Tryptophan Repressor of Escherichia Coli Shows Unusual

Proc. Nat!. Acad. Sci. USA Vol. 85, pp. 6731-6732, September 1988 Biophysics Tryptophan repressor of Escherichia coli shows unusual thermal stability (protein denaturation/scanning calorimetry) SONG-JA BAE*, WEI-YUAN CHOUt, KATHLEEN MATTHEWSt, AND JULIAN M. STURTEVANT* *Departments of Chemistry and of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511; and tDepartment of Biochemistry, Rice University, Houston, TX 77251 Contributed by Julian M. Sturtevant, June 13, 1988

ABSTRACT Differential scanning calorimetry demon- strates that the tryptophan repressor of Escherichia coli is unusually resistant to thermal denaturation. The dimeric protein undergoes reversible dissociative unfolding at pH 7.5 I-w centered at about 900C. The thermal stability may be due in part to the unusual structure of the protein, which is composed U. of two identical intertwined polypeptide chains. LU

u) The tryptophan (trp) repressor from Escherichia coli is Cf) involved in regulating the expression of biosynthetic en- CU) zymes for tryptophan in response to the level of this amino x acid in the cell. Tryptophan binds to the repressor and increases its affinity for the operator target site within the trp promoter, thereby preventing transcription initiation. The trp repressor protein is a dimer of molecular weight 24,700 (1) TEMPERATURE,,C with two binding sites for tryptophan and a single operator DNA binding site. The three-dimensional structure of this FIG. 1. Tracings ofDSC scans oftrp repressor after deduction of protein has been determined (2). The monomer structure the instrument baseline. Protein concentration was 255 ,uM in 0.05 M consists of six helical regions, which are intertwined with the phosphate buffer, pH 7.0/0.1 M NaCl. The scan rate was 1 Kemin - 1. corresponding helices ofthe companion monomerto form the The first scan (curve A) was interrupted at 100TC, the protein solution dimeric structure. The significant intersubunit was cooled in situ over a period of about 30 min and then was contacts seen rescanned (curve B). The dashed lines are the pre- and post- in the crystallographic analysis suggest that a stable dimeric transition baselines. The tracings illustrate realistically the very low structure might be anticipated for this protein. We report here noise level of the original recordings. the results of differential scanning calorimetric (DSC) exper- iments that show trp repressor to be remarkably thermally weight; no contaminants were detected when the gel was stable and will report elsewhere more detailed data of the overloaded. effects on the thermal denaturation of variations in pH, the Before introduction into the calorimeter, the protein was addition ofligands, and other experimental conditions as well dialyzed against 10 mM potassium phosphate/100 mM KCl, as the replacement of single amino acid residues. pH 7.5. The dialysate served as the reference solution in the calorimeter. Protein concentrations were estimated by taking MATERIALS AND METHODS the absorption of a 1 mg-ml-1 solution to be 1.20. The tryptophan repressor was isolated in the following The DSC experiments were run at a scan rate of 1 K-min' manner. Bacterial cells containing a multicopy plasmid in a DASM4 scanning microcalorimeter (5) purchased from (pJPR2) that overproduces the trp repressor were obtained Mashpriborintorg, Moscow. from C. Yanofsky (Stanford University). Procedures for cell growth were those described by Paluh and Yanofsky (3). The RESULTS AND DISCUSSION protein was purified by using the procedure ofJoachimiak et al. (4) with the changes delineated below. The cell suspension A tracing of a typical DSC curve is shown in Fig. 1. As was sonicated 30 ml at a time for a total duration of3 min (the indicated in the tracing, no noise was visible in the original cycle was sonication for 30 s followed by cooling for 30 s). recording. The initial scan (curve A) was interrupted at The ammonium sulfate pellet was suspended in 0.01 M 100TC, the material was cooled over a period of 30 min in the potassium phosphate, pH 7.4/0.1 mM EDTA/0.2 M KCl, and calorimeter, and then it was rescanned (curve B). It is evident the suspension was dialyzed against this buffer. After dialy- that the denaturation was essentially fully reversed during the sis, the sample was applied directly to a phosphocellulose cooling, showing this to be a most unusually thermally stable column equilibrated in sample buffer, and the column was protein. It seems likely that this stability is at least in part due developed with the same buffer. The trp repressor was eluted to the unusual structure of the protein, with intertwined by sample buffer containing 0.5 M KCl. The fractions polypeptide chains. containing the repressor were pooled and concentrated to :8 The curves in Fig. 1 are asymmetric, being sharper on the mg/ml. Polyacrylamide gel electrophoresis of the purified high-temperature side than on the low-temperature side. A protein yielded a single component ofthe expected molecular possible cause of this type of asymmetry is a decrease in the extent of oligomerization during the transition (6). It was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: DSC, differential scanning calorimeter (calorimetric, in accordance with 18 U.S.C. §1734 solely to indicate this fact. calorimetry). 6731 Downloaded by guest on September 29, 2021 6732 Biophysics: Bae et al. Proc. Natl. Acad. Sci. USA 85 (1988) Table 1. The thermal unfolding of tryptophan repressor at pH 7.5 AHvH, kcal mol Protein, AHc,,, Curve Ace, SD, AuM tmi, C tA2, 0C kcal-mol-' fitting Eq. 1 cal-K-g-1 % cexa 70.4 90.8 90.80 74.0 126 128 -0.034 1.8 102.0 91.6 91.55 71.0 135 131 -0.037 3.7 136.0 92.6 90.25 100.8 103 102 0.035 3.5 142.8 92.2 88.47 97.3 91 88 0.136 2.8 204 92.7 90.26 87.3 108 108 0.081 1.3 255 93.5 90.14 106.0 100 100 0.086 2.2 340 93.5 90.39 100.8 98 98 0.087 4.1 676 94.0 90.41 95.3 95 95 0.118 2.5

Mean ± SEM 90.30 ± 0.35 91.6 ± 5.3 107 ± 6 106 ± 6 0.059 ± 0.027 2.6 ± 0.5 found that the data could be fit with reasonably small standard concentrations, values for AHVH (column 6) calculated by deviations to the model A2 a± 2B, using the calculational means of the equation procedure outlined earlier (7). Fig. 2 illustrates the closeness of fit obtained. In Fig. 2 the solid line represents the observed data, AHVH = ARTi12 &/29 [l] the filled circles represent the calculated values, and the dashed A/Rcal curve represents the calculated baseline. The initial and final where the factor A has the value 6 for the dissociative baselines (not shown) were evaluated by linear least-squares denaturation ofa dimeric species, agree reasonably well with treatment ofthe data obtained at temperatures below 67TC and AH,.,; In this expression Ti,2 is the absolute temperature at above 1050C. half completion of the denaturation, c",1/2 is the apparent excess specific heat at this temperature, and Ah~a is the Data for eight experiments are summarized in Table 1. For specific denaturational enthalpy. unknown reasons, the calorimetric enthalpies were low and If the assumed model is' appropriate for this system, an the van't Hoffenthalpies were high for the experiments at the additional value for AHVH should be given by -RS, where S two lowest protein concentrations, and negative values were is the slope of a van't Hoff plot of ln[P]. vs 1/Tm or 1/Ti/2. observed for Ace, the excess specific heat. The validity ofthe [P]0 is the total protein concentration, and Tm is the absolute assumed model is supported by the close agreement between temperature at which the excess specific heat is maximal. It the values for the van't Hoffenthalpy, AHvH, obtained in the is evident that the values of ti,2 = Ti,2 - 273.15 remain curve fitting (column 6 in Table 1) with those for the constant within experimental uncertainty. A van't Hoff plot calorimetric enthalpy, AHIaI (column 5). Further support for using Tm leads to AHVH = 169 kcalmol- 1, with a coefficient the model is given by the fact that, at the six highest protein of determination r2 = 0.90. This enthalpy value is nearly twice as large as expected. We can offer no explanation at 0.40 present for this discrepancy or for the apparent insensitivity of ti,/ to protein concentration. It is evident in Fig. 2 that the post-transition baseline has a negative slope, as was observed also in nearly all the other experiments. It is unusual for a protein to have its apparent specific heat decrease with increasing temperature. In view 0.3) F of the fact that the behavior of'the calorimeter is somewhat 8cm less reliable at very high temperatures than in the more usual ,..d temperature range, further work is required to establish the 9-2 validity of the -a observation of an abnormal temperature co -W dependence of the specific heat. Li 0.20 F We have mentioned several abnormalities encountered in C-) the present work which we CL) hope will be resolved by further C=L cn research. Despite these complications, we have firmly es- U) CL. tablished that the stability ofthe trp repressor against thermal Li D.< LL.j unfolding is most unusual and that the unfolded protein also resists irreversible degradation of its separated polypeptide D.to01 chains to an unusual degree. This research was supported in part by grants from the National Institutes of Health (GM04725 and GM22441), the National Science Foundation (DMB8421173), and the Robert A. Welch Foundation (C576). 0.0( 0 60 70 80 90 100 110 1. Gunsalus, R. P. & Yanofsky, C. (1980) Proc. Natl. Acad. Sci. USA Temperature, 'C 77, 7117-7121. 2. Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L. & Sigler, P. B. (1985) Nature (London) 317, 782-790. FIG. 2. A DSC scan of trp repressor illustrating the curve fitting 3. Paluh, J. L. & Yanofsky, C. (1986) NucleicAcids Res. 14,7851-7860. according to the model A2 ;± 2B. Protein concentration was 204 ,uM. 4. Joachimiak, A., Schevitz, R. W., Kelley, R. L., Yanofsky, C. & The experimental curve is the solid curve, the filled circles are Sigler, P. B. (1983) J. Biol. Chem. 258, 12641-12643. calculated points, and the dashed curve is the baseline calculated in 5. Privalov, P. L. (1980) Pure Appl. Chem. 52, 479-497. the curve-fitting process. The calculated points deviate from the 6. Manly, S. P., Matthews, K. S. & Sturtevant, J. M. (1985) Biochem- experimental curve with a standard deviation of1.3% ofthe maximal istry 24, 3842-3846. value of the excess specific heat. 7. Sturtevant, J. M. (1987) Annu. Rev. Phys. Chem. 38, 463-488. Downloaded by guest on September 29, 2021