RESTRICTION IN THE CONFORMATIONAL FLEXIBILITY OF APOPROTEINS IN THE PRESENCE OF ORGANIC COSOLVENTS - A CONSEQUENCE OF THE FORMATION OF NATIVE-LIKE CONFORMATION

The influence of n-propanol on the overall alpha-helical conformation of beta-globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of alpha-helical conformation is induced into these proteins at pH 6.0 and 4-degrees-C in the presence of relatively low concentrations of n-propanol. The induction of alpha-helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of alpha-globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The alpha-helical conformation induced into alpha- and beta-globin by n-propanol decreased as the temperature is raised from 4 to 24-degrees-C. In contrast, the alpha-helical conformation of both alpha- and beta-chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced "alpha-helical conformation" of globins and the "alpha-helical conformation" of alpha- and beta-chains. A cross-correlation of the n-propanol induced increase in the fluorescence of beta-globin with the corresponding increase in the alpha-helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the alpha-helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a "native-like structure." The induction of alpha-helical conformation into these proteins in the presence of n-propanol and the consequent generation of "native-like conformation" is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an alpha-helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume alpha-helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high alpha-helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of alpha-helical proteins. Consistent with this concept, the induction of alpha-helical conformation into shorter polypeptide fragments of 30 residues, (e.g., alpha1-30, which exists in an alpha-helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.