A novel method of peptide sequencing by mass spectrometry is described. Metastable decay of laser-desorbed ions, taking place in the first field-free drift region of a reflection time-of-flight mass spectrometer, has been monitored to get structural information from larger peptides. Fragment ions from metastable decay are mass analysed by adjusting the potentials of the ion reflectron according to the kinetic energies of the ions. The features of the technique and its significance for future applications are outlined.

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of P-sheet A. This is five-stranded in the intact molecules and six-stranded after cleavage by insertion of strand s4A whose C-terminus has become free [Lobermann, H., Tokuoka, R., Deisenhofer, J. is accompanied by changes in spectral properties and an increase in thermal stability. We show here that an Naacetyl-tetradecapeptide with the amino acid sequence of strand s4A, residues 345 -358 of human a,-antitrypsin, associates with intact a,-antitrypsin and forms a stoichiometric complex with properties very similar to cleaved a,-antitrypsin. Complex generation has the characteristics of a folding process. a,-Antitrypsin is a member of a superfamily of proteins, the serpins (serine protease inhibitors). Most of these proteins are serine protease inhibitors, others have lost this function and serve in extracellular body fluids as carrier proteins or as hormone precursors (for a review see [l]). The establishment of al-antitrypsin as the archetype of the serpins was strengthened by the determination of its crystallographic structure, in modified form, by Lobermann et al. [2]. The deduced structure readily explained that the proteolytically cleaved species represents an irreversible structural and functional alternative to small protease inhibitors which react reversibly (for a review see [3]). In cleaved a,-antitrypsin the Nand C-termini generated by limited proteolysis of the Met358-Ser359 bond are incorporated into P-sheets A and C, respectively. As only the structure of cleaved al-antitrypsin had been determined at the time, alternative reconstructions of the intact species were considered, but removal of strand s4A from the middle of the large P-sheet A was considered most likely [2]. The recent crystal and molecular structure of a model of an uncleaved serpin, plakalbumin, confirmed this hypothesis, and showed that cleaved and intact serpins differ essentially by the insertion of strand s4A between strands s5A and s3A into P-sheet A [4].The molecular structures of cleaved a,-antitrypsin and plakalbumin are shown for comparison in Fig. 1.Comparative measurements of CD and NMR spectra [5, 61 and heat denaturation [7] had indicated that cleaved serpins are more rigid and thermostable than the intact species, suggesting a strained conformation (S) in the intact form which relaxes (R) after limited proteolysis [2, 71. The S -+ R transition may be related to the inhibitory function, as two non-inhibitor serpins, ovalbumin and angiotensinogen, . The functional and structural implications of the S -+ R transition which involved an expansion of the P-sheet deserved further study which we pursued guided by previous observations of the trypsinogen -trypsin transition [lo]. In this system, peptides sequentially related to the N-terminus of trypsin induce the structural transition of trypsinogen to a trypsin-like state. Similarly exogenous peptides sequentially related to strand s4A induce the S -+ R transition in intact...

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SynopsisThe conformational properties of a series of gastrin-related peptides in aqueous solution and in 2,2,2-trifluoroethanol (TFE) have been investigated by CD measurements. In aqueous solution the peptides Leu3*-HG-34 (human big gastrin), Nle15-HG-17 (human little gastrin), and Nlell-HG-13 assume a random-coil structure in the pH range 3-7. In TFE the three hormones fold into partially ordered structures, consisting of mixtures of a-helix, @-form and random coil. Comparison with the CD properties of the shorter gastrin peptides HG-4 (tetragastrin), NU-Boc-HG-5 (pentagastrin), and HG-7 (heptagastrin) indicates that the biologically important C-terminal seqcence Trp-Met-Asp-Phe-NHz in TFE does not maintain the same geometry upon elongation of the chain a t the N-terminus from 4 to 34 residues. Thus, the various conformations in solution of the gastrin peptides examined do not provide a structural explanation for their very similar biological activity. Therefore, we hypothesize that the C-terminal tetrapeptide amide folds into an "active" structure only upon interaction with the receptor.

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Autoantibodies from patients with systemic rheumatic diseases were used to map antigenic sites on the 68-kD autoantigen (p68) associated with (U1)RNA-containing small nuclear ribonucleoprotein (snRNP) particles. With truncated recombinant fusion proteins and synthetic peptides, a subset of anti-p68 autoantibodies was found to recognize the amino acid sequence motif Glu-Arg-Lys-Arg-Arg (ERKRR). To investigate the possible involvement of epitopes shared by microbial antigens and host self-components in initiation of autoimmunity (molecular mimicry), a sequence data bank was screened for proteins containing an amino acid motif identical or related to ERKRR. The identical motif was found on the M1 matrix protein of influenza B viruses, and affinity-purified human anti-ERKRR autoantibodies recognized this epitope also in the viral amino acid sequence context. The common epitope recognized by human autoantibodies suggests that influenza B virus infection may play a role in initiation of the anti-p68 and anti-(U1)RNP autoimmune response.