Conformationally Dependent Low-Frequency Motions of Proteins by Laser Raman Spectroscopy

Abstract

Low-frequency Raman bands (lower than 50 cm-') exist in certain proteins. They are dependent upon the conformation of the protein molecule, but are relatively independent of the form of the sample, i.e., whether it is a film or a crystal.Low-frequency Raman spectra were obtained from samples of a-chymotrypsin that had been prepared in several ways. A peak at about 29 cm-' was found for all samples except the one that had been denatured with sodium dodecyl sulfate. Such low frequency motions must arise from vibrations that involve all, or very large portions, of the protein molecule.In the past few years the technique of laser Raman spectroscopy has been used with considerable success to obtain the Raman active vibrations of several proteins (1-4). However, if one examines the published spectra, it is apparent that it has been impossible in the past to obtain the Raman bands in proteins that lie below 150 cm-'. The reason for this is that the scattering due to the Rayleigh component is too large for a double-grating monochromator to discriminate against. Recently, in this laboratory, we have shown how to obtain Raman spectra only a few wave numbers from the exciting line on synthetic polymers, such as polyethylene and poly-i-alanine, by the iodine filter technique (5-7) and a Spex double-grating monochromator. More recently, we have also found it possible to obtain these low frequency bands using a Cary triple-grating monochromator. For the work reported here, we have used both of these instruments and have obtained equivalent results on each. This has been of the greatest help in elimination of the possibility of experimental artifacts. From our work with these instruments it is possible to show, for the first time, that definite low-frequency motions exist in many common proteins and that these vibrations appear to be sensitive to the conformation of the protein. As we will discuss below, such low-frequency motions must arise from vibrations that involve either all or very large portions of the protein molecule. Thus, it appears from our measurements that large portions of the protein molecule are constantly undergoing a coherent periodic vibration. The existence of such vibrations is of considerable interest even though the exact assignment of the motion is not possible at present.Figs. la and b show the low-frequency Raman spectra of samples of a-chymotrypsin that were prepared in several ways. In every case, except the sample that had been denatured with SDS, a pronounced peak at about 29 cm-' is found. It is apparent that there is some splitting of the peak in the single crystal and also some slight change in the shape of the peak with deuteration and acylation. However, upon denaturation with SDS the peak at 29 cm-' vanishes. Rather intense Raman scattering throughout the region of 20-150 cm-' is observed on the denatured material, but it is broad and structureless-a fact that probably reflects the decrease in the order of the protein conformation.The fact that this low-frequency band is dependent ...

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