Bacteriophage lambda cro mutations: effects on activity and intracellular degradation.

Abstract

Following random mutagenesis of the bacteriophage X cro gene, we have isolated missense mutations that affect approximately half of the 66 residue positions of Cro. About two-thirds of the mutations change residues involved in the maintenance of Cro structure and stability. The corresponding mutant proteins are severely degraded in the cell but often have specific activities near that of wild-type Cro. The remaining mutations affect residues involved in DNA binding. These mutant proteins are present at moderately reduced intracellular levels, but their specific activities are much lower than that of wild type.The study of missense mutants provides a means of testing and adding to our understanding of the determinants of protein structure, stability, and function. In this paper, we describe mutations that affect the intracellular level and activity of bacteriophage X Cro, a small repressor with a known three-dimensional structure (1-3). We find that a Crophenotype results from replacing any 1 of at least half of the 66 residues of Cro; some of the mutations change residues on the DNA binding surface, some change solvent-exposed residues distant from the proposed binding surface, and some affect residues in the hydrophobic core. Mutations that would be expected to destabilize the folded structure of Cro generally result in extreme sensitivity to intracellular proteolysis. In many cases, the resulting reduction in concentration is severe enough to account fully for the observed mutant phenotype. MATERIALS AND METHODSPlasmids, Phage, and Bacteria. Three related Cro-producing plasmids-pAP100, pAP101, and pAP104-were constructed for this work. Each plasmid is a pBR322 derivative, and each bears the same fusion of the lac UV5 promoter to the phage X cro gene. This fusion was derived from plasmid pTR214 (4). Plasmid pAP100 was constructed by inserting the 353-base-pair EcoRI/Nae I fragment from pTR214 between the EcoRI and filled-in Cla I sites of pBR322 (5). Plasmid pAP101 was constructed by replacing the 391-base-pair Cla I/BamHI fragment of pAP100 with the 375-base-pair EcoRI/BamHI fragment from pBR322. pAP101 has an intact tet promoter and, thus, confers a significantly higher level of tetracycline resistance than does pAP100. pAP104 is similar to pAP101 but contains the phage M13 origin of replication. pAP104 was constructed by replacing the small EcoRI/Bam-HI fragment of pZ150 (6) with that from plasmid pAP101. Strains containing pAP100, pAP101, or pAP104 express the same level of phage X Cro, as judged by the resistance of the strain to phage infection and by competitive radioimmunoassay. Thus, Cro-mutations isolated in any of the three plasmid backgrounds can be directly compared.X200 is an immunity 21 bacteriophage that carries the lacZ gene under the control of the phage X PR promoter (7). Phage M13 appl was constructed by deletion in vitro of the lac promoter and operator sequences of M13 mp8 (8). DNA between the unique Ava II and EcoRI sites was deleted, the DNA was ligated? and candidates were scree...

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain

“…SDS-PAGE analysis (Fig. S3) of the soluble and insoluble fractions of the Escherichia coli cells expressing these PYP derivatives indicated the insolubility of some mutants and proteolytic degradation (little PYP detected) of others, similar to published results on bacteriophage λ Cro (34).…”

Genetic Constraints on Protein Evolution

“…In these studies, misfolding was found to be the main cause of protein inactivation rather than loss of catalytic activity (Loh et al, 2007b;Pakula et al, 1986). The role of thermodynamic stability in limiting mutation tolerance has been confirmed in later studies using thermostable proteins and modeling in silico.…”

The denaturation and degradation of stable enzymes at high temperatures

“…A variety of studies [27][28][29][30][31] have shown that the majority of point mutations have little effect on stability. For example, of over 2000 mutations made to T4 lysozyme, 91 % had no significant effect on thermal stability [24,25].…”