Spectroscopic and hydrodynamic studies reveal structural differences in normal and transforming H-ras gene products

Abstract

We have recorded the circular dichroism spectra of the cellular and the viral H-ras gene products both in the absence and in the presence of guanine nucleotides and analyzed these spectra in terms of the secondary structure composition of these proteins. It is shown that the GTP complex of the ras proteins has a different secondary structure composition than the GDP complex and, furthermore, that there are differences in the secondary structure of the viral ras protein and the cellular ras protein. We have also recorded and analyzed the circular dichroism spectrum of the isolated guanine nucleotide binding domain of the Escherichia coli elongation factor Tu (EF-Tu), which has been considered as a model for the tertiary structure of the ras proteins [McCormick, F., Clark, B.F.C., LaCour, T.F.M., Kjeldgaard, M., Norskov-Lauritsen, L., and Nyborg, J. (1985) Science (Washington, D.C.) 230, 78-82]. Our data show that the guanine nucleotide binding domain of EF-Tu (20% .alpha.-helix and 16% .beta.-pleated sheet for the GDP complex) has a quite different secondary structure composition than the ras proteins (e.g., the cellular ras protein has 47% .alpha.-helix and 22% .beta.-pleated sheet for the GDP complex) indicating that the protein core comprising the guanine nucleotide binding site might be similar but that major structural differences must exist at the portion outside this core. Normal and transforming ras proteins also differ slightly in their hydrodynamic properties as shown by sedimenation velocity runs in the analytical ultracentrifuge. The cellular ras protein has an s20,w value of 2.4 S and the viral homologue has an s20,w value of 2.2 S, indicating that the former is somewhat more compact than the latter. The structural differences between the cellular and the viral ras proteins do not affect the stability of these proteins toward guanidinium chloride induced or temperature-induced denaturation, as shown by circular dichroism spectroscopy and GDP exchange activity measurements.