Conformation of immunologically-active fragments of sperm whale myoglobin in aqueous solution

J. Mol. Biol. (1967) 26, 143-146

Conformation of Immunologically-active Fragments of Sperm Whale Myoglobin in Aqueous Solution An attempt has been made to determine the conformations of two immunologically active fragments of sperm whale myoglobin (peptides Bl and Cla; Crumpton & Wilkinson, 1965) by comparing their optical rotatory properties. Peptide Bl, which contained 14 amino acids (residues D6 to El2 inclusive; Kendrew et al., 1961) has 12 consecutive helical residues in metmyoglobin, whereaspeptide Cla, which contained 13 amino acids (residues E20 to F4 inclusive) has eight consecutive non-helical residues and only a few helical residues at either end; bothpeptides contained “corners” between either successive helical, or helical and non-helical segments of metmyoglobin. If an octapeptide is the smallest peptide capable of forming a stable cc-helix (Doty & Lundberg, 1956; Goodman, Schmitt & Yphantis, 1960) and if the conformations of Bl and Cla in aqueous solution are the same as those of the corresponding regions of metmyoglobin, then these peptides would possess different optical rotatory properties. Peptides Bl and Cla were prepared as described previously except that peaks B and C (Fig. 3; Crumpton & Wilkinson, 1965) were pooled, passed through a column of DEAE-Sephadex A25 equilibrated with O-01 M-ammonium bicarbonate (pH 8-O) and the unadsorbed material was fractionated on CM-Sephadex C25; peptide BI was eluted with a gradient from 0.01 M- to 0.15 M-sodium chloride in O-01 M-phosphate buffer at pH 6.5 and peptide Cla with a further gradient to 0.5 M-sodium chloride. Each peptide was then eluted from a column of Sephadex 625 with O-05 M-ammonium bicarbonate (pH 8,O). Peptide solutions were concentrated by rotary evaporation in vcrcuoat 2°C and clarified by centrifuging; the solvent concentration was determined from the conductivity and the concentration of peptide was calculated from the amino acid a,nalysis of a known volume of solution. No impurities were detected in either peptide by amino acid analysis. Optical rotations within the spectral range 366 to 577 rnp were measured with a Rudolph 80 spectropolarimeter, jacketed cells of 2 dm light-path and solutions of Bl and Cla containing 0.623 and O-686 g/l00 ml., respectively, in 0.20 M-ammonium bicarbonate (pH 8-l). Measurements between 215 and 250 rnp were made with a Gary model 60 recording spectropolarimeter, jacketed cells of 0.02 drn light-path and solutions of Bl and Cla containing 0.078 and O-086 g/100 ml., respectively, in O-05 M-ammonium bicarbonate (pH 8-O). Reduced mean residue rotations ([m’ln; Urnes & Doty, 1961) were calculated using 108.8 and 116.4 for the mean residue weights of Bl and Cla, respectively; the refractive index of the solvent was assumed to be 1.37 at 233 rn,u and 1.33 between 366 and 577 mp. The dispersion parameters of peptides Bl and Cla at various temperatures are shown in Table 1. A comparison of the values obtained at similar temperatures indicated that the peptides possessed similar dispersion parameters; peptide Bl, however, bad a lower -a,, and a slightly higher - b, and h, than peptide Cla. In each 143

144

M. J. CRUMPTON

AND TABLE

P. A.

SMALL,

JR.

1

Optical rotatory dispersion parameters for queous solutions of peptides Bl and Gla at various temperatures Dispersion parameter & -a0 4,

Peptide

Bl

Peptide

TV

55%

ll°C

33°C

221 464 52.0

228 404 88.8

216 620 46.8

218 586 69.8

Cla 48°C 222 561 79.6

63OC 222 548 85.2

The dispersion constant (X,) was calculated from measurements of optical rotation at 366, 406, 436 and 547 rnp using the one-term Drude equation (Yang t Doty, 1957). Values of afi and b. were estimated from measurements of optical rotation at 366,406,436, 547 and 5’77 rnp using the were obtained equation of Moffitt & Yang (1956) and a value of 212 for h,. Linear relationships when the data were plotted according to the above equations. Experimental details are given in the text.

case, increase in temperature caused a slight increase in the optical rotation at 436 rnp. The change in [albS6 with rise in temperature was linear and was similar for both peptides (f0.4” to O@/“C); the original rotations at 436 ml* were restored on cooling the heated peptide solutions. The optical rotatory dispersion curves between 215 and 250 rnp of the peptides are shown in Fig. 1. The peptides possessed similar dispersion curves. No curve had a significant trough at 233 rnp; the values of [m1]233 for Bl and Cla at 25°C were - 2610 and - 1920, respectively. In each case, the shape of the curve between 230 and 250 m,u was independent of the temperature,

210

220

230

240

250

h(mpd

Fro. 1. Optical rotatory dispersion curves of aqueous solutions of (a) peptide Bl and (b) peptide Cla at 25% (0) and 75’C (A), and of solutions of Bl and Cla which had been heated at 75°C and then cooled to 10°C and 33”C, respectively (a). The width of the curves below 230 rnp indicates the reproducibility of the measurements. The optical rotations of samples which had been heated and then cooled were corrected for any changes in conoentration due to evaporation of the solvent. Experimental details are given in the text.

LETTERS

TO THE

EDITOR

145

but a temperature-dependent reversible change in shape occurred below 230 mp. Although the reason for this change is not known, it presumably reflects some reversible conformational change. The above values of - b, and [m’],,, for peptide BP were similar to those obtained (R. H. Pain, personal communication) using a different preparation of Bl and a different instrument (- b,, 38; [TTL’]~~~,- 2580; 25°C). A comparison of the results reveals that peptides Bl and Cla had similar optical rotatory properties. The dispersion parameters, the negligible amplitude of the Cotton effect at 233 rnp and the effect of temperature on the dispersion parameters indicate that the peptides possess similar conformations which resemble that of a random coil (Urnes & Doty, 1961; Simmons, Cohen, Szent-Gyorgyi, Wetlaufer 8~ Blout, 1961). It was concluded that peptides Bl and Cla possessed very little helical conformation in aqueous solution. In contrast, twelve of the amino acid residues of peptide Bl were included’ in a helical segment of metmyoglobin. Consequently, the helical conformation of the portion of the metmyoglobin molecule that corresponds to peptide Bl depends on stabilizing interactions with other parts of the molecule. 0n the other hand, the temperature-dependent reversible change in shape of the dispersion curves between 215 and 23Omp suggests that peptides Bl and Cla possessed some non-random conformation, which may be due to the presence of the “corners” that both peptides contained when part of metmyoglobin. Although the conformations of peptides Bl and Cla in aqueous solution were essentially non-helical, the peptides inhibited the precipitation of metmyoglobin or apomyoglobin by antisera to metmyoglobin. Furthermore, the conformations of the peptide antigenic sites when combined with antibody were probably the same as those of the corresponding sites of the native protein (Crumpton & Wilkinson, 1965). This suggests that the antibodies combined with a limited region of unchanged conformation, such as the “corners” of the peptides. Alternatively, if each peptide possessed a variety of conformations in continuous interchange (cf. Craig, 1964), the antibodies may have reacted only with those molecules whose conformations were the same as that of the corresponding region in metmyoglobin. The optical rotatory properties of peptide 51 do not disprove this possibility, but they suggest that at any one moment a small fraction of the molecules only may possess a helical conformation. Indeed, the values of h,, - b, and - [n”v’lZ5afor Bl, which were greater than those for Cla, may indicate that about 5% of the molecules of peptide Bl have a helical conformation. If this interpretation is correct, it is possible that the helical conformation of peptide Bl was stabilized by interaction with antibody. In summary, the results show that the conformation of a tetradecapeptide, which corresponded to a predominantly helical segment of metmyoglobin, was essentially non-helical in aqueous solution. The peptide may, though, possess some non-random conformation. In spite of the apparent difference in helical conformation, the peptide combined with antibody to metmyoglobin. Some explanations of this phenomenon have been considered. ?Ve are indebted to Dr R. Resnik for his advice and for the use of the Cary polarimeter, and to Dr R. H. Pain for the determination of some of the optical dispersion parameters. The technical assistance of Mr P. B. Gill&t is gratefully ledged. This investigation was supported by grants from the National Science tion, the U.S. Public Health Service, the Wellcome Trust and the Medical Comxil.

spectrorotatory acknowFoundaResearch

146

M. J. CRUMPTON

Department of Immunology St Mary’s Hospital Medical London, W.2, England Laboratory of Neurochemistry National Institute of Mental Bethesda, Maryland, U.S.A. Received

13 February

AND

P. A. SMALL,

JR.

M.J.CRUMPTON~ School

PARKERA.SMALL,

JR.$

Health

1967 REFERENCES

Craig, L. C. (1964). Science, 144, 1093. Crumpton, M. J. & Wilkinson, J. M. (1965). Biochem. J. 94, 545. Doty, P. & Lundberg, R. D. (1956). J. Amer. Chem. Sot. 78, 4810. Goodman, M., Schmitt, E. E. & Yphantis, D. (1960). J. Amer. Chem. Sot. 82, 3483. Kendrew, J. C., Watson, H. C., Strandberg, B. E., Dickerson, R. E., Phillips, D. C. & Shore, V. C. (1961). Nature, 190, 666. Moffitt, W. & Yang, 5. T. (1956). Proc. Nat. Acad. Sci., Wash. 42, 596. Simmons, N. S., Cohen, C., Seent-Gyiirgyi, A. G., Wetlaufer, D. B. & Blout, E. R. (1961). J. Amer. Chem. Sot. 83, 4766. Urnes, P. J. & Doty, P. (1961). Advanc. Protein Chem. 16, 401. Yang, J. T. & Doty, P. (1957). J. Amer. Chem. Sot. 79, 761.

t Present address: National Institute for Medical Research, London, N.W.7, England. f Present address: Department of Microbiology, University of Florida, Gainesville, Florida,

U.S.A.