THE PHAGE-LAMBDA TERMINASE ENZYME - 2 - REFOLDING OF THE GPNU1 SUBUNIT FROM THE DETERGENT-DENATURED AND GUANIDINIUM HYDROCHLORIDE-DENATUREDSTATE YIELDS DIFFERENT OLIGOMERIZATION STATES AND ALTERED PROTEIN STABILITIES

The terminase enzyme from bacteriophage lambda is responsible for pack aging a single genome within the viral capsid. Gold and co-workers hav e developed a scheme for the solubilization of the small terminase sub unit (gpNu1) from inclusion bodies using the strong detergent sarkosyl and purification of the protein to homogeneity (gpNu1(SRK)) (Parris e t al., J Biol Chem 1994,269:13564-13574). We have developed a similar purification scheme except that guanidinium hydrochloride was used to denature the insoluble protein (gpNu1(GDN)). The circular dichroism (C D) spectra of both protein preparations suggest that they are predomin antly a-helical when purified and stored in Tris buffers. Moreover, th ermal denaturation of the proteins thus purified yielded similar therm odynamic parameters for unfolding (T-m, Delta H-m and Delta S-m of unf olding of approximate to 306 K, approximate to 22 kcal/mol and approxi mate to 70 cal/mol.K, respectively). Interestingly, however, when the proteins were purified and stored in imidazole buffers, the gpNu1(SRK) preparation lost a significant amount of secondary structure and was more stable to both thermally-induced and guanidinium HCl-induced dena turation than was gpNu1(GDN). The purified gpNu1 monomers oligomerize into apparent tetramers and hexamers in solution and the distribution between these two oligomeric states and into higher order aggregates d epends upon buffer composition, salt concentration and protein concent ration. Moreover, differences in the oligomerization state of gpNu1(SR K) and gpNu1(GDN) under identical buffer conditions were observed. The significance of these results with respect to the biological role of the phage lambda gpNu1 protein are discussed. (C) 1998 Elsevier Scienc e B.V. All rights reserved.