig. 7) indicates that, for each Trp, the temperature had only a minor effect on the lifetimes but a large effect on the amplitudes. Arrhenius factors could be determined from the temperature dependence of ti : 1/ti Å k0 / Aiexp(0E*i /RT),

[7]

TABLE III

Comparison of the Lifetime Values Obtained from the Determination of the x2 Error Surface and with the MEM Trp

Method

162

ESa MEMb ES MEM ES MEM

163 165

a

FIG. 3. Weighted residuals for the best MEM fit of a typical fluorescence decay of Trp-162 (a), Trp-163 (b), and Trp-165 (c). The decay data are the same as in Fig. 2. The corresponding lifetime distributions are reported in Fig. 4.

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t1 6.1 /0.5/00.4 6.12 { 0.39 7.0 /3.6/02.0 6.83 { 1.47 5.8 /2.2/01.2 6.23 { 1.85

t2 2.4 /0.4/00.3 2.52 { 0.23 3.2 /0.3/00.4 3.18 { 0.23 2.8 /0.3/00.3 2.93 { 0.30

t3 0.65 /0.1/00.1 0.79 { 0.03 0.8 /0.05/00.1 0.92 { 0.04 0.9 /0.05/00.1 1.00 { 0.04

The lifetime values and the uncertainties corresponding to the 67% confidence limits were determined from the x2 error surfaces shown in shown in Fig. 4. b The lifetime values and the uncertainties determined by the MEM correspond to the lifetime distributions shown in Fig. 4.

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235

that each decay component had the same kr . The radiative decay rates ranged from 0.040 1 109 s01 for Trp162 to 0.047 1 109 s01 for Trp-165, in agreement with the average value determined by Ricci (15) for various indole derivatives (0.044 1 109 s01). The intersystem crossing rate was taken as equal to the value determined for Trp and NATA, 0.050 1 109 s01 (16). Nonlinear least-squares analysis of 1/ti as a function of T (Table IV) allowed determination of the Arrhenius factors Ai and E*i (Table IV). The long lifetimes of Trp-163 and Trp-165 were not taken into account because of the large uncertainties in their determination (Table III). This low precision might explain the anomalous behavior of the long component of Trp-165 whose lifetime increased with temperature, although we cannot exclude a temperature-dependent conformational change in the protein altering its quenching mechanism. The temperature-dependent nonradiative decay rate is the composite of several decay pathways with differ-

FIG. 4. Lifetime distributions obtained with the MEM (a, c, e) and triexponential x2 error surfaces (b, d, f) for the fluorescence decay of Trp-162 (a, b), Trp-163 (c, d), and Trp-165 (e, f). The drawn horizontal lines correspond to the 67% (broken line) and the 90% (continuous line) confidence levels. Measurements were carried out in 10 mM HEPPS, pH 8.0, 100 mM NaCl, 5 mM MgCl2 , 10 mM b-ME, at 207C.

where k0 is the total temperature-independent decay rate, Ai is the frequency factor and E*i is the activation energy. The temperature-independent decay rate k0 is equal to kr / kisc , where kr is the radiative decay rate and kisc the intersystem crossing rate. The radiative decay rate was calculated from the ratio of the quantum yield to the average lifetime, under the assumption

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FIG. 5. Wavelength dependence of the lifetimes and corresponding decay-associated spectra for Trp-162 (a, b), Trp-163 (c, d), and Trp165 (e, f) (t1 , diamonds; t2 , squares; t3 , triangles). Measurements were carried out in 10 mM Tris, pH 8.0, 100 mM NaCl, 5 mM MgCl2 , 10 mM b-ME, at 207C. Decay data were analyzed as sums of exponential components.

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FIG. 6. Lifetime distribution obtained with the MEM at 107C (continuous line) and 357C (broken line) for Trp-162 (a), Trp-163 (b), and Trp-165 (c). Measurements were carried out in 10 mM HEPPS, pH 8.0, 100 mM NaCl, 5 mM MgCl2 , 10 mM b-ME. Weighted residuals are shown in the insets.

ent temperature dependence. The emission maxima of the Trp residues studied here, which are in the 345- to 348-nm range, indicate that these residues are largely exposed to solvent. Although a precise estimate is not

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possible, the Arrhenius factors in Table IV probably take into account some contribution from the very temperature-dependent water quenching (17). In any case, they indicate low activation energies and frequency fac-

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FLUORESCENCE STUDY OF TetR LOOP 154–167 TABLE IV

Arrhenius Parameters of the Nonradiative Decay Rate A (s01)

Trp 162

163 165

t1 t2 t3 t2 t3 t2 t3

9.9 4.0 1.0 2.0 9.7 9.2 1.8

1 1 1 1 1 1 1

1010 109 1010 1010 108 108 109

Ea (kcal) 4.2 1.5 1.2 2.7 1.8 0.7 0.3

Note. The Arrhenius parameters have been determined from nonlinear least-squares analysis of 1/ti as a function of T. The lifetimes correspond to those reported in Fig. 7.

of the temperature (Fig. 8) allowed determination of the differences in standard enthalpy and entropy, DH7i, j and DS7i, j , using a two-parameter least-squares analysis (DH7 and DS7 independent of temperature):

DG7i, j Å DH7i, j 0 TDS7i, j.

[9]

FIG. 7. Temperature dependence of the lifetimes and corresponding amplitudes for the fluorescence decay of Trp-162 (a, b), Trp-163 (c, d), and Trp-165 (e, f) (t1 , a1 , triangles; t2 , a2 , squares; t3 , a3 , circles). Data correspond to averages ({standard deviation) of two experiments. When no error bar is seen, the standard deviation is smaller than the symbol. Measurements were carried out in 10 mM HEPPS, pH 8.0, 100 mM NaCl, 5 mM MgCl2 , 10 mM b-ME. Decay analyses were carried out with the MEM.

tors (less than 5 kcal/mol and 1011 s01, respectively) which are compatible with charge transfer as the main quenching mechanism (16). Since, for each Trp, there was no significant difference in the decay-associated spectra, under the assumption that each component had the same radiative decay rate and molar extinction coefficient, the relative concentrations of the lifetime-associated classes were proportional to their amplitudes at 350 nm. The thermodynamic parameters of the equilibrium between the three Trp classes were estimated from

DG7i, j Å 0RT ln ai /aj ,

[8]

where DG7i, j is the difference in standard Gibbs free energy between the classes associated with the decay components i and j and ai(j) are the corresponding amplitudes at 350 nm. The study of DG7i, j as a function

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FIG. 8. Differences in standard Gibbs free energy between the lifetime-defined classes for Trp-162 (a), Trp-163 (b), and Trp-165 (c). DG7i, j Å 0RT ln(ai/aj) is plotted as a function of T (DG71,2 , squares; DG71,3 , triangles; DG72,3 , circles). When no error bar is seen, the standard deviation is smaller than the symbol. The solid lines correspond to the linear least-squares fits of the data.

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ALBERTI ET AL. TABLE V

Thermodynamic Parameters of the Equilibrium between the Lifetime Defined Classes Trp

Species

162

1,3 1,2 2,3 1,3 1,2 2,3 1,3 1,2 2,3

163

165

DG7 (207) 0.42 0.23 0.19 0.83 1.08 00.25 1.20 1.23 00.03

{ { { { { { { { {

0.05 0.05 0.02 0.09 0.09 0.05 0.12 0.12 0.01

DH7 (kcal/mol) 03.6 02.5 01.1 010.3 06.8 03.8 018.8 015.9 02.8

{ { { { { { { { {

0.5 0.6 0.3 3.9 3.8 0.2 3.8 2.9 0.3

DS7 (e.u.) 013.7 09.6 04.2 038.9 027.9 012.0 067.8 058.0 09.2

{ { { { { { { { {

1.7 2.0 0.9 13.2 12.9 0.8 13.0 10.2 1.2

Note. The differences in standard enthalpy and entropy between the lifetime defined classes were determined by linear least-squares analysis of the differences in standard Gibbs free energy as a function of T reported in Fig. 8.

Such an analysis assuming a heat capacity change of zero is useful to give qualitative information on the changes in standard enthalpy and entropy. The limited temperature range and uncertainties on data do not allow reliable three-parameter analysis in order to estimate heat capacity changes. Thermodynamic parameters are reported in Table V. At 207C, the differences in standard Gibbs free energies between the different classes of a same Trp residue were less than 1.3 kcal/mol, due to compensation of enthalpy and entropy terms. For each Trp, the classes associated with components 2 and 3 had similar free energies (ÉDG72,3É £0.3 kcal/mol), whereas the class associated with component 1 had the highest free energy due to an unfavorable entropic term. DH71,2(3) and DS71,2(3) of W163 and W165 were very large. The plot of DH7i, j as a function of DS7i, j (Fig. 9) shows a linear relationship, typical of the enthalpy–entropy compensation effect.

In this model, each rotamer has a different proximity and orientation to protein functional groups which act as fluorescence quenchers and thus nonradiative decay rates (18, 19). In a few cases, it has been possible to correlate lifetime components with rotamers observed by NMR (20, 21) or calculated by molecular mechanic computations (22). Several nonradiative decay mechanisms can compete with the radiative emission of the indole moiety: excited-state charge and proton transfer, solvent quenching, and intersystem crossing. The intersystem crossing rate should not depend on the peculiar environment (16). Solvent quenching, which depends on the accessibility of the indole moiety and may be altered by the presence of charges in its environment, should have a marginal effect on lifetimes when more efficient nonradiative decay pathways are possible, since the water quenching rate of NATA at 207C is only 2.3 1 107 s01 (16). Proton transfer should be possible when the indole moiety is in the vicinity of a proton donor (23). Electron transfer from the excited state of the indole moiety to the backbone carbonyl groups is usually considered the main nonradiative decay pathway of Trp in proteins (16, 19, 24). Both proton and electron transfers are characterized by low activation energies and frequency factors (16, 23) and might be consistent with the observed values for these parameters. A preliminary modeling study of the loop (not shown) indicates that the Ne atom of Arg158 and Arg-171 might contact some of the rotamers of Trp-162 and Trp-163, respectively. On the other hand, Trp-165 should not be at the proximity of a proton donor. The similarity in lifetime values between the three Trp suggests that proton transfer should be marginal compared to electron transfer to carbonyl groups. At least for external residues, the stable rotameric positions of a side chain are mainly determined by the local secondary structure (25, 26). Consequently, lifetime values will depend mainly on the distance be-

DISCUSSION

The time-resolved fluorescence decay of the three Trp could be described by a triexponential decay law, with lifetimes in the 0.7- to 1.0-ns, 2.5- to 3.2-ns, and 6to 7-ns ranges (Tables II and III). Whatever the Trp position, the three lifetimes did not show significant dependence on the emission wavelength (Fig. 5). This is consistent with the hypothesis that the three decay components are related to the existence of three lifetime-associated classes of Trp residues. The absence of lifetime redistribution with temperature (Fig. 4) indicates that these classes do not interconvert on the fluorescence timescale (nanosecond). The tryptophan multiexponential fluorescence decay is usually interpreted as arising from the existence of different rotameric orientations of the Trp side chain.

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FIG. 9. Correlation between the differences in standard enthalpy and entropy for the three lifetime-defined classes of Trp-162, Trp163, and Trp-165. The solid line corresponds to the linear leastsquares fit of the data.

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FLUORESCENCE STUDY OF TetR LOOP 154–167

tween Trp rotameric minima and carbonyl groups, i.e., on the secondary structure. This is consistent with the assumption that Trp fluorescence decay could act as a probe of the protein secondary structure (27–29). Whatever the Trp studied here, the lifetime values had only a very weak temperature dependence. Within the framework of the rotamer model, this indicates no change in the relative positions of rotamers and quenchers and strongly suggests that the local secondary structure of the loop, at least in the 162–165 range, is not altered by temperature. A striking result of our temperature study is the large differences in standard enthalpy and entropy between the lifetime-associated Trp classes (Table V). A NMR study has shown that the differences in enthalpy and entropy (TDS) between the three x1 rotamers of a Trp side chain within oligopeptides typically ranged from less than 1 to 3 kcal/mol, depending on the peculiar sequence of the oligopeptide bearing the Trp side chain (30). The values of DH71,2(3) and DS71,2(3) for Trp163 and Trp-165 are very high (Table V). For comparison purposes, the standard enthalpy change associated with the formation of a b-cyclodextrin–indole complex is 04 kcal/mol (31). The standard enthalpy change associated with solvation of a Trp side chain is estimated to be in the 010 to 015 kcal/mol range (32, 33). However, the decay-associated spectra (Fig. 5) indicate that the different rotamers of each Trp have a similar solvent accessibility and rule out large changes in the solvation of the Trp side chain to explain the thermodynamic parameters measured. In addition, the absolute value of the enthalpy change for a Trp side chain moving from a buried to a fully solvent-exposed environment should be markedly lower than that of the enthalpy change related to solvation because of the loss of intramolecular interactions (32). The very high values of DH71,2(3) and DS71,2(3) for Trp163 and Trp-165 strongly suggest that the changes in rotamer distribution are coupled to a temperature-dependent conformational change in the protein. This conformational change would alter the rotamer distribution due to different tertiary environments. The observed DG7i, j would depend on the equilibrium constant for the protein conformational change and on the equilibrium constants for the rotamer distribution in each conformation. The protein would be shifted at high temperature toward a conformation unfavorable to the rotamer 1 of the Trp-163 and Trp-165 side chains for steric reasons. The differences in standard enthalpy and entropy between rotamers 2 and 3 are much lower than those between rotamers 1 and 2 (3), indicating that the equilibrium between conformers 2 and 3 does not depend markedly on the protein conformational change. The different behavior of Trp-162 might be due to the fact that rotamer 1 of Trp-162 would be possible for both protein conformations or to a specific effect

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of the T162W mutation on the protein conformational change. The linear relationship between DH7 and DS7, analogous to that observed for families of reactions in water (32, 34), corroborates the hypothesis of a coupling between rotamer distribution and protein conformational change. From a microscopic point of view, a sufficient condition to have a linear relationship between DH7 and DS7 for a family of reactions is that the number of thermal modes (i.e., degrees of freedom at thermal equilibrium at temperature T) undergoing a transformation varies systematically among the family members (35). The thermal modes which vary in a family of reactions do not involve the reaction centers directly, but secondary, or background, structural components which are coupled to the primary reaction centers (35). This strongly suggests that the overall thermodynamics of the rotamer equilibrium are mainly determined by background structural regions of the protein (and interacting solvent) coupled to the rotamer equilibrium. The very high values of DH71,2(3) and DS71,2(3) observed for the equilibrium between rotamers 1 and 2 (3) of Trp-163 and Trp-165 should reflect a temperature-dependent conformational change of a large part of the protein. Although we cannot exclude involvement of other parts of the protein, the plainest candidate for such a conformational change is the loop 154–167. As a matter of fact, the structure of the loop where the Trp studied are located has not been resolved (2). In addition, time-resolved anisotropy measurements on the three single Trp mutants indicate high-order parameters for the loop backbone motion in solution (S2 ú 0.75) (Vergani et al., to be published elsewhere). This strongly suggests the existence of several discrete conformations of the loop. The assumption of an equilibrium between (at least) two conformations of the loop is not inconsistent with the assumption that the secondary structure of the 162–165 sequence is not altered with temperature (see above). Hinged ‘‘lid’’ motion has already been observed for, e.g., the triosephosphate isomerase loop (36). Our time-resolved fluorescence data would be consistent with such a model, at least for the C-terminal half of the loop 154–167. It is noteworthy that the loop segment studied here is delimited by two proline residues (Pro-161 and Pro167), whose cis–trans isomerization could act as a hinge motion. Such a loop motion might regulate the accessibility of the tetracycline binding site and be crucial for the mechanism of allosteric regulation of TetR. ACKNOWLEDGMENTS We thank Pr. Nardelli (Milan) for stimulating discussions and Dr. W. Hinrichs (Berlin) for providing us with the coordinates of class D TetR in the complex with Tc. We acknowledge the support provided by the Erasmus scholarship program of the ECC to P. Alberti and E. Bombarda.

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