Polypeptide optical rotatory dispersion

Tanford (1968) reviewed early studies of protein denaturation and concluded that high concentrations of Gdm-HCl and, in some cases, urea are capable of unfolding proteins that lack disulfide cross-links to random coils. This conclusion was largely based on intrinsic viscosity data, but optical rotation and optical rotatory dispersion (ORD) [reviewed by Urnes and Doty (1961) ] were also cited as providing supporting evidence. By these same lines of evidence, heat- and acid-unfolded proteins were held to be less completely unfolded, with some residual secondary and tertiary structure. As noted in Section II, a polypeptide chain can behave hydrodynamically as random coil and yet possess local order. Similarly, the optical rotation and ORD criteria used for a random coil by Tanford and others are not capable of excluding local order in largely unfolded polypeptides and proteins. The ability to measure the ORD, and especially the CD spectra, of unfolded polypeptides and proteins in the far UV provides much more incisive information about the conformation of proteins, folded and unfolded. The CD spectra of many unfolded proteins have been reported, but there have been few systematic studies. 

Helical fraction is the only quantity that can be estimated experimentally among the various quantities characterizing the conformation of polypeptides. There are several means of estimating helical fraction (15,16). The most commonly used is based on the assumption that the Moffitt-Yang parameter b0 derived from optical rotatory dispersion measurement is a linear function of fN. Thus... 

Blout ER, Carver JP, Shechter E (1967) ORD of Polypeptides and Proteins. In Snatzke G (ed) Optical Rotatory Dispersion and Circular Dichroism in Organic Chemistry. Heyden, London, p 224... 

Optical Rotatory Dispersion of Polypeptides and Some Proteins "... 

A Some ambiguity must be attached to this statement in view of the conclusions reached by Luzzati el al. (1961) on the basis of small angle X-ray scattering studies from dilute solutions of polybenzyl-L-glutamate in dimethylformamide, pyridine, and TO-cresol. These authors conclude that the conformation of the polypeptide in these solutions is that of the 3io-helix (Donohue, 1953) and not that of the a-helix. The value of 6o = —630° in Eq. (13) was obtained from optical rotatory dispersion measurements with similar solutions, and therefore if the conclusions of Luzzati et al. are correct, this value characterizes the 3io-helix. It would then be necessary to determine whether the 6o value for the a-helix is significantly different. 

Fiq. 1. Optical rotatory dispersions for ordered and disordered forms of two synthetic polypeptides and a globular protein, PBG poly-7-benzyl-L-glutamate. Helical... 

Fig. 4. Optical rotatory dispersion of helical synthetic polypeptides in the ultraviolet spectrum. Poly-y-benzyl-L-glutamate in dioxane solution, --- poly-L-...

In summary, we have therefore seen that poly-L-lysine presents a valuable model for a partially helical polypeptide chain, one which is amenable to conformational analysis by optical rotatory dispersion. The method by which residues in a helical conformation may be discerned and counted against a background of disordered regions has been illustrated with this polypeptide under almost ideal conditions. The adequacy of the method is corroborated by copolymers a step closer to proteins in complexity, but some of the limitations that will be encountered in its application to proteins are already foreshadowed. Before this application is discussed, however, two other phenomena relevant to protein structure that are clearly exhibited in synthetic polypeptides, the helix-coil transition and the /3-conformation, will be considered. 

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. 

Absorption, circular dichroism and optical rotatory dispersion of polypeptides, proteins, prosthetic groups and biomembranes... 

The transition can perhaps most conveniently be followed polarimet-rically. Figure 1 shows the change in specific optical rotation of a 3% PBG solution (solvent, 70 volume % DCA-30 volume % DCE) as the temperature is varied through the transition range. From optical rotatory dispersion measurements one may obtain the Moffitt parameter, bQ, and from this it has been shown that for PBG the high temperature form (with positive [o ]d) corresponds to the helical conformation of the polypeptide (12). 

An extensive but unfortunately, as yet, unpublished study by Yamaoka (28) was concerned with the mode of orientation of several polypeptides in varied solvents under the influence of a rectangular voltage pulse. While measurements could be made in most organic solvents, he was unable to obtain steady-state values for the birefringence of PBLG dissolved in benzene and dioxane, except at low concentrations in dioxane. Extremely long rise times were observed in these solvents, and the 1.4-millisecond limit on his pulse width prevented establishment of equilibrium. Yamaoka showed by means of optical rotatory dispersion that PBLG assumes a helical conformation in benzene. 

Optical activity of natural products may depend on chemical factors such as asymmetric carbon atoms, restricted rotation, etc. These may be termed primary structural features. There are also secondary structures, e.g., helices or random coils, that may confer chirality to a natural product. Optical rotatory dispersion (ORD, i.e., rotation of plane-polarized radiation over a range of wave-lengths usually from approximately 200 to approximately 500/im) has been used in studies of the conformations of many different molecules, including polymers, proteins, and polypeptides [90]. 

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