Residual structure in large fragments of staphylococcal nuclease: effects of amino acid substitutions.
In an attempt to develop a model of the denatured state of staphylococcal nuclease that can be analyzed experimentally under physiological conditions, a series of four large fragments of this small protein which extend from residues 1 to 103, 1 to 112, 1 to 128, and 1 to 136 have been generated through the overexpression of nuclease genes containing stop codons at defined positions. Large amounts of protein fragments were accumulated in induced cells and were purified by carrying out all fractionation steps in the presence of 6 M urea. The far-ultraviolet circular dichroism spectra of all four fragments suggested the presence of small to moderate amounts of residual structure. When the CD spectra were monitored as a function of concentrations of the tight-binding ligands Ca2+ and thymidine 3',5'-bisphosphate and the known affinity constants for wild-type nuclease (1-149) were used, apparent equilibrium constants of 160 and 2000 for the reversible denaturation reaction for fragments 1-136 and 1-128, respectively, were estimated. Four single and two double mutations, all of which exhibit unusual behavior in the full-length protein on solvent denaturation [Shortle, D., & Meeker, A. K. (1986) Proteins: Struct., Funct., Genet. 1, 81-89] and thermal denaturation [Shortle, D., Meeker, A. K., & Freire, E. (1988) Biochemistry 27, 4761-4768], were recombined into the 1-136 and 1-128 fragment expression vectors, and purified mutant fragments were characterized.(ABSTRACT TRUNCATED AT 250 WORDS)
Shortle
D
D
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Meeker
A K
AK
D002942:Circular Dichroism; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013816:Thermodynamics; D014452:Ubiquitins / Q000302:isolation & purification / Q000378:metabolism*
D004274:DNA, Recombinant; D006657:Histones; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014452:Ubiquitins; D008836:Micrococcal Nuclease