Temperature-sensitive folding (tsf) mutants of the phage P22 coat protein prevent newly synthesized polypeptide chains from reaching the conformation competent for capsid assembly in cells, and can be rescued by the GroEL chaperone (Gordon, C., Sather, S., Casjens, S., and King, J. (1994) J. Biol. Chem. 269, 27941-27951). Here we investigate the stabilities of wild-type and four tsf mutant unpolymerized subunits. Wild-type coat protein subunits denatured at 40 degrees C, with a calorimetric enthalpy of approximately 600 kJ/mol. Comparison with coat protein denaturation within the shell lattice (Tm = 87 degrees C, delta H approximately 1700 kJ/mol) (Galisteo, M.L., and King, J. (1993) Biophys. J. 65, 227-235) indicates that protein-protein interactions within the capsid provide enormous stabilization. The melting temperatures of the subunits carrying tsf substitutions were similar to wild-type. At low temperatures, the tsf mutants, but not the wild-type, formed non-covalent dimers, which were dissociated at temperatures above 30 degrees C. Spectroscopic and calorimetric studies indicated that the mutant proteins have reduced amounts of ordered structure at low temperature, as compared to the wild-type protein. Although complex, the in vitro phenotypes are consistent with the in vivo finding that the mutants are defective in folding, rather than subunit stability. These results suggest a role for incompletely folded subunits as precursors in viral capsid assembly, providing a mechanism of reaching multiple conformations in the polymerized form. Study holds ProTherm entries: 5167, 5168, 5169, 5170, 5171 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:28 p.m.
|Number of data points||10|
|Proteins||Major capsid protein|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm|
|Libraries||Mutations for sequence MALNEGQIVTLAVDEIIETISAITPMAQKAKKYTPPAASMQRSSNTIWMPVEQESPTQEGWDLTDKATGLLELNVAVNMGEPDNDFFQLRADDLRDETAYRRRIQSAARKLANNVELKVANMAAEMGSLVITSPDAIGTNTADAWNFVADAEEIMFSRELNRDMGTSYFFNPQDYKKAGYDLTKRDIFGRIPEEAYRDGTIQRQVAGFDDVLRSPKLPVLTKSTATGITVSGAQSFKPVAWQLDNDGNKVNVDNRFATVTLSATTGMKRGDKISFAGVKFLGQMAKNVLAQDATFSVVRVVDGTHVEITPKPVALDDVSLSPEQRAYANVNTSLADAMAVNILNVKDARTNVFWADDAIRIVSQPIPANHELFAGMKTTSFSIPDVGLNGIFATQGDISTLSGLCRIALWYGVNATRPEAIGVGLPGQTA|