Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.

The plasmid-located gene cafl encoding the capsular antigen fraction I (Fl) of Yersiniu pestis was cloned and sequenced. The gene codes for a 170 amino acid peptide with a deduced M, of 17.6 kDa. The signal peptide sequence was highly homologous to the E. co/i consensus signal sequence. The Fl was assumed to have p-sheet structure for the most part. The region located between amino acids 100 and 150 was suggested to contain putative antigenic determinants and to stimulate T cells. 1, INTRODUCTIONThe ultimate goal of infectious disease research is their prevention. Vaccination is one of the most effective ways in which that goal can be attained. It is necessary to know the gene structure and putative immunogenic surface structures of antigens to create recombinant vaccines.More than 10 antigens have been isolated from Yersinia pestis. The capsular antigen fraction 1 (Fl) was shown to be a highly protective antigen among such thermolabile antigens as D, Fl, T, V and W [I). Some properties of the Fl structure have been studied recently [2], but the nucleotide and amino acid sequences have been unknown so far. Here, we report the cloning and sequencing of the Y. pestis cafl gene coding for the Fl , and the predicted secondary structure with potential antigenic determinants. MATERIALS AND METHODS Bacterial strains, plasmids and DNA manipulafionsThe E. co/i strains LE392 and HBIOI were used as transistent hosts for cosmid pHC79 [3], and pUC18 or f9 [4], respectively. Y. pestisF1 positive vaccine strain EV was obtained from the Culture Collection, All-Union Antiplague Institute 'Microb', USSR. Cultures were grown overnight while shaking at 37°C in liquid LB or on solid medium supplemented with the relevant antibiotics for plasmid selection. Construction of a gene library, screening, and subcloningThe Y. pestis plasmid pFra DNA (about 110 kb in size) was partially digested with EcoRI, ligated with EeoRIdigested cosmid pHC79 and packaged in vitro. The library was amplified in E. coli LE392 and ApRTcR colonies selected were further screened for Fl production by enzyme immunoassay. The isolated cosmid ~153 containing a 40-kb fragment of pFra DNA was then digested with EcoRl and an 8.6kb fragment was cloned into pHC79. The resulting cosmid pFS2 was digested with SalI and Hind111 and a 4.5kb fragment was cloned into pUCI9. The plasmid pFS2-13 generated was used for gene sequencing. The 1 .O-kb Alul fragment of pFS2-I 3 was cloned into the SmaIdigested pUCl8 (plasmid pF18L) and sequenced. Protein sequencingThe Fl protein was isolated from culture medium and purified by polyacrylamide gel electrophoresis as described [2]. The N-terminus of the mature protein was identified by a PTH-amino acid analyzer (Model 120A, Applied Biosystems). Secondary structure and anrigenic determinant analysis

The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a K d of (4.77p0.50)i10 −* M for the intact protein to one of (3.68p0.68)i10 −) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in i o was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule,

No abstract

The cooperative structure of Fc fragments prepared from myeloma human IgG1 was studied using scanning microcalorimetry and fluorescence at pH 4.2-8.0. It was shown that the first to be melted are CH2 domains whose interaction with each other is rather weak, while that with CH3 domains is strong. Then CH3 domains which form a single cooperative block are melted. The data for the structure of the Fc fragment in solution agree with the X-ray data according to which the interaction between CH2 domains is mediated by the carbohydrate moiety while the two CH3 domains are strongly associated. The presence of intensive CH2-CH3 interaction is a distinctive feature of the state of the Fc fragment in the given pH region as compared to that at pH <4.1 [Tischenko, V. M., et al. (1982) Eur. J. Biochem. 126, 517-521; Ryazantsev, S., et al. (1990) Eur. J. Biochem. 190, 393-399]. First, cis interactions greatly increase the free energy of the native structure stabilization in CH2 domains. Second, they decrease this energy for CH3 domains when compared to the state of the latter at pH 3.8 or within the Fc' fragment (the dimer of CH3 domains). The temperature and enthalpy of melting of CH2 domains coincide in all the samples studied despite heterogeneity of the carbohydrate moiety. Thus, it may be postulated that the conservative part of CH2 domains makes a cardinal contribution to the interaction of these domains with the carbohydrate moiety.