Effect of single amino acid replacements on the ... - Semantic Scholar

Proc. Nail. Acad. Sci. USA Vol. 81, pp. 5685-5689, September 1984 Biochemistry

Effect of single amino acid replacements on the thermal stability of the NH2-terminal domain of phage X repressor (scanning calorimetry/circular dichroism/proteolysis/temperature-sensitive mutants)

MICHAEL H. HECHT*, JULIAN M. STURTEVANTt,

AND ROBERT T. SAUER*

*Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and tDepartment of Chemistry and Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511

Contributed by Julian M. Sturtevant, May 29, 1984

The thermal stabilities of mutant phage X reABSTRACT pressors that have single amino acid replacements in the NH2terminal domain have been studied by means of circular dichroism and differential scanning calorimetry. The variations in stability determined by these physical methods correlate with the resistance to proteolysis at various temperatures and can be compared with the temperature-sensitive activity of the mutants in vivo. In general, mutant proteins bearing solventexposed substitutions have thermal stabilities identical to wild type, whereas buried substitutions reduce stability. In one case, a single amino acid replacement increases the thermal stability of the repressor.

10% of their total protein. Mutant proteins were purified by procedures similar to those described for wild-type repressor

(8).

Thermal Stabilities. CD measurements were made with a Cary model 60 spectrapolarimeter equipped with a Cary model 6001 CD accessory. A water-jacketed cylindrical cell with a 1-cm pathlength was used for protein concentrations