Polypeptides conformation, optical rotation

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. 

Optical Rotation and the Conformation of Polypeptides and Proteins Peter Urnes and Paul Doty... 

Polymer molecules in solution can be found in many different geometric conformations, and there exist a variety of experimental methods (e.g., viscosity, light scattering, optical rotation) for obtaining information about these conformations. In this experiment, the measurement of optical rotation will be used to study a special type of conformational change that occurs in many polypeptides. 

Optical rotation and chain conformation in polypeptides and proteins have their common ground in the peptide bond, for it is the spatial disposition of this generic chemical group that gives rise to the principal rotatory characteristics of these snbstances and at the same time defines their secondary structure. Changes in the mutual orientation of peptide groups in the polymeric backbone are thus tantamount to conformational changes, so that the special sensitivity of optical rotatory power to steric form renders it uniquely suited to the study of conformation. 

The synthetic polypeptide most completely studied in the solid state by optical rotation is poly-n-alanine, which Elliott et al. (1957b, 1958) treat as though it were an L-polypeptide with opposite helical sense in order to make straightforward comparison of the results with other studies. The 6o for the pure L-polymer is —475, and for two helical DL-copolymers, —500 and —505 extrapolated values for the meso-polypeptide lead to a fio of — 560. These compare favorably with Pasman s result of about —425 in 30% dichloroacetic acid in chloroform, a solvent in which the conformation is helical (I asman, 1961a). The [m ] values were found to be less than those obtained for solution, a result probably attributable to environmental changes, yet of meso-poly-DL-alanine is -f 70° at 578 m/u, which is... 

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). 

However, a distinction between the two types of structure is useful in some cases, for example the evaluation of certain physical or chemical studies. Some methods used to study the conformation are sensitive mainly to the local arrangements of the backbone, and specifically to the presence of regular structures, but do not reveal the overall folding of the molecule. These methods are considered to be probes of the secondary structure [e.g., optical rotation (ORD) and circular dichroism (CD), infrared (IR) spectrophotometry]. Some techniques are sensitive to the nature of the overall folding of the polypeptide chain and thus to the changes in the conformation, but do not reveal the details of the local conformation of the polypeptide chain. They reflect the changes of the overall conformation and are probes of the tertiary structure (e.g., changes in solubility, or viscosity.) A more precise definition is preferred which terms these two types of structures as backbone and side chain conformations. 

Nuclease behaves like a typical globular protein in aqueous solution when examined by classic hydrodynamic methods (40) or by measurements of rotational relaxation times for the dimethylaminonaphth-alene sulfonyl derivative (48)- Its intrinsic viscosity, approximately 0.025 dl/g is also consistent with such a conformation. Measurements of its optical rotatory properties, either by estimation of the Moffitt parameter b , or the mean residue rotation at 233 nin, indicate that approximately 15-18% of the polypeptide backbone is in the -helical conformation (47, 48). A similar value is calculated from circular dichroism measurements (48). These estimations agree very closely with the amount of helix actually observed in the electron density map of nuclease, which is discussed in Chapter 7 by Cotton and Hazen, this volume, and Arnone et al. (49). One can state with some assurance, therefore, that the structure of the average molecule of nuclease in neutral, aqueous solution is at least grossly similar to that in the crystalline state. As will be discussed below, this similarity extends to the unique sensitivity to tryptic digestion of a region of the sequence in the presence of ligands (47, 48), which can easily be seen in the solid state as a rather anomalous protrusion from the body of the molecule (19, 49). 

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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