Analysis of deamidation of small, acid-soluble spore proteins from Bacillus subtilis in vitro and in vivo.
Deamidation of one specific asparagine residue in an alpha/beta-type small, acid-soluble spore protein (SASP) of Bacillus subtilis took place readily in vitro (time for 50% deamidation [t(1/2)], approximately 1 h at 70 degrees C), and the deamidated SASP no longer bound to DNA effectively. However, DNA binding protected against this deamidation in vitro. A mutant alpha/beta-type SASP in which the reactive asparagine was changed to aspartate also failed to bind to DNA in vitro, and this protein did not restore UV radiation and heat resistance to spores lacking the majority of their alpha/beta-type SASP. When expressed in Escherichia coli, where it is bound to DNA, the alpha/beta-type SASP deamidated with a t(1/2) of 2 to 3 h at 95 degrees C. However, the alpha/beta-type SASP was extremely resistant to deamidation within spores (t(1/2), >50 h at 95 degrees C). A gamma-type SASP of B. subtilis also deamidated readily in vitro (t(1/2) for one net deamidation, approximately 1 h at 70 degrees C), but this protein (which is not associated with DNA) deamidated fairly readily in spores (t(1/2), approximately 1 h at 95 degrees C). Total spore core protein also deamidated in vivo, although the rate was two- to threefold slower than that of deamidation of total protein in heated vegetative cells. These data indicate that protein deamidation is slowed significantly in spores, presumably due to the spore's environment. However, alpha/beta-type SASP are even more strongly protected against deamidation in vivo, presumably by their binding to spore DNA. Thus, not only do alpha/beta-type SASP protect spore DNA from damage; DNA also protects alpha/beta-type SASP.
Hayes
C S
CS
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030, USA.
Setlow
P
P
D001216:Asparagine / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D006358:Hot Temperature; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012808:Sigma Factor; D012995:Solubility; D013171:Spores, Bacterial / Q000378:metabolism; D014157:Transcription Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D001224:Aspartic Acid; D001216:Asparagine