Crystallization and preliminary X-ray diffraction studies of the signal recognition particle receptor FtsY from Mycoplasma mycoides.

The prokaryotic signal recognition particle (SRP) pathway comprises two proteins, Ffh and FtsY, homologous to the SRP54 and SRalpha proteins in the more complex eukaryotic system. All four proteins are part of a unique subfamily of GTPases. Four truncated versions of the 412 amino-acid FtsY receptor protein from Mycoplasma mycoides have been cloned, expressed in Escherichia coli and purified. Purified proteins from all constructs and the full-length FtsY protein were subjected to crystallization trials. Crystals were obtained for the construct which comprised residues 98-412 corresponding to the conserved NG-domain (residues 194-497 in E. coli). A native data set at 1.9 A resolution has been collected at 100 K using synchrotron radiation. The crystals belong to the space group P2(1)2(1)2, with unit-cell parameters a = 68.7, b = 101.1, c = 42.5 A and one molecule in the asymmetric unit.

The role of the P2' position of Bowman-Birk proteinase inhibitor in the inhibition of trypsin. Studies on P2' variation in cyclic peptides encompassing the reactive site loop.

The role of the P2' residue in proteinase inhibitors of the Bowman-Birk family was investigated using synthetic cyclic peptides based on the reactive site loop of the inhibitor. A series of 21 variants having different P2' residues was tested for inhibition of trypsin, and the rate at which they were hydrolysed by this enzyme was also measured. Variation at P2' was found to result in marked differences in inhibitory potency, with the best sequence (Ile) having a Ki value of 9 nM. Peptides with P2' Gly, Pro or Glu failed to demonstrate any measurable inhibition (Ki>1 mM). The peptides also displayed significant differences in the rates at which they were hydrolysed, which varied by over three orders of magnitude between the difference sequences. There was found to be overall correlation between the Ki value and the rate of hydrolysis, with peptides that inhibited best also being hydrolysed more slowly. The results are discussed in light of the sequence information for Bowman-Birk inhibitor proteins.

Stability of protease inhibitors based on the Bowman-Birk reactive site loop to hydrolysis by proteases.

Bowman-Birk proteinase inhibitor proteins contain two inhibitory regions, each of which is encapsulated within nine-residue disulfide-linked loops. It is known that short cyclic peptides that retain the nine-residue disulfide-bridged motif have inhibitory activity, and can be used as models of the natural inhibitor protein. Two factors are important in determining the effectiveness of such inhibitor peptides: the value of the inhibition constant, and rate at which the inhibitor peptide is hydrolyzed by the proteinase. In this paper we report a study of the inhibitory properties and stability towards proteolytic hydrolysis of a family of synthetic peptides derived from the trypsin reactive site loop of the Bowman-Birk inhibitors. The addition of a single amino acid residue to each end of the nine-residue disulfide-linked loop is found to reduce the rate at which the peptide is hydrolyzed. In addition, changing the P2' residue from Asn-->Ile gives inhibitors with considerably enhanced stability to proteolysis, as well as reduced values of Ki. The implications of these factors for the design of inhibitors based on this loop motif is discussed.