Photochemically Induced Dynamic Nuclear Polarization Investigation Of

Proc. Natl. Acad. Sci. USA Vol. 77, No. 9, pp. 5145-5148, September 1980 Biochemistry

Photochemically induced dynamic nuclear polarization investigation of complex formation of the NH2-terminal DNA-binding domain of lac repressor with poly[d(AT)] (lac repressor DNA binding/photochemically induced dynamic nuclear polarization 'H NMR) F. BUCK*, H. ROTERJANS*, R. KAPTEINt, AND K. BEYREUTHERt *Institut fur Physikalische Chemie der Universitit, Mfinster, West Germany; tFysisch Chemisch Laboratorium der Rijksuniversiteit, Groningen, The Netherland; and tInstitut fur Genetik der Universitit, K6ln, West Germany Communicated by Robert G. Shulman, June 9,1980

ABSTRACT The interaction of the NH2-terminal DNA- ification or substitution of tyrosines may lead to a loss of the binding domain of Iac repressor with synthetic oligold(AT)J was DNA-binding capacity in an indirect way simply by impairing studiedby a photo-CIDNP technique (CIDNP is chemically induced dynamic nuclear polarization). Three of the four ty- the structure of the DNA-binding domain (14). rosines of the NH-terminarregion were found to be accessible In order to establish which of the four tyrosine residues of the to the photosensitive dye. The corresponding ring proton reso- DNA-binding domain of lac repressor is directly involved in nances were enhanced in the photo-CIDNP 'H NMR spectrum, the DNA binding, we have made use of the photo-CIDNP and the only histidine (histidine 29) was located at the surface method (CIDNP is chemically induced dynamic nuclear po- of the domain, which is supposed to be linked to the core protein larization) (15, 16). In this method the NMR signal intensities of lac repressor by a fle e hinge region. After complex formation of the NH-terminal region with oligo[d(AT)J, two of the of the aromatic amino acids tyrosine, histidine, and tryptophan three tyrosine residues were no longer accessible to solvent or can be selectively enhanced if their side chains are accessible to photosensitive dye, which is strong evidence that the two to attack by a photoexcited dye (16). Hence, it is possible to tyrosines are part of the contact region. distinguish between aromatic amino acid side chains that are located at the surface of a protein and internal side chains that Specific binding of lac repressor to lac operator and nonspecific are buried and thus protected against attack by the dye. A binding to nonoperator DNA and to synthetic polynucleotides protective effect may also occur upon complexation of a protein have been studied by various methods (for reviews see refs. 1-3). with ligands. Disappearance of the photo-CIDNP effect then It was shown that the NH2-terminal region (residues 1-59) of identifies residues involved in the interaction. Thus, we have the lac repressor has a strong nonspecific affinity for DNA (4, previously demonstrated the involvement of gene-5 protein 5) and binds specifically to the operator (6, 7). with oligonucleotides (17). Here we wish to report a photo- It has been demonstrated by 1H NMR spectroscopy (8, 9) that CIDNP analysis of the tryptic NH2-terminal region of the lac this NH2-terminal region forms an independent structural repressor protein and of its complex with oligo[d(AT)]. Because domain whether it is part of the complete repressor or separated the isolated NH2-terminal region retains its structural integrity, from the core by proteolytic cleavage. This was concluded from the results have bearing on the nonspecific DNA-binding a comparison of the 1H NMR spectrum of the intact tetrametric properties of the native lac repressor. lac repressor with that of the isolated monomeric NH2-terminal domain (residues 1-51 and 1-59) and of the remaining tetra- MATERIALS AND METHODS meric repressor core (8, 9). The narrowness of the linewidths shows that the DNA-binding domains have a faster tumbling Preparations. The lac repressor of Escherichia coli (strain rate than would be expected if they were an integral part of the BMH 593) was isolated and purified as described (8). The repressor. Thus, it is likely that residues 50-60 form a flexible NH2-terminal region was prepared by tryptic digestion of lac hinge which allows the DNA-binding domain the observed high repressor under the conditions reported by Geisler and Weber degree of motion with respect to the core. (4). In order to decrease the ionic strength of the solution a Amino acids 1-59 of the lac repressor contain four tyrosine 10-fold volume of equilibration buffer (50 mM K2HPO4/ residues at positions 7, 12, 17, and 47 and a single histidine at KH2PO4, pH 7.0) was added. The lac repressor fragments were position 29 (10). From a study of mutant lac repressors with separated on phosphocellulose (Whatman p. 11; 2.2 X 25 cm) amino acid substitutions, tyrosine-17 and, to a lesser extent, equilibrated with the same buffer. The lac repressor core pro- tyrosine-7 have been implicated in DNA binding (11). Chem- tein does not bind to phosphocellulose, whereas the NH2-ter- ical modification (iodination and nitration) of tyrosine-7 and minal region is retained under these conditions. The column -17 also resulted in loss of DNA binding (12, 13). However, both was washed with equilibration buffer until the absorbance of residues 7 and 17 are simultaneously affected by chemical the effluent at 230 nm was equal to that of the buffer. Elution modification so that it is not clear whether the effect is due to was with a 400-ml linear gradient containing 0-1 M KCI in the one or both of these residues. Also, these experiments do not equilibration buffer. The fraction containing the NH-terminal constitute a clear proof of the direct participation of these two was eluted at 0.44 M KCl. Because the pH of the phosphate tyrosine residues in the DNA-binding process; chemical mod- buffer is decreased due to the addition of neutral salt, a pH gradient is obtained. The elution diagram is shown in Fig. 1. The publication costs of this article were defrayed in part by page Because contamination with small amounts of trypsin was charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviation: CIDNP, chemically induced dynamic nuclear polar- this fact. ization. 5145 Downloaded by guest on October 2, 2021 5146 Biochemistry: Bucket al. Proc. Natl. Acad. Sci. USA 77 (1980)

B O

02-

0.1-

20 40 60 80 Fraction FIG. 1. Separation of the tryptic NH2-terminal region and core of lac repressor (30 mg) on phosphocellulose. The NH2-terminal region is found in fractions 73-80 and the core in fractions 3-32. The gradient was applied at fraction 41; fraction size was 4.0 ml. possible, the fraction containing the NH2-terminal region was also filtered through a lima bean trypsin inhibitor affinity column (volume of 2 ml) prepared as described (18). Thus, large amounts of a mixture containing the large (residues 1-59) and the small (residues 1-51) NH2-terminal regions can be isolated in a convenient way. Because there is no indication of structural . I differences between the large and small NH2-terminal regions 9 8 7 according to previous 'H NMR investigations (8, 9), the two 6, ppm regions were not separated. FIG. 3. (A) Aromatic region of the 360 MHz 1H photo-CIDNP Poly[d(AT)] of high molecular weight (5 X 106) was pur- difference spectrum (light minus dark) of0.5 mM NH2-terminal re- chased from Boehringer Mannheim. Oligo[d(AT)] of an average gion of lac repressor in 2H20 (pH 4.60, 0.1 M NaCl); 20 scans were chain length of about 40 base pairs was prepared by extensive accumulated. (B) Dark spectrum. sonification (19) and characterized by polyacrylamide gel electrophoresis (20). of the pH meter readings were made for the 2H effect of the NMR Spectra. 1H NMR measurements were carried out at electrode. 270 MHz with a Brucker WH 270 spectrometer in the Fourier Photo-CIDNP Measurements. 1H photo-CIDNP spectra transform mode with a deuterium lock system. Probe diameter were taken at 360 MHz on a Bruker HX-360 NMR spectrom- was 10 mm. All chemical shifts were determined relative to the eter. Samples to which a flavin dye (3-N-carboxymethyllumi- [2,2,3,3-2H4]trimethylsilylpropionic acid signal. pH was mea- flavin) was added were irradiated in the NMR probe by 0.6-sec sured with a Radiometer pH meter (PHM 26) in connection light pulses from a Spectra Physics model 171 argon ion laser with a combined glass electrode (Ingold, Frankfurt, W. Ger- (multiline, 7 W) prior to data acquisition. Alternating "light" many). All spectra were taken in 2H20 solution. No corrections and "dark" free induction decays were collected, and sub-

r T j 7, 17

47 12

9.0 8.0 7.0 6.0 a, ppm FIG. 2. Aromatic region of the 270 MHz lH NMR spectrum of the NH2-terminal region of lac repressor (0.4 mM in 2H20, pH 4.60). As- signments of the tyrosine lines (made by 0. Jardetzky, see text) are indicated. Downloaded by guest on October 2, 2021 Biochemistry: Bucket al. Proc. Natl. Acad. Sci. USA 77 (1980) 5147 traction yielded the photo-CIDNP difference spectrum. In- cluding delays, a full light-dark cycle took 15 sec; typically 20 A scans were accumulated. A more complete description of the experimental method has been given in ref. 16. RESULTS AND DISCUSSION Free NH2rterminal region In Fig. 2 the aromatic region of the 270 MHz 'H NMR spectrum of the NH2-terminal region of lac repressor is shown. Histidine-29 shows singlets at 8.65 and 7.31 ppm for the C2 and C4 protons, respectively. The four tyrosines give rise to pairs of doublets, indicating that their rings are free to make rapid 180° flips about the C,6-Cy bonds. The tyrosine lines have re- B cently been tentatively assigned to specific residues by 0. Jar- detzky (personal communication) using a chemical modification procedure. This assignment is also indicated in Fig. 2. In Fig. 3A the photo-CIDNP difference spectrum of the NH2-terminal region is shown; for comparison the dark spectrum is shown in Fig. SB. All aromatic residues are polarized. Strong emission effects are observed for tyrosine-47 at 6.88 ppm and for tyrosine-7 and -17 at 6.5 ppm. The strong polarized lines must be assigned to 3,5-ring protons and indicate that the three tyrosines, at 7, 17, and 47, are fully accessible to the photoexcited dye. The shoulder of the line at 6.5 ppm, which becomes more pronounced at higher pH, shows that, indeed, both tyrosine-7 and -17 contribute to this line, as was further sub- stantiated by the measurements on the complex between the NH2-terminal region and oligofd(AT)] discussed below. The

emission at 7.1 ppm belongs to the 2,6-protons of tyrosine-7, -17, I I and -47. Small negative polarization for tyrosine 2,6-protons 9 8 7 6 6, ppm FIG. 5. Aromatic region of the 360 MHz 1H photo-CIDNP difference spectrum of0.5 mM NH2-terminal region of lac repressor with about 2.0 mM oligo[d(AT)J at pH 7.00. (A) With 0.1 M NaCI; (B) with 1 M NaCL. A is usually observed (16), and results from transfer of polarization from the C2 and C4 protons of histidine-29 show that this is also a surface residue. A small but distinct emission is observed at 6.68 ppm for tyrosine-12. However, its origin is not clear. Either tyrosine-12 is directly polarized to a small extent or the emission B arises from polarization transfer from tyrosine-7 or -17 (or both). In any case, the accessibility of tyrosine-12 is much smaller than that of the other three tyrosines. NH2rterminal region-oligo[d(AT)] complex In Fig. 4A the aromatic region of the 1H NMR spectrum of the oligo[d(AT)] sample is shown. The chemical shift values are in agreement with those observed by Patel and Cannel (2-1) for double-helical poly[d(AT)] of higher molecular weight at ele- vated temperatures. Linewidths of about 20 Hz for the signals of the aromatic ring protons of the bases and the anomeric CI protons of the sugar are observed. From these observations it can be concluded that the present oligo[d(AT)] sample pref- erentially exists in the double-helical form. In the spectrum of the NH2-terminal region-oligo[d(AT)] complex (Fig. 4 C and D), the resonances of the NH2-terminal region, as far as they can be observed despite overlaps with the broad and strong signals of oligo[d(AT)], are broadened in the complex due to 9.0 8.0 7.0 6.0 the binding to double-helical oligo[d(AT)]. Only minor 6, PPm chemical shift changes of the order of 0.2 ppm are observed. Fig. 5 shows the photo-CIDNP difference spectra of the FIG. 4. 1H NMR absorption region of aromatic amino acid resi- NH2-terminal region-oligo[d(AT)] complex in 0.1 M and in 1 dues of poly[d(AT)] (A), the NH2-terminal region of lac repressor (B), and the poly[d(AT)J-NH2-terminal region complex at two pH values M NaCL. At an ionic strength of 0.1, the complex is stable, (C and D). pH values: A, pH 7.10; B, pH 5.64; C, pH 7.18; D, pH whereas in 1 M NaCl it dissociates. At low ionic strength (Fig. 5.44. 5A) the photo-CIDNP spectrum still shows a strong emission Downloaded by guest on October 2, 2021 5148 Biochemistry: Bucket al. Proc. Natl. Acad. Scd. USA 77 (1980)

at 6.88 ppm, the position of the 3,5-protons of tyrosine-47, We are grateful to Dr. 0. Jardetzky for communicating to us the whereas the polarization for tyrosine-7, -12, and -17 and for tyrosine assignments of the NH2-terminal region of lac repressor prior histidine-29 has largely disappeared. Hence, access to the side to publication. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ru 145/4) and by the Netherlands Foun- chains of tyrosines-7 and -17 and of histidine-29 must be dation of Chemical Research (SON) with financial aid from the blocked in the complex (the situation of tyrosine-12 is not clear), Netherlands Organization for the Advancement of Pure Research and tyrosine-47 remains freely accessible. Upon dissociation (ZWO). of the complex in 1 M NaCL (Fig. 5B), the polarizations for tyrosine-7 and -17 and, albeit weakly, for histidine-29 reappear. The lines of the 3,5-protons of tyrosine-7 and -17 are now separated, whereas for the NH2-terminal region alone (Fig. 3A) only a shoulder was observed. This small increase in chemical shift difference leads to the conclusion that even at high ionic 1. Muller-Hill, B. (1975) Prog. Biophys. Mol. Biol. 30,227-252. strength, weak residual interactions between the NH2-terminal 2. Bourgois, S. & Pfokl, M. (1976) Adv. Protein Chem. 30, 1-99. and exist. 3. Miller, J. H. & Reznikoff, W. S., eds. (1978) The Operon (Cold region oligo[d(AT)] Spring Harbor Laboratory, Cold Spring Harbor, NY). Conclusions 4. Geisler, N. & Weber, K. (1977) Biochemistry 16,938-943. Because for tyrosine and histidine the primary step for the 5. Muller-Hill, B., Heiderker, G. & Kania, J. (1976) in Proceedings photoreaction most probably is hydrogen atom abstraction by of the Third John Innes Symposium, Structure and Function W. the excited flavin, the photo-CIDNP experiments on the of Relationship of Proteins, eds. Markham, R. & Home, R. 167-179. region of lac repressor have established that (North-Holland, New York), pp. NH2-terminal 6. Ogata, R. T. & Gilbert, W. (1978) Proc. Nati. Acad. Sci. USA 75, tyrosine-7, -17, and -47 and histidine-29 have exposed OH (or 5851-5854. NH) groups. Possibly this is true for tyrosine-12 as well, but to 7. Ogata, R. T. & Gilbert, W. (1979) J. Mol. Biol. 132,709-728. a lesser extent. Both the 1H NMR shifts (as was noted in ref. 9) 8. Buck, F., Ruiterjans, H. & Beyreuther, K. (1978) FEBS Lett. 96, and the photo-CIDNP results suggest that tyrosine-7, -12, and 335-3. -17 are in a stacking arrangement whereby tyrosine-7 and -17 9. Wade-Jardetzky, N., Bray, R. P., Conover, W. W., Jardetzky, O., are fully exposed and tyrosine-12 is sandwiched between the Geisler, N. & Weber, N. (1979) J. Mol. Biol. 128,259-264. other two. 10. Beyreuther, K., Adler, K., Geisler, N. & Klemm, A. (1973) Proc. The photo-CIDNP experiments on the NH2-terminal re- Nati. Acad. Sd. USA 70,3576-3580. gion-oligo[d(AT)] complex show that tyrosine-7 and -17 and 11. Miller, J. A., Coulondre, U., Schmeisser, U., Schmitz, U. & Lu, histidine-29 are involved in DNA binding. The CIDNP evi- P. (1975) in Protein-Ligand Interactions, eds. Sund, H. & Blauer, dence is more direct than that previously obtained because the G. (de Gruyter, Berlin), pp. 238-252. experiment was carried out on the intact NH2-terminal region 12. Fanning, T. (1975) Biochemistry 14,2512-2530. from native lac repressor and does not rely on repressors that 13. Alexander, M., Burgum, A., Noall, R., Shaw, M. & Matthews, K. are chemically or genetically modified. Interestingly, histi- (1977) Biochim. Biophys. Acta 493,367-379. dine-29 had not previously been implicated in DNA binding, 14. Beyreuther, K. (1978) in The Operon, eds. Miller, J. H. & although its neighbor, glutamine-26, has (11). The mode of Reznikoff, W. S. (Cold Spring Harbor Laboratory, Cold Spring interaction of the tyrosines cannot be established with certainty. Harbor, NY), pp. 123-154. 15. Kaptein, R., Dijkstra, K. & Nicolay, K. (1978) Nature (London) Lack of significant chemical shift changes upon complex for- 274,293-294. mation argues against strong stacking interactions with nucleic 16. Kaptein, R. (1978) in NMR Spectroscopy in Molecular Biology, acid bases. In this respect the situation is quite different from ed. Pullman, B. (D. Reidel, Dordrecht, Holland), pp. 211-229. the case of the gene-5 protein binding to single-stranded DNA. 17. Garssen, G. J., Kaptein, R., Schoenmakers, J. G. G. & Hilbers, C. There, both a suppression of the CIDNP effect and large shifts W. (1978) Proc. NatI. Acad. Sci. USA 75,5281-5285. of the tyrosine protons indicate that intercalation with the bases 18. Otsuka, A. & Price, P. (1974) Anal. Biochem. 62,180-187. does occur (17). With the NH2-terminal region of lac repressor 19. Davis, A. & Phillips, D. (1978) Biochem. J. 173, 179-183. some interactions could still occur with fortuitous cancelling 20. Maniatis, T., Jeffrey, A. & van de Sande, H. (i975) Biochemistry of ring-current shifts, but hydrogen bonding of the hydroxyl 14,3787-3794. groups of tyrosine-7 and -17 could also explain the present 21. Patel, D. & Cannel, L. (1976) Proc. Nati. Acad. Sci. USA 73, photo-CIDNP results. 674-678. Downloaded by guest on October 2, 2021