Protein mutagenesis with monodispersity-based quality probing: selective inactivation of p53 degradation and DNA-binding properties of HPV E6 oncoprotein.
Interpretation of protein mutagenesis experiments requires the ability to distinguish functionally relevant mutations from mutations affecting the structure. When a protein is expressed soluble in bacteria, properly folded mutants are expected to remain soluble whereas misfolded mutants should form insoluble aggregates. However, this rule may fail for proteins fused to highly soluble carrier proteins. In a previous study, we analysed the biophysical status of HPV oncoprotein E6 fused to the C-terminus of maltose-binding protein (MBP) and found that misfolded E6 moieties fused to MBP formed soluble aggregates of high molecular weight. By contrast, preparations of properly folded E6 fused to MBP were monodisperse. Here, we have used this finding to evaluate the quality of 19 MBP-fused E6 site-directed mutants by using a light scattering assay performed in a fluorimeter. This assay guided us to rule out structurally defective mutants and to obtain functionally relevant E6 mutants selectively altered for two molecular activities: degradation of tumour suppressor p53 and DNA recognition.
Ristriani
Tutik
T
Laboratoire d'Immunotechnologie, UMR CNRS 7100, Ecole Supérieure de Biotechnologie de Strasbourg, 67400 Illkirch, France.
Nominé
Yves
Y
Laurent
Cécile
C
Weiss
Etienne
E
Travé
Gilles
G
D000595:Amino Acid Sequence; D002352:Carrier Proteins; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D027383:Papillomaviridae; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C118860:E6 protein, Human papillomavirus type 6; D058619:Maltose-Binding Proteins; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA