Protein SRP54 is an essential component of eukaryotic signal recognition particle (SRP). The methionine-rich M-domain (SRP54M or 54M) interacts with the SRP RNA and is also involved in the binding to signal peptides of secretory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP-mediated protein targeting, we studied the human 54M polypeptide. The recombinant human protein was expressed successfully in Escherichia coli and was purified to homogeneity. Our studies determined the sites that were susceptible to limited proteolysis, with the goal to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced by V8 protease or chymotrypsin, 54MM-2 was the shortest (120 residues; Mr = 13,584.8), but still contained the conserved amino acids suggested to associate with the signal peptide or the SRP RNA. 54MM-2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presence of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM-2 were established. Examination of the crystals by X-ray diffraction showed an orthorhombic unit cell of dimensions a = 29.127 Å, b = 63.693 Å, and c = 129.601 Å, in space group P212121, with reflections extending to at least 2.0 Å.

The consequences of amino acid substitutions at the dimer interface for the strength of the interactions between the monomers and for the catalytic function of the dimeric enzyme alkaline phosphatase from Escherichia coli have been investigated. The altered enzymes R10A, R10K, R24A, R24K, T59A, and R10A/R24A, which have amino acid substitutions at the dimer interface, were characterized using kinetic assays, ultracentrifugation, and transverse urea gradient gel electrophoresis. The kinetic data for the wild-type and altered alkaline phosphatases show comparable catalytic behavior with kcat values between 51.3 and 69.5 s−1 and Km values between 14.8 and 26.3 μM. The ultracentrifugation profiles indicate that the wild-type enzyme is more stable than all the interface-modified enzymes. The wild-type enzyme is dimeric in the pH range of pH 4.0 and above, and disassembled at pH 3.5 and below. All the interface-modified enzymes, however, are apparently monomeric at pH 4.0, begin assembly at pH 5.0, and are not fully assembled into the dimeric form until pH 6.0. The results from transverse urea gradient gel electrophoresis show clear and reproducible differences both in the position and the shape of the unfolding patterns; all these modified enzymes are more sensitive to the denaturant and begin to unfold at urea concentrations between 1.0 and 1.5 M; the wild-type enzyme remains in the folded high mobility form beyond 2.5 M urea. Alkaline phosphatase H370A, modified at the active site and not at the dimer interface, resembles the wild-type enzyme both in ultracentrifugation and electrophoresis studies. The results obtained suggest that substitution of a single amino acid at the interface sacrifices not only the integrity of the assembled dimer, but also the stability of the monomer fold, even though the activity of the enzyme at optimal pH remains unaffected and does not appear to depend on interface stability.