Role for cysteine residues in the in vivo folding and assembly of the phage P22 tailspike.
The predominantly beta-sheet phage P22 tailspike adhesin contains eight reduced cysteines per 666 residue chain, which are buried and unreactive in the native trimer. In the pathway to the native trimer, both in vivo and in vitro transient interchain disulfide bonds are formed and reduced. This occurs in the protrimer, an intermediate in the formation of the interdigitated beta-sheets of the trimeric tailspike. Each of the eight cysteines was replaced with serine by site-specific mutagenesis of the cloned P22 tailspike gene and the mutant genes expressed in Escherichia coli. Although the yields of native-like Cys>Ser proteins varied, sufficient soluble trimeric forms of each of the eight mutants accumulated to permit purification. All eight single Cys>Ser mature proteins maintained the high thermostability of the wild type, as well as the wild-type biological activity in forming infectious virions. Thus, these cysteine thiols are not required for the stability or activity of the native state. When their in vivo folding and assembly kinetics were examined, six of the mutant substitutions--C267S, C287S, C458S, C613S, and C635S--were significantly impaired at higher temperatures. Four--C290S, C496, C613S, and C635--showed significantly impaired kinetics even at lower temperatures. The in vivo folding of the C613S/C635S double mutant was severely defective independent of temperature. Since the trimeric states of the single Cys>Ser substituted chains were as stable and active as wild type, the impairment of tailspike maturation presumably reflects problems in the in vivo folding or assembly pathways. The formation or reduction of the transient interchain disulfide bonds in the protrimer may be the locus of these kinetic functions.
Department of Biology, Masschusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
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Haase-Pettingell
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Betts
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Raso
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Stuart
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Robinson
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King
D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics; D017100:Bacteriophage P22 / Q000737:chemistry*; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry* / Q000502:physiology*; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D012694:Serine / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
D018829:Adhesins, Bacterial; D004220:Disulfides; D012694:Serine; D003545:Cysteine; D010100:Oxygen