Polypeptides helical content

Quantification of ORD and CD Data. In principle ORD and CD can be used to calculate the amounts of a, / , and random conformations in protein, but in practice such estimates are subject to large errors. The Moffitt-Yang plot is probably the best estimate of percentage of a-helicity, but it is unable to distinguish between the ft and random structures. A detailed analysis of CD bands and their resultant Cotton effects, combined with infrared data, is the most promising approach even here the limits of error are large (82). Traditional estimates have been based on combinations of a-helix and random coil, and attention has been centered upon estimation of helical content. Consideration of j3 structure has been introduced more recently. The technique must be calibrated empirically with synthetic polypeptides of known conformation, and the proper choice of reference is not obvious. The /3 structure seems to be particularly variable in its rotational properties (27, 82). 

It has recently been found (Simmons et al., 1961) that a pronounced Cotton effect exists in solutions of helical polypeptides and proteins in the wavelength region from about 260 mg to 220 mg. A large trough in rotation occurs at 233 mg which is removed when the helix is disrupted. The magnitude of [a]233 may therefore be found to serve as an independent measure of the helical contents of proteins. This effect has not yet been extensively applied to proteins in nonaqueous solvents, but it should prove to be of great interest for proteins dissolved in those solvents which are sufficiently transparent in this region of the spectrum. 

Inasmuch as this form of rotatory dispersion is in principle characteristic of the interaction of like chromophores, one would wish to employ it in the investigation of ordered polypeptide structures. But, as will become evident in the experimental use to which Eq. (18) has been put, it possesses in addition the distinct practical advantage that Xo of helices is close to Xc for disordered pol3q)eptide chains, that is, about 220 m/j, so that one equation can characterize mixtures of disordered chains and helices and at the same time yield a parameter, 6o, uniquely related to helical content. This feature is highlighted by a lack of similar adaptability displayed by alternative empirical equations proposed to describe helical dispersion. Yang and Doty (1957) have fitted data for helical poly-y-benzyl-L-gluta-mate to an abbreviated two-term Drude equation. 

Equation (38) shows that all three parameters may be expected to vary in a systematic manner with helical content when compared to a common reference state, the disordered polypeptide chain. 

The aim of this final section is twofold. It will first examine the basis upon which the rotatory dispersions of proteins may be treated in the same manner that has been employed for synthetic polypeptides and, secondly, assess the extent to which the dispersive properties of proteins may be quantitatively explained in terms of known conformations of a polypeptide chain. A pattern of conformational analysis embodying the hypothesis that a polypeptide chain is partitioned into standard helical segments and disordered regions has been shown capable of a rather precise estimate of partial helical content in polymers such as poly-L-lysine and copoly-L-... 

If water replaces dichloroacetic acid or trifluoroacetic acid as the hydrogen-bonding component in mixtures of decreasing polarity, then the helical content of disordered water-soluble polypeptide chains will increase continuously, as is, for example, the case with equimolar copoly-L-lysine-L-... 

The initial decrease in optical rotation found in aqueous solutions of /3-lactoglobulin and ovalbumin is not, however, sufficient to differentiate globular proteins from simpler synthetic polypeptides in their transition behavior, for neither ribonuclease nor human serum albumin appear to exhibit it. The specific rotation of ribonuclease in water-2-chloroethanol mixtures becomes steadily less levorotatory as the proportion of nonpolar solvent increases (Weber and Tanford, 1959). In the case of human serum albumin Bresler (1958) and Bresler el al. (1959) find that only progre.ssive increases in specific rotation occur as the concentration of 2-chloroethanol is increased and that this change is accompanied by a steady rise in viscosity and the corresponding axial ratios characteristic of the formation of rodlike particles. If these proteins do have some initial helical content in water, as can be argued from their optical rotatory dispersion, then it appears that hydrophobic forces are not required for the stability of these regions. 

What accommodations, then, must be made in the pattern of analysis developed for standard synthetic polypeptides if it is to yield quantitative estimates of partial helical content in proteins The requirements are much the same as those set out for poly-L-lysine (see Section III, G, 3), yet since globular proteins, like copolymers of L-lysine and L-glutamic acid, cannot be made completely helical in aqueous solution, a helical reference conformation must be taken either from other standard molecules or from the nonaqueous behavior of the protein in question. This latter procedure involves solvent changes with little bearing on native conditions and may be impossible to carry out in the face of restraints imposed by proline and cross-links, so that standard helical dispersion can more feasibly serve as this reference conformation. 

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