Intragenic suppression of a capsid assembly-defective P22 tailspike mutation.

The tailspike protein of bacteriophage P22 assembles with mature capsids during the final reaction in phage morphogenesis. The gene 9 mutation hmH3034 synthesizes a tailspike protein with a change at amino acid 100 from Asp to Asn. This mutant form of trimeric tailspike protein fails to assemble with capsids in vivo. By using in vitro quantitative tailspike-capsid assembly assays, this mutant tailspike trimer can be shown to assemble with capsids at very high tailspike concentrations. From these assays, we estimate that this single missense mutation decreases by 100-500-fold the affinity of the tailspike for capsids. Furthermore, hmH3034 tailspike protein has a structural defect which makes the mature tailspike trimers sensitive to SDS at room temperature and causes the trimers to "partially unfold." Spontaneously arising intragenic suppressors of the capsid assembly defect have been isolated. All of these suppressors are changes at amino acid 13 of the tailspike protein, which substitute His, Leu or Ser for the wild type amino acid Arg. These hmH3034/sup3034 mutants and the separated sup3034 mutants form fully functional tailspike proteins with assembly activities indistinguishable from wild type while retaining the SDS-sensitive structural defect. From the analysis of the hmH3034 mutant and its suppressors, we propose that in the wild-type tailspike protein, the Asp residue at position 100 and the Arg residue at position 13 form an intrachain or interchain salt bridge which stabilizes the amino terminus of the tailspike protein and that the unneutralized positive charge at amino acid 13 in the hmH3034 protein is the cause of the assembly defect of this protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of protein phosphorylation state by a combination of vertical slab gel isoelectric focusing and immunoblotting.

Conditions have been established for one-dimensional isoelectric focusing using vertical slab gel electrophoresis, followed by immunoblotting, for the measurement of the phosphorylation state of proteins. The method provides a less time-consuming alternative to two-dimensional gel electrophoresis combined with radiolabeling or immunoblotting. The main advantage of the method is that many samples can be analyzed simultaneously. The technique is applied here to the study of a mammalian initiation factor for protein synthesis, eukaryotic initiation factor 2 (eIF-2). The method allows good separation and quantitation of the different phosphorylated forms of the alpha subunit of eIF-2, when used to analyze either purified eIF-2 or eIF-2 contained in complex mixtures. The method is shown to be well adapted to the measurement of rapid phosphorylation/dephosphorylation kinetics in cell extracts, as well as the measurement of the phosphorylation state of eIF-2 in cultured cells. In addition, the method is shown to confirm the existence of a second phosphorylation site on eIF-2. Although eIF-2 has been used for this demonstration of the efficacy of the method, the technique is applicable to a study of the regulation of covalent modification of any polypeptide for which antibodies are available.