Eur. J. Biochem. 29,537-541 (1972)

Optical Properties of the Hexon of Adenovirus

Loren A. DAY, Richard M. FRANKLIN, Ulf PETTERSSON, and Lennart PHILIPSON The Public Health Research Institute of the City of New York,

and Microbiologiska Institutionen, Wallenberglaboratoriet, Uppsala Universitet

(Received June 14, 1972)

The ultraviolet absorbance, circular dichroism (CD) and optical rotatory dispersion of the hexons of adenovirus type 2 have been measured and analyzed in terms of secondary structural features. A curve-fitting method for the CD spectra from 190 nm to 240 nm indicates 17O/, a-helical, 2601, t9, and 5601, “random” conformations ; the classical parameter b, indicates from 15 to 20°/, a-helical conformation. The far ultraviolet absorbance indicates considerable hypochromicity in comparison to the absorbance expected from the chromophore composition of the protein, which indicates very high a-helical content. We have applied corrections for absorbance flattening in the analysis of all the spectra and demonstrate the relatively large magnitude of such corrections in the regions of intense absorbance for this protein complex of molecular weight around 400000. In spite of such corrections, the discrepancy in apparent a-helical contents indicated by ultraviolet hypochromism and by optical rotatory methods persists.

This study was undertaken to obtain additional structural information on the hexon of adenovirus type 2, an icosahedral animal virus cootaining DNA. The hexons form the bulk of the outer capsid and are located a t the local six-fold symmetry axes. The hexon ha0 been and is being studied by X-ray crystallography and electron microscopy [I -51. In this study we have used ultraviolet absorbance, circular dichroism (CD) and optical rotatory dis- persion (ORD). Since these methods are still under- going testing and development, it is important to apply them to proteins, the structures of which are known or will eventually be known. In the analysis we have applied correction factors for the absorbance flattening [6-81 of the spectra.

MATERIALS AND METHODS Preparation and Handling of Hexon Protein

Hexons were obtained from infected cells accord- ing to the method of Pettersson et al. [l] with the modifications described by Franklin et al. [4]. The samples used for the present study had been stored at 4 “C in 0.01 M Tris-acetate, O.Olo/o sodium azide, pH 8. The samples were dialyzed exhaustively against 0.01 M sodium phosphate buffer pH 7.0 for measure- ments in the dispersed state. In this buffer, the hexons are present as monomeric capsomers of molecular weight 320000 to 360000 [4].

Abbreviations. CD, circular dichroism; ORD, optical rotatory dispersion.

Instrumental Methods The absorption coefficient for hexons in 0.01 M

sodium phosphate, pH 7 was obtained by deter- mining the refractive increment at room temperature of a hexon solution which had been dialyzed for two days, the last 6 h of which were at room temper- ature. A Brice-Phoenix differential refractometer, calibrated against standard KC1 solutions, was used. From the measured refractive increment, the pro- tein concentration was obtained with an assumed value of dn/dc at 546 nm of 0.185 & 0.005 ml/g (cf. compilation by Timasheff [9]). The protein con- centration so determined was considered accurate

Absorbance measurements were made with a Cary 14 recording spectrophotometer. Wavelength calibration in the visible and near ultraviolet regions was carried out with a mercury spectral line source. Oxygen band spectra were used for wave- length calibration below 200 nm. For absorbance measurements in the low ultraviolet region, special care was taken in the cleaning and handling of the cuvettes and solutions. Absorbance amplitudes of test samples recorded by our Cary 14 were the same as those given by a Zeiss PMQII single-beam spectrophotometer at 260 nm and at 365 nm, within the legibility of the amplitude scales of the two instruments. To ascertain that stray-light levels were within acceptable limits for measurements in the low ultraviolet range with our Cary 14, we placed a wire mesh screen which had a measured trans-

to 4’10.

538 Optical Properties of the Hexon of Adenovirus Em. J. Biochem.

mittance of very nearly loo/, (absorbance = 1) throughout the range from 210 nm down to 185 nm in the reference beam of the instrument and a 1-cm cuvette containing isopropanol in the sample beam, and then recorded the apparent absorbance of the isopropanol down to 185 nm. The slide wire registered its maximum reading of 2 absorbance units through- out the range, hence the stray light was considered to be less than 0.lo/,. The importance of this type of test and other aspects of such measurements has been discussed by Gratzer [lo]. The slit widths used for recording the spectra of the samples did not exceed those used in the calibration procedure. Base lines were obtained before and after the absorbance scans.

Circular dichroism and optical rotatory disper- sion measurements were done with Cary 14 and Cmy 60 instruments. Wavelengths were calibrated with the mercury spectral line source and were known to within the legibility of the wavelength indicators, approximately 0.2 or 0.3 nm. National Bureau of Standards sucrose was used as the rotation standard, and based on this and on the calibration data of Cassim and Yang [ll] for d-camphor-sulfonic acid, the correct ellipticity amplitudes relative to our rotation amplitudes were established to within 2O/,. Optical cells of 1-mm and 10-mm path length were used and the total absorbance of the measured samples, including buffer absorbance, did not ex- ceed 1.2 units.

Computational Methods Corrections for light-scattering contributions

to the near ultraviolet absorption spectrum were obtained as usual by plotting the log absorbance us log wavelength in the nonabsorbing range from 400 nm down to 320 nm and making a linear extra- polation into the absorbing range. The extrapolations were extended into the range below 200nm, and the apparent light-scattering contributions indicated were less than 2O/, of the maximum low ultraviolet absorbance amplitudes measured. However, no corrections in this range were applied.

Corrections for flattening of spectral amplitudes were applied for the hexon in the 0.01 M phosphate buffer [7]. The observed amplitudes are related to the corrected amplitudes as (obs) = QAA;')~ (corrected), [Fl(obs) = QB [PI (corrected) and [e](obs) = Q B [ ~ ] (corrected). The correction factors QA and QB, for absorbance and optical activity, respectively, were computed for a cylinder of radius 4.4nm and height 11 nm, the size and shape of the hexon as determined by low-angle X-ray scattering (Lind- quist, I., personal communication) according to the method of Day and Hoppensteadt [8], which is a direct extension of the methods of Duysens [6] and or Gordon and Holzwarth [7] to the case of randomly

oriented cylinders. The values of QA and QB calcu- lated for the hexon as a function of absorbance A?)m were found to fit the empirical relations QA = exp (- 1.27 x lo-* A:@$m) QB = exp (- 2.57 x Aiabm). Very nearly the same relations would have been obtained from the results of Duy- sens [6] and Gordon and Holzwarth [7] had we approximated this particular cylinder as a sphere of equivalent volume [8]. Absorption coefficients per unit length of path within the material of the protein necessary for the computation [8] were obtained as y = In ( Io/I) per nanometer = 2.303 x where the partial specific volume B = 0.727 [4]. No corrections have been applied for various distortions to the spectra introduced by light scattering [18,19], but their possible effects will be discussed below.

Transformation of the observed and corrected circular dichroism to optical rotatory dispersion was carried out according to the Kronig-Kramers relationship [12]

and

[p1 (A)] = $ J [ O (A')] [A'/(A2 - X2)] d L'

by approximating the integral as a sum over the CD spectrum a t intervals of A' of 0.5nm. The summation was done by computer.

RESULTS Absorbance Xpectrum of Adenovirus Hexom

The complete absorbance spectrum in 0.01 M sodium phosphate pH 7 is given in Fig.l. The ab- sorption coefficient corrected for light scattering as described above was found to be 279nm = 14.6 & 0.6. From the amino acid composition, the absorption coefficients for N-acetyl methyl esters of tyrosine, tryptophan and phenylanine compiled by Gratzer [13], and the data of Yanari and Bovey [14] for spectral shifts of these chromophores in detergent micelles relative to H,O, we compute values of AFPm = 14.8 and 14.3 a t 280 nm for model spectra in detergents and water, respectively, in agreement with the measured value. The measured spectrum clearly shows a shoulder a t about 291 nm characteristic of tryptophan, and appears red- shifted relative to the calculated one, probably the result of localization of the chromophores in hydrophobic regions of the hexon.

An analysis of the far ultraviolet absorbance data according to the method of Rosenheck and Doty [17] is presented in Table 1. The corrections for side-chain contributions to low ultraviolet absorbance were taken from the data of McDiar- mid [15]. The unusual result is the extreme apparent hypochromicity calculated for the peptide chromo- phores of the protein backbone, even after the experimental absorbance has been increased by

Vo1.29, No.3, 1972 L. A. DAY, R. M. FRANKLIN, U. PETTERSSON, and L. PHILIPSON 539

12

10

8

aez q 6

Table 1. Analysis of the far-ultraviolet-absorbance spectrzcm of hexons in 0.01 M sodium phosphate pH 7.0

Quantity Wavelennth

190nm 197nm 205nm

Measured A:'~m QA

Corrected Atopm &(total per amino acid

residue) a

&(side chains) b &(side chains, '/* amidated) &(peptide bond) = &(total)-

&(peptide bond) = &(total)-

&(random coil) d

&(a-helix) d

&(side chains)

&(side chains, 'la amidated)

1020

0.884 1150

12900

7 903

(8 560)

5 000

(4 340) 6 950

4 300

848 0.900 943

10550

6 877 (7 110)

3 670

(3440) 6 550

3400

428

0.950 450

5050 4698

(4740)

350

(310) 3 400 2 200

~~~~~~~~

a Based on b Side-chain contribution to molar absorption per amino acid as

computed from the amino acid composition given in [I81 and the molar absorption coefficients for side chains in 0.1 M sodium dodecylsulfate given in (151.

C Same as (b) but with the assumption that '/, of the carboxylic acid groups are amidated.

d Reference data from polypeptide spectra reported by Rosenheck and Doty [17].

corrected and a mean residue weight of 112.

Fig. 1. Ultraviolet-absorbance spectrum of adenovirus type-2 hexon in 0.01 M sodium phosphate pH 7.0. -, Spectrum after corrections for flattening have been made; ---- , in the near ultraviolet is the computed spectrum, in the far ultraviolet is the measured spectrum prior to corrections for flattening, with an extrapolation (-.---.- ) used for com- puring flattening corrections to the circular dichroism. The measured absorbance did not exceed 1 unit at any wave- length, and the amplitudes at wavelengths longer than 188 nm were independent of instrumental spectral band widths below 1 nm. The ratio A191/A2,9 = 65 1 from six scans on two different samples. The concentrations were

coneidered to be known to -+4O/, (see text)

corrections for flattening. Corrections for light scattering, which have not been applied, would lead to an even greater lack of additivity in the direction of more apparent hypochromicity.

Circular Dichroism and Optical Rotation The measured and the corrected CD are presented

in Fig.2, together with the measured ORD and the ORD calculated from both the uncorrected and cor- rected CD spectra €or hexons in 0.01 M phosphate. The rotations computed from corrected CD have been reduced by the flattening coefficients to allow direct comparison with the uncorrected experimental curve. The large corrections indicated are required because of the intense absorbance and the size of

the hexons. Not shown in Fig.2 is a symmetric ellipticity band centered a t 292 nm with a half- height band width of 5 nm and a negative maximum of 81 degrees x dmol-1 x cm2 based on moles per liter of amino acid residues. The small CD bands above 250 nm were not included in the CD to ORD transformation.

From the measured optical rotation in the range from 320nm to 550nm we obtain a value of b, = 100 f 50 in agreement with - 85 computed from the corrected CD spectrum. The a-helical content according to the 6 , method is in the range

The CD spectra in 0.01 M phosphate have been subjected to it curve-fitting analysis to ascertain /?-pleated sheet and random conformations. The reference data used and hence, implicitly, the defini- tions of these structures from the interpretation of X-ray data was that of Saxena and Wetlaufer [16]. Their reference spectra are based on an analysis of the CD spectra of proteins the structures of which have been determined by X-ray crystallography. The actual computer output data points for the reference curves were kindly provided to us by Dr Saxena. The analysis is shown in Fig.2, and the best fit by this method would indicate 17O/, a-helix, 26 ,I,, /? structure and 57 random conforma- tion. The a-helical content by this method is in good agreemet with that obtained from b,.

10 to 2001,.

Optical Properties of the Hexon of Adenovirus Eur. J. Biochem. 540

12 - -

10 -

- 8 - -

6 -

I I

170 180 190 200 210 220 2.30 240 Wavelength (nm)

Fig.2. CD and ORD of hexon in 0.01 ikl sodium phosphate pH 7.0. The spectrum with a positive maximum at 189 nm and a negative maximum a t 208 nm (-) is the circular dichroism corrected for flattening. The spectrum below it (----) is . the uncorrected experimental curve. The spectra with positive maxima a t 198nm and negative maxima a t 231 nm are the computed ORD spectra for corrected (-) and uncorrected (----) CD spectra. A, The experi- mental ORD spectrum. The corrected CD for hexon was best fit by a computer program solving systems of linear equations defined as z[O], + y[O]b + ~[Olrandorn = [el hexons a t various wavelengths. The best solution was ob- tained with 17O/, 01, 25O/, B, 57OlO random conformation and the computed spectrum for this combination is indicated (0). The signal/noise ratios a t principal CD wavelengths were 8, 15, and 10 a t 192 nm, 210 nm and 292 nm, respec-

tively

DISCUSSION

The principal structural results of the present study are the low apparent contents of a-helix and b-structure and the more extensive amount of less defined structures, as estimated from optical rotatory properties. The &-helical content from the curve- fitting procedure agrees with that from the classical parameter b,. Unfortunately, the large amount of aromatic chromophores hinders the assignment of structural composition, and in fact is probably the source of a large discrepancy between the type of structure indicated by the far ultraviolet absorbance method and by rotatory methods. For example, the hypochromicity at 197 nm, suggested as the most useful single wavelength for such an analysis by Rosenheck and Doty[l7], if assigned to the

peptide chromophore would indicate an almost perfect &-helix (Table l ) , in contrast to the curve fitting analysis of the CD spectrum and the low magnitude of b,.

The present analysis provides a demonstration of the magnitude of flattening effects which might be encountered even in relatively small multisubunit structures, the hexon being only 320000 to 360000 in molecular weight with a cylindrical shape, 4.4nm in radius and 11.0 nm in height. At 190 nm, the wavelength of maximum absorbance, the measured absorbance had to be increased 1301, and the ellipti- city 2601,. These effects have been considered separately from distortions in the measured spectra due to light scattering, a source of distortions equal in importance to flattening which, in a complete and general treatment of the problem, should not be separately considered, for the phenomena are highly interrelated [19 -211. At present, a satisfactory and convenient way of accurately correcting measured spectra for both effects is not available, although progress is being made in a number of laboratories. Nevertheless, the purposes of the present paper have been served by the flattening correction alone since our primary reason for applying the correction was to resolve the discrepancy between the apparent high a-helical content on the basis of ultraviolet hypochromism and the low &-helical content on the basis of optical rotatory properties. Scattering correc- tions to the ultraviolet absorbance data, if applied, would have increased the apparent helical content still further. The principal scattering correction to the circular dichroism spectra enters through a term proportional to n1 - nr, the difference in refractive indices for left and right circularly polarized light, and therefore, to first approximateion, proportional t o the measured optical rotation spectrum. Examples of relative scattering and absorption contributions to the measured circular dichroism calculated for spheres of &-helical polyglutamic acid are given by Gordon in Fig.3 of [20] and by Urry in Table VIII of [21]. From theoretical work to date [19-211 we might expect that the differential scattering contribu- tions to the measured circular dichroism of hexons correspond in magnitude to approximately the differ- ence between the solid and dashed optical rotation spectra in Fig. 2. It can be seen by inspection of these curves and the reference spectra of Saxena and Wetlaufer [I61 that such corrections to the CD spectrum would be small, and the effect on the con- formational assignments would be of the order of & 5 ,lo, although exact calculations have not been done. It is therefore expected from these considera- tions that although proper correction for differential scattering effects may give a more suitable circular dichroism spectrum for conformational analysis, there is no possible way in which such corrections could have resolved the large discrepancy in

Vo1.29, No.3.1972 L. A. DAY, R. M. FRANKLIN, U. PETTERSSON, and L. PHILIPSON 541

apparent &-helical contents given by the two meth- ods used in this study.

Accepting the conformational results from rota- tory power for the hexon, and that peptide bonds in non-a-helical conformations have molar absorp- tions about 7000 a t 190 and 197 nm (cf. Gratzer [13]) we must conclude that in the far ultraviolet the side-chain chromophores in the hexon absorb some 30 to 40°/, less than in the isolated state.The aromatic side chains probably have optically active electronic transitions in the far ultraviolet region, but it is difficult to estimate the contributions of such transitions to the total CD and ORD of a given pro- tein, hence the optical activity assigned to the peptide backbone must remain somewhat uncertain. Although the application of these spectroscopic methods does not allow a completely satisfactory interpretation in terms of protein conformation, the spectra are highly characteristic of the protein and can be used for control of chemical modification and heavy atom substitution work in conjunction with X-ray structural studies. It is hoped that future X-ray diffraction results for the hexons of type-2 adenovirus will help resolve the problems encounter- ed in this spectroscopic study.

Boulanger and Loucheux [22] recently published CD and ORD results on isolated adenovirus type-2 hexon and fiber proteins. Although exact quanti- tative comparison should not be made with our results because they indicated neither the method used for determining concentrations, nor the pH of their samples and their measurements on hexon were done a t ten-times higher ionic strength, the overall result of low a-helical content by optical rotatory methods reported by them is in qualitative agreement with the results presented here.

This work was supported by grants A109049 and A1 07645 from the U.S. Public Health Service and by grants from the Swedish Cancer Society and the Swedish Medical Research Council.

REFERENCES 1. Pettersson, U., Philipson, L. & Hoglund, S. (1967)

Virology, 33, 575. 2. Philipson, L., Pettersson, U., Hoglund, S., Prage, L. &

Lonberg-Holm, K. (1968) Nobel Symp. 11, 329-340.

3.

4.

5.

6. 7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18. 19.

20. 21. 22.

Cornick, G., Sigler, P. B. & Ginsberg, H. S. (1971) J . Mol. Biol. 57, 397.

Franklin, R. M., Pettersson, U., Akervall, K., Strand- berg, B. & Philipson, L. (1971) J . Mol. Biol. 57, 383.

Franklin, R. M., Harrison, S. C., Pettersson, U., Philip- son, L., Briinden, C. I., &, Werner, P. E. (1971) Cold Spring Harbor Symp. Quant. Biol. 36, 503.

Duysens, L. N. M. (1956) Biochim. Biophys. Acta, 19, 1. Gordon. D. J. & Holzwarth. G. (1971) Arch. Biochem. , . ,

Biophys. 142, 481. Day, L. A. & Hoppensteadt, F. (1972) Biopolymers, in

press. Timasheff, S. N. (1970) in Handbook of Biochemistry

(Sober, H . A., ed.) p. C-67, The Chemical Rubber Co., Cleveland.

G. D., ed.) p. 117, Marcel Dekker, New York.

1947.

Gratzer, W. B. (1967) in Poly a-Amino Acids (Fasman,

Cassim, J. Y. & Yang, J. T. (1969) Biochemistry, 8,

Moffit, W. & Moscowitz, A. (1959) J . Chem. Phys. 30, 648.

Gratzer, W. B. (1970) in Handbook of Biochemistry (Sober, H. A., ed.) p. B-74, The Chemical Rubber Co., Cleveland.

Yanari, S. & Bovey, F. A. (1960) J . BioE. Chem. 235, 2818.

McDiarmid, R. (1965) Thesis, Harvard University (as cited by Gratzer [lo]).

Saxena, V. & Wetlaufer, D. B. (1971) Proc. Nat. Acad. Sci. U . 8. A. 68, 969.

Rosenheck, K. & Doty, P. (1960) Proc. Nut. Acad. Sci. U . 8. A. 47, 1775.

Pettersson, U. (1970) Thesis, Uppsala University. Gordon, D. J. & Holzwarth, G. (1971) Proc. Nat. Acad.

Gordon, D. J. (1972) Biochemistry, 11, 413. Uny, D. W. (1972) Biochim. Biophys. Acta, 265, 115. Boulanger, P. A. & Loucheux, M. H. (1972) Biochem.

Sci. U . 8. A. 68, 2365.

Biophys. Res. Commun. 47, 194.

L. A. Day Department of Biochemistry The Public Health Research Institute, City of New York 455 First Avenue, New York, N.Y. 10016, U.S.A.

R. M. Franklin’s present address: Biozentrum der UniversitPt, KlingelbergstraBe 70 CH-4056 Basel, Switzerland

U. Pettersson and L. Philipson Mikrobiologiska Institutionen Wallenberglaboratoriet, Uppsala Universitet Dag Hammarskjolds Vag 21, 5-75237 Uppsala, Sweden