Mutational analysis of kinetic partitioning in protein folding and protein-DNA binding.
Kinetic partitioning between competing routes is present in many biological processes. Here, we propose a methodology to characterize kinetic partitioning through site-directed mutagenesis and apply it to parallel routes for unfolding of the TI I27 protein and for recognition of its target DNA by the human papillomavirus E2 protein. The balance between the two competing reaction routes can be quantified by the partitioning constant K(p). K(p) is easily modulated by point mutations, opening the way for the rational design of kinetic partitioning. Conserved wild-type residues strongly favor one of the two competing reactions, suggesting that in these systems there is an evolutionary pressure to shift partitioning towards a certain route. The mutations with the largest effects on partitioning cluster together in space, defining the protein regions most relevant for the modulation of partitioning. Such regions are neither fully coincident with nor strictly segregated from the regions that are important from each competing reaction. We dissected the mutational effects on partitioning into the contributions from each competing route using a new parameter called pi-value. The results suggest how the design of kinetic partitioning may be approached in each case.
Sánchez
Ignacio E
IE
Protein Structure-Function and Engineering Laboratory, Fundación Instituto Leloir and IIBBA-CONICET, Av. Patricias Argentinas 435, 1405 Buenos Aires, Argentina. isanchez@leloir.org.ar
Ferreiro
Diego U
DU
Gay
Gonzalo de Prat
Gde P
D064211:Connectin; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
D064211:Connectin; D004268:DNA-Binding Proteins; D009124:Muscle Proteins; D009856:Oncogene Proteins, Viral; C577285:TTN protein, human; C076036:oncogene protein E2, Human papillomavirus type 1; D004247:DNA; D011494:Protein Kinases