Inl. J . Peptide Protein Rex 33, 1989, 256-262

Determination of the contributions of individual aromatic residues to the CD spectrum of IL-lD using site directed mutagenesis

STEWART CRAIG’+, ROGER H. PAIN’, URSULA SCHMEISSNER, RICHARD VTRDEN’ and PAUL T. WINGFIELD’?

‘Dept. of’ Biochemistry and Genetics, University of Newcastle upon Tyne, UK; ’Biogen S.A. , Carouge, Geneva, Snitzerland

Circular dichroism (CD) spectra have been measured in the aromatic region for recombinant human interleukin IL-IP and for site specific mutants in which each of the four tyrosines and the single tryptophan residue have been replaced one at a time by phenylalanine. These mutant proteins have been shown to have conformations that are closely similar to that of the wild type protein. By comparing the spectrum of each mutant with that of the wild type human protein it has been possible to assess the contribution of each aromatic residue to the CD spectrum of the latter. It has been shown that the spectrum is largely a result of contributions from Trp 120 and from Tyr 68.

Kej words: circular dichroism; interleukin IL- I p; protein circular dichroism; site specific mutagenesis; spectroscopic assignment: tryptophan dichroism: tyrosine dichroism

The technique of site directed mutagenesis is being increasingly used in studies of enzyme catalysis and in stabilising protein structures (for a review, see ref. I ) . Commercially it has a great potential, allowing changes to be engineered into a protein molecule in order to improve structure and function of drugs and enzymes and make them more effective.

Interleukin- Ip is an interesting molecule, being a cytokine displaying a wide range of biological activi- ties involved in immune and inflammatory responses (2). The human gene for this molecule has been cloned and large amounts of pure recombinant protein can be produced which are chemically identical to the auth- entic human protein (3). The conformation and stabil- i ty of the wild type recombinant molecule has been characterized as well as its folding and unfolding properties (4). Single site mutants of IL-IP have been constructed in which each of the four tyrosines (re- sidues 24, 68,90 and 121)* and the single tryptophan

Present addresses: + British Biotechnology Ltd, Watlington Road, Cowley, Oxford, OX4 5LY, UK; tGlaxo Institute for Molecular Biology, 3 Route des Troinex, F-1227 Carouge, Geneva, Switzer- land. Abbreviations: IL-lp, interleukin-l/3; CD. circular dichroism; u.v.. ultra-violet; MES. 2-[N-morpholino]ethanesulphonic acid; TRIS, tris(hydroxymethy1)-aminoethane; EDTA, ethylenediaminetetra- acetic acid. *The residue numbers used in this paper are those for the mature protein, residue 1 of which corresponds to Ala I17 from the cDNA scquence as listed previously (5).

residue 120 has been substituted by phenylalanine. The sequence specific ‘H-n.m.r. assignments for most of these mutants have been previously reported (6) and their conformational similarity to the wild type protein noted.

Circular dichroism is frequently used to charac- terise the conformation of proteins in solution, the near U.V. CD spectrum in particular providing a sen- sitive “finger print” of the native state (see for example ref. 7). In addition this spectrum provides a useful parameter by which to follow unfolding and refolding transitions. It is generally the case that the whole spectrum changes uniformly in intensity during such transitions, which, given the assumption that different aromatic residues will make different contributions to the spectrum (8), provides support for the cooperat- ivity of unfolding. For this reason, however, it has been difficult to separate out the contributions of individual aromatic residues in a protein. This study makes use of site specific mutagenesis to provide evidence for different contributions from the four tyr- osine and one tryptophan residue in IL-IB.

EXPERIMENTAL PROCEDURES

All chemicals used were Analar grade (B.D.H. Poole, Dorset, UK) unless otherwise stated.

Buffers. 0.01 M MES, 0.09 M NaCI, 1 mM Na,EDTA, pH 6.5, were used unless otherwise stated.

256

Aromatic residue contributions to CD

increases in the phenylalanine fine structure to varying degrees, the most notable being the Trpl2O --+ Phe substitution (data not shown). Comparison of the second derivative spectrum of wild type human IL- 1 (Fig. 1, spectrum 3) with those for mutants containing substituted tyrosyl residues (Tyr -+ Phe; Fig. 1, spectra 1 , 2, 4, and 6) indicates increases in the inten- sities of the spectral bands corresponding to Phe (250- 270nm). The increase is greatest for the Tyr 121 --+ Phe, suggesting a perturbation of residue 121 by the vicinal Trp 120. The mutant containing the Trp,,, -+ Phe substitution (Fig. 1, spectrum 5 ) also

Mutagenesis of interleukin-la was carried out as previously described (6).

Pur$cation of human wild type and mutant recom- binant derived IL-1B. Wild type and mutant recom- binant proteins were produced and purified to hom- ogeneity by the methods reported previously (3, 6).

Circular dichroism spectra were measured using a Jobin-Yvon Dichrograph IV. Spectra shown are averages of 4-10 scans with an average of an equal number of baseline scans subtracted. The resulting spectra were smoothed using a digital low-pass filter (9). All software used in the data collection and hand- ling was carried out using a BBC+ microcomputer linked to the Dichrograph. Protein concentrations were determined using calculated values of A;:; at 280nm as shown in Table 1. The molecular weight used to calculate molar ellipticities on a single residue basis was 17377. All solutions were filtered through 0.22 pm pore size filters (Millipore) prior to use.

Ultra-violet absorption spectra and derivative spec- tra were recorded using a Hewlett Packard 8450 u.v./ vis scanning spectrophotometer. IL-lp and mutants were dissolved (1-1.5 mg/mL except IL-1B Trp,,, -+ Phe which was 3.0 mg/mL) in 100 mM TRIS/ HCl pH 7.5. Table 1 summarizes some details from the zero order absorbance spectra.

RESULTS AND DISCUSSION

Biological activity of the mutant proteins The IL-lB mutant proteins used in this investigation contained substitutions of individual tyrosines 24,68, 90, and 121 and also of tryptophan 120 by phenylala- nine. All show full activity as measured by the recep- tor binding assay (10) and by the lymphocyte acti- vation assay (6).

Absorbance spectroscopy of the mutant proteins The absorbance spectra of the various mutants show

TABLE 1

Summary of zero order absorption spectra for IL-lp

Wild type human 6.3 277 1.04 250 Tyr, --t Phe 5.6 277 1.02 250 Tyr,, -+ Phe 5.6 277 1.02 250 Tyr, -+ Phe 5.6 277 1.03 250 Tyr,,, -+ Phe 5.6 278 1.01 250 Trp,,, -t Phe 3.0 276 1.12 250

Calculated from the protein Tyr and Trp content and based on M, = 17400.

@.I rc T )

6 \

cu U

0

0

0

250 260 270 280 290 300 310 320

WAVELENGTH (NM)

FIGURE 1 Second derivative absorption spectra of aromatic residue sub- stituted IL-la. Proteins were dissolved in 0.1 M TRIS/HCl pH 7.5. (1) Tyr,, + Phe; (2) Tyrg, 4 Phe; (3) IL-IB wild type; (4) Tyr,, + Phe; (5) Trp,,, --* Phe and (6) Tyr,,, --* Phe. The spectra indicated by dashed lines (2,4, and 6) have zero intensities similarly indicated by dashed lines. The dZA/di2 scale is arbitrary.

257

S . Craig et al.

shows an increased intensity of d' Ald12 in the phenyl- alanine region but is lacking the characteristic Trp contributions, namely, two maxima at 287 and 295 nm and two minima at 283 and 291 nm.

2000 1

Far U.V . circular dichroism spectra of the mutants All five of the aromatic mutant proteins have far U.V. CD spectra similar to that of human IL-Ip. A re- presentative spectrum of IL-IP (Tyr90 + Phe) is shown with that of the human protein for comparison (Fig. 2). The intensity of the spectrum for this P type protein (4) is weak and any possible contribution of aromatic residues could therefore lead to small dis- crepancies. There is no evidence from the n.m.r. results (6) for changes in secondary structure in any of the mutants.

Near U . V . circular dichroism of the aromatic mutants The near U.V. CD spectrum of each mutant protein (with baseline subtracted) was taken in turn and sub- tracted from the similarly corrected spectrum for human IL- If i . The difference spectrum thus obtained provides a direct quantitative estimate of the contri- bution of the substituted residue to the aromatic CD spectrum of human IL-lP. The difference spectrum

1000

-3000

-4000 200 210 220 230 240 250

WAVELENGTH Inm)

FIGURE 2 Far U.V. circular dichroism spectra of human interleukin-lfl and IL-lfl mutant (Tyr 90 * Phe). Spectra are averages of 16 scans with baselines subtracted. Protein concentration 0.8-1.2 mg/mL in 0.01 M MES, 0 . 0 9 ~ NaCl, 1 M NaZEDTA pH 6.5. Spectra were recorded using a 0. I mm pathlength cell and 2 nm bandwidth at 20".

258

resulting from the replacement of a tyrosine residue by phenylalanine will comprise the intrinsic contribution of that tyrosine to the wild type CD spectrum together with any contribution that may arise from its possible influence on the other aromatic residues. In addition, the incoming phenylalanine will make a contribution to the difference spectra but only below 270nm and only then if the intrinsic ellipticity of the sidechain is considerably enhanced.

Individual contributions of tyrosine residues to the aromatic CD spectrum of IL-lP The difference spectra representing the contribution of each tyrosine residue to the CD spectrum of human IL-lB computed by using the single mutants are shown in Fig. 3 and Table 2. The range of values of

for a single tyrosine residue in a protein has been estimated to be 2.0, based on model compound spectra (8). By this criterion tyrosines 24 and 90 each make only a minor contribution to the near U.V. CD

250 260 270 280 290 300 310 YRVELENGTH Inn1

'0

FIGURE 3 Individual contributions of tyrosine residues to the near U.V. CD spectrum of human interleukin-lp. Difference CD spectra of IL-1fl mutants containing Tyr + Phe substitutions were obtained as de- scribed in the text, from original spectra which were averages of 4-8 scans wIth baseline subtracted. Protein concentration 0.9-1.7mg/ mL in 0.01 M MES, 0 . 0 9 ~ NaCI, 1 mM Na2EDTA pH 6.5. Spectra were measured at 20" using a 1 cm pathlength cell and 1 nm band- width. Each mutant contribution is labelled by the sequence pos- ition of Tyr in human interleukin-lp. The molar ellipticity relates to mole individual aromatic residue.

Aromatic residue contributions to CD

are compared with their contributions assessed by using the single mutants and wild type protein as described above.

The quantitative differences between the dichroic contributions of Trp 120 and Tyr 121 as assessed by these two different experiments are not great. Tyr 121 makes only a minor contribution to the overall spec- trum. In the absence of Trp 120, the spectrum of Tyr 121 is negative at longer wavelengths (Fig. 6A; Table 2) with the band at 287 nm, usually observed only as a shoulder (1 1, 12), being clearly resolved and prob- ably indicating a p - p interaction (8). Bands from phenylalanine at 260 and 266 nm are superimposed on the tyrosine band.

With Trp 120 present, on the other hand, the spec- trum is qualitatively different. The tyrosine and phenylalanine contributions to the spectrum are invert- ed, with negative bands at 260 and 266nm (phenyl- alanine) superimposed on a positive tyrosine spectrum with maxima at 279 and 286nm again visible but in this case more intense. Evidence for coupling between Tyr 12 1 and Trp 120 is seen in the I L, bands at 292 nm

TABLE 2 Contributions of tyrosine and tryptophan residues to the CD spectrum

of IL-IB ~~

Residue [O],,, x 10-'/deg.cm2.dmol~' AE/I*mol-' mcm-'

Tyr 24 - 1.45 Tyr 68 - 5.69 Tyr 90 - 1.82 Tyr 121" c: 2.8 Tyr 121' - 1.25 Trp 120" 17.4 Trp 120h 15.9

- 0.44 - 1.72 - 0.55 < 0.83

5.28 4.82

- 0.38

'Data from single mutants (see Fig. 6 legend). bData from double mutant (see Fig. 6 legend).

spectrum of IL- 18 and are presumed therefore to be in a relatively low asymmetry environment. The dif- ference spectrum generated by IL-1B (Tyr 121 -, Phe) is also weak but complicated by the location of Tyr 121 vicinal to Trp 120. This is discussed below. Tyr- osine 68, on the other hand, exhibits a large negative value of A& comparable to the anticipated maximum value, and is likely therefore to be involved in a rela- tively strong coupling reaction with another aromatic or a peptide bond chromophore (8).

Contribution of tryptophan 120 to the aromatic CD spectrum of IL-IB As previously suggested (4), Trpl20 is the major con- tributor to the aromatic CD of IL-la. Fig. 4 shows the difference spectrum for Trpl20 with the strongest tyr- osine contribution (Tyr 68) for comparison. The high intensity at the low wavelength end of the spectrum (A,,, = 272 nm) suggests the presence of a strong 'La band compared with the longer wavelength I L, bands at 285nm and above. The value of AE,,, (Table 2) is comparable with the maximum expected value of 5 for the 'La band of a single tryptophan residue in a protein (8).

Contribution of the residue pair Trpl20 and Tyrl2l It is of interest to establish the contributions of the neighbouring residues Trpl20 and Tyrl21 and to assess the effect, if any, that they exert on each other's contribution to ellipticity. A double mutant in which both residues are replaced by Phe was made. By sub- tracting spectra of the appropriate mutants, i.e. [Trp 120.Phe 121]-[Phe 120.Phe 1211 and [Phe 120.Tyr 1211- [Phe 120.Phe 1211 it is possible to obtain difference spectra for the contribution of Trp I20 and of Tyr 121, each with its neighbour replaced by Phe.

The far U.V. CD and the biological assays all show that the conformation of the double mutant is not perturbed from that of the human wild-type IL-lp. The difference spectra for the contributions of Trp 120 and Tyr 121 are shown in Fig. 6A and B, where they

18, I I I

WAVELENGTH (nm)

FIGURE 4 Contribution of tryptophan 120 to the near U.V. CD spectrum of human interleukin-I/?. The difference spectrum was obtained as described in the text from original spectra which were averages of 4-8 scans with baseline subtracted. Protein concentration 1.4 mg/ mL in 0.01 M MES, 0 . 0 9 ~ NaC1, 1 mM Na,EDTA pH 6.5. Spectra were measured at 20" using a 1 cm pathlength cell and 1 nm band- width. Also shown for comparison is the contribution of Tyr 68. Molar ellipticity as for Fig. 3.

259

S. Craig et al.

and probably at 286 nm, together with a negative band out at 304nm. It is implicit from the difference spec- trum that similar coupling between Trp 120 and Phe 121 does not occur.

The presence or absence of tyrosine at position 121, in contrast to the above, makes no qualitative dif- ference to the spectral contribution of Trp 120. The presence of Tyr 121 induces a small increase in ellip- ticity between 250 and 295nm (Fig. 6B; Table 2) probably due to n-n coupling associated with the 'La band, accompanied by a slight blue shift of the fine structure associated with the tryptophan 'L, band and the phenylalanine. There is a noticeable increase in the ellipticity of the band at 287 nm that implies a speci- ficity for the coupling within the transitions of the 'L, band.

Reconstruction of the human IL-lp aromatic CD spectrum from the individual aromatic contributions As the near U.V. difference spectra for each mutant

250 260 270 280 290 300 310 320 WAVELENGTH (nrn)

FIGURE 5

Theoretical human interleukin-lp near U.V. circular dichroism spec- trum obtained by the summation of individual aromatic contribu- tions. A theoretical near U.V. CD spectrum of human IL-11 was calculated by computer summation of the individual contributions of tyrosine residues 24,68,90 and 121 and of tryptophan 120. This reconstructed spectrum is shown superimposed on the near U.V. CD spectrum of human IL-lp (labelled HUMAN). Molar ellipticity relates to mole protein.

265 280 295 310 WAVELENGTH (nm)

18

16

14

12

- - 0

TI E 10

N

5 TI z 8 PI

z 6 X -

0 4

2

a

-2

FIGURE 6

I

265 280 295 310

WAVELENGTH (nm)

Influence of neighbouring aromatic groups on the contributions of Trp 120 and Tyr 121 to the CD spectrum of IL-IB. Difference spectra were computed by subtraction of CD spectra for the dif- ferent proteins as follows: A. (i)[Trp 12O.Tyr 121]-[Trp 120.Phe 1211; (ii)[Phe 12O.Tyr 121]-[Phe 12O.Phe 1211; B. (i) [Trp 12O.Tyr 121]-[Phe 12O.Tyr 1211; (ii)[Trp 120.Phe I2l]-[Phe 120.Phe 1211.

260

Aromatic residue contributions to CD

biological activity (6) and receptor binding affinity (10) as the wild type protein, supporting the above conclusion that the changes detected by n.m.r. are local perturbations only. The second derivative ab- sorbance spectra show no wavelength shifts, the changes being limited to small changes in intensity and to changes associated with the mutated residues, in particular the tryptophan. It is evident therefore that, even when n.m.r. spectra show some change, i.e. for the Tyr,, + Phe mutant, there is no change in ex- posure to solvent of the aromatic chromophores.

The evidence from the near U.V. CD spectra present- ed here shows that the only major spectral pertur- bation occurs on substitution of phenylalanine for Tyrl21. This is not surprising considering its position adjacent to tryptophan residue at position 120 with which strong coupling could take place (8). The above evidence supports the view that the perturbations ob- served are not the result of gross conformational change.

It is concluded that, of the four tyrosine residues, only Tyr68 makes a major contribution to the near U.V. dichroic spectrum of human interleukin IL-IB. When such an analysis is coupled with a known crys- tallographic structure it will be possible to compare calculated CD bands with the experimental bands for specific aromatic residues.

contain contributions from the intrinsic ellipticity of each respective aromatic residue and from the in- fluence of each of these on the others, summation of the spectra, with subtraction of the contribution to each of the substituted phenylalanine, should lead to a spectrum identical to that of the wild type protein. As the contributions of the phenylalanine residues in situ are not known, the summation has been carried out directly as shown in Fig. 5.

The difference between the spectrum of the wild type protein and that computed from the five dif- ference spectra provides an indication of the relatively small effects of the protein environment on the phenyl- alanine residues and of the influence of these phenyl- alanines on the other chromophores, in each case summed over the five mutant proteins.

CONCLUSIONS

It has been recognised (8) that each aromatic residue in a protein will make a different contribution to the near U.V. CD spectrum depending on the specific en- vironment but until now it has not been possible to assign these contributions experimentally. A notable example where contributions of tyrosine residues to the CD spectrum have been calculated from the three- dimensional structure is that of ribonuclease-S (8) and this has received partial confirmation from an evo- lutionary variant (8, 13). Serial replacement of re- sidues by site directed mutagenesis offers the possibil- i ty of investigating these individual contributions in more detail.

It is important first to ensure that the substitutions do not significantly affect the overall conformation of the protein. The results from far U.V. CD spectra show that any changes are minor. The mutant proteins behaved similarly to the wild type protein during pur- ification, indicating that no extensive structural alter- ations affecting either solubility or protein charge are involved (3). N.m.r. spectroscopy (6) has shown that the aliphatic regions of the spectra of the mutant proteins are virtually superimposable on that for the wild type protein. The aromatic regions are also superimposable for the Tyr,, -+ Phe mutant save for the resonances assigned to those two residues.

The n.m.r. spectra for the Tyr,, + Phe and for the Trp,,, -+ Phe/Tyr,,, -+ Phe double mutant have again been shown to be very similar to that of the wild type protein. The Tyr,, -+ Phe mutant shows some marked upfield shifts of aromatic residues interpreted as a noticeable rearrangement of the local protein structure involving Tyr 68, Tyr 90, and a phenylala- nine. Apart from this it is clear that the overall folding of the protein is unaffected and that the changes are limited to these three residues, possibly involving changes in flipping of the aromatic ring for Tyr 68.

Further, the mutant proteins all exhibit the same

ACKNOWLEDGMENTS

This work was supported by BIOGEN S.A., Geneva. The assistance of Tony Pickard in data processing is acknowledged.

REFERENCES

1. Leatherbarrow, R.J. & Fersht, A.R. (1986) Profein Engineer- ing I , 7-16

2. Oppenheim, J.J., Kovacs, E.J., Matshushima, K. & Durum, S.K. (1986) Immunol. Today I, 45-56

3. Wingfield, P., Payton, M., Taverneir, J., Barnes, M., Shaw, A,, Rose, K., Simona, M.G., Demezuk, K., Williamson, K.W. & Dayer, J.M. (1986) European J . Biochem. 160, 491-497

4. Craig, S . , Schmeissner, U., Wingfield, P. & Pain, R.H. (1987) Biochemistry 26, 3570-3576

5. March, C.J., Mosley, B., Larsen, A., Cerretti, D.B., Braedt, G., Price, V., Gillis, S., Henney, C.S., Kronheim, S.R., Grabs- tein, K., Conlon, P.J., Hopp, T.P. & Cosman, D. (1985) Nature 315, 641-647

6. Gronenborn, A.M., Clore, G.M., Schmeissner, U. & Wing- field, P. (1986) European J . Biochem. 161, 37-43

7. Craig, S., Hollecker, M., Creighton, T.E. &Pain, R.H. (1985) J . Mol. Biol. 185, 681-687

8. Strickland, E.H. (1974) C.R.C. Crif. Rev. Biochem. 2, 113-175 9. Lynn, P.A. (1973) An introduction to the analysis andprocess-

ing of signals, pp. 197-199, Macmillan Press, London 10. MacDonald, H.R., Wingfield, P., Schmeissner, U., Shaw, A,,

Clore, G.M. & Gronenborn, A.M. (1986) FEBS Lett. 209, 295-298

26 1

S. Craig et al.

1 1 . Horwitz, J. & Strickland. E.H. (1971) J . Bid. Chem. 246, Address:

Professor R . H . Pain Department of Biochemistry, Genetics University of Newcastle upon Tyne Newcastle upon Tyne, NEI 7RU UK

3749-3152 12.

13.

Bewley, T.A., Sairam, M.R. & Li, C.H. (1972) Biochernls/ry

Klec, W.A. & Streat3, R.A. (1970) J . Biol. Chant. 245, 1227- 1232

11, 932-936

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