NMR analysis of cleaved Escherichia coli thioredoxin (1-73/74-108) and itsP76A variant: Cis/trans peptide isomerization

Inspection of high resolution three-dimensional (3D) structures from the pr otein database reveals an increasing number of cis-Xaa-Pro and cis-Xaa-Yaa peptide bonds. However, we are still Car from bring able to predict whether these bonds will remain cis upon single-site substitution of Pro or Yaa an d/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfami ly with a single alpha/beta dol nain and cis P76 to determine the effect of single-site substitution and/or cleavage on this isomer. Standard two-dime nsional (2D) NMR analysis were performed on cleaved Trx (1-73/74-108) and i ts P76A variant. Analysis of the NOE connectivities indicates remarkable si milarity between the secondary and supersecondary structure of the noncoval ent complexes and Trx. Analysis of the 2D version of the HCCH-TOCSY and HMQ C-NOESY-HMQC and C-13-filtered HMQC-NOESY spectra of cleaved Trx with unifo rmly C-13-labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31. A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in bo th the packing of I75 and the interstrand connectivities between its most p rotected hydrophobic strands (Pl and PI). Indeed, a molecular simulation mo del for the trans P76A variant of Trx shows only subtle local changes aroun d the substitution site, In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single-site substi tution (P76A) favors the trans isomer.