Role of proline in polypeptide chain configuration of proteins.

Optical rotation can be used to detect the presence of helical configurations of the polypeptide chains in proteins, to determine screw sense, and to estimate the extent of helical regions ( 1 ) . Moffitt's theory ( l a ) of rotatory dispersion for helical macromolecules has been successfully applied to synthetic polypeptides ( 2 ) and to proteins (2, 3 ) , and at the present time it may be applied empirically to estimate a-helix content. Globular proteins have been shown to have relatively low ( 2 ) and fibrous proteins to have relatively high ( 3 ) helix content. We have examined the amino acid composition of proteins which belong to the keratin-myosin-epidermin-fibrinogen class (KMEF proteins) to account for the wide variation in amount of a-helix present; in this report we demonstrate a striking correlation between proline content and extent of helical configuration. Table 1 lists data obtained by proline determinations using the method of Troll and Lindsley ( 4 ) . Helix content is based on rotatory dispersion measurcmcnts reported previously ( 3 ) . Wc assume for this discussion that light mcromyosin fraction I is 100-percent helical and that the helices have a single sense of twist. Fragmentation of myosin by tryptic digestion into light meromyosin and heavy meromyosin corresponds to a fractionation into one component relatively poor and one relatively rich in proline. Furthermore, the light meromyosin may be separated by ethanol into two fractions differing in prolinc content, one of which (light meromyosin fraction I ) remains soluble after treatment with ethanol concentrations of 50 percent (volume by volume) or higher and represents about 25 percent of the intact myosin by weight ( 5 ) . In each case, the higher the a-helix content, the lower the amount of proline present. These results suggest that proline interferes ~v i th the formation of the a-helical configuration. By adopting a very simple model for the a-proteins consisting of helical and nonhelical regions, one may estimate the disordering effect of a single proline residue, assuming that the proline is distributed statistically. Table 1 shows that, on the average, each proline residue is associated with 15 to 20 residues (hence several helical turns) not participating in the right-handed a-helical configuration in aqueous solution. In nonaqueous solution, this effect may be decreased ( 2 ) . These obsenlations are supported by model building from which it is seen that the pyrrolidines do not fit well into a right-handed a-helix. Available data on proteins other than the KMEF series show a generally similar correlation of proline and helix content. On the basis of dispersion studies (2, 3 ) , onc may take the specific rotation of n ~ t i v e proteins in aqueous solution as inversely proportional to the a-helix content. Thus, a fully-coiled right-handed a-helix may be characteri7ed by [a',, r O 0 ( 2 ) and nonhelical chains by ;a], LZ 100' 16). Globular proteins have rotations [a:, r30' to 60°, correspondin? to low helix content of about 30 to 40 percent ( 2 ) . Tristram's compilations ( 7 ) show that these proteins contain from 3 to 8 percent proline, corresponding to values expected from the data given above on the KMEF series. Exceptions such as lysozyme, insulin, and avidin having less than 2 percent proline might be accounted f o r by sulfur or phosphorus cross-linka~es which may interfere with a-helix formation. I t should be noted that such cross-linkages, as well as sidechain interactions, may stabili~e or disrupt helical confiqurations (8 ) . Thus the KhfEF proteins discussed above, having few if any sulfur cross-linkages (fibrinown cxccpted), provide simpler systems for thiq correlation than do thc "globular" proteins ( 9 ) . Proline content higher than about 8 percent would be expected to cause almost completc absence of thc a-helix, provided that the prolinc residues do not exiit as "blocks" in the polvpeptide chain. The rotation of casein [a], r 100' 11'1) sunporti: thii idca, and rotatory dispersion data on casein and the prolamines chould bc of considerable interest ( 11\ . Collagen has an exceptionall) high pyrrolidine content (about 25-percent), but rather than assuming a nonhelical configuration, the polypeptide chains in collagen comprise a cable of three left-handed helices, each similar Table 1. Helix content and proline concentration of KMEF proteins.