Unfolding of bacteriophage P22 tailspike protein: enhanced thermal stability of an N-terminal fusion mutant.
The tailspike protein (TSP) of bacteriophage P22 is a homotrimeric multifunctional protein responsible for cell attachment and hydrolysis of the Salmonella typhimurium host cell receptor. Despite the folding of TSP involves the formation of thermolabile intermediates, the mature protein is extremely resistant to heat and detergent denaturation. We have analyzed the thermal resistance and unfolding pathway of two mutant, functional TSPs carrying end-terminal peptide fusions. Whereas the C-terminal fusion has minor effects on the TSP stability, the presence of a 23-mer foreign peptide at the N terminus (protein ATSP) results in a significant enhancement of the thermal resistance by retarding the first transition step of the unfolding process. At 65 degrees C and in 2% SDS, the unfolding rate constant for the transition from the native to the unfolding intermediate is 9.3 x 10(-4) s(-1) for ATSP versus 1.7 x 10(-3) s(-1) for wild-type TSP. On the other hand, the electrophoretic mobility of ATSP intermediates is greatly affected, proving structural modifications induced by the fused peptide. These results suggest a critical participation of the N-terminal domain in the unfolding kinetic barriers generated during the TSP denaturation pathway.
Carbonell
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Institut de Biologia Fonamental and Department de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Villaverde
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D017100:Bacteriophage P22 / Q000737:chemistry* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D016254:Mutagenesis, Insertional / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics; D017376:Viral Tail Proteins / Q000737:chemistry* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D017376:Viral Tail Proteins; D006026:Glycoside Hydrolases; C083268:tailspike protein, bacteriophage