Andrzej Galat

Complete amino acid sequence of the FK506 and rapamycin binding protein, FKBP, isolated from calf thymus

FKBP, an 11.8 kD intracellular protein that binds the immunosuppressants FK506 (Kd=0.4 nM) and rapamycin (Kd=0.2 nM) with high affinity, was purified to homogeneity from calf thymus. The complete amino acid sequence has been determined by automated Edman degradation of the intact molecule and overlapping fragments generated by proteolytic and chemical cleavage. The analysis revealed a 107 amino acid peptide chain with the following sequence: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFVLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPNATLIFDVELLKLE. The molecular weight, calculated from the amino sequence to be 11,778 D, was confirmed by electrospray ionization mass spectrometry. Thus, naturally isolated bovine FKBP does not appear to have any residues modified by glycosylation, phosphorylation, or other post-translational derivatization processes. Bovine FKBP has only three amino acid residues that differ from human FKBP, whose sequence was elucidated by cloning and sequencing complementary DNA (Standaertet al., 1990). The protein has a substantial number of hydrophilic peptide segments with prevalent β-strand type of chain fold. Understanding the biological function of FKBP and other members of the immunophilin class and their respective complexes with immunosuppressive drugs may provide insights into cytoplasmic signalling mechanisms, protein folding and translocation, and other cellular processes.

Conserved structural determinants in three-fingered protein domains.

The three‐dimensional structures of some components of snake venoms forming so‐called ‘three‐fingered protein’ domains (TFPDs) are similar to those of the ectodomains of activin, bone morphogenetic protein and transforming growth factor‐β receptors, and to a variety of proteins encoded by the Ly6 and Plaur genes. The analysis of sequences of diverse snake toxins, various ectodomains of the receptors that bind activin and other cytokines, and numerous gene products encoded by the Ly6 and Plaur families of genes has revealed that they differ considerably from each other. The sequences of TFPDs may consist of up to six disulfide bonds, three of which have the same highly conserved topology. These three disulfide bridges and an asparagine residue in the C‐terminal part of TFPDs are essential for the TFPD‐like fold. Analyses of the three‐dimensional structures of diverse TFPDs have revealed that the three highly conserved disulfides impose a major stabilizing contribution to the TFPD‐like fold, in both TFPDs contained in some snake venoms and ectodomains of several cellular receptors, whereas the three remaining disulfide bonds impose specific geometrical constraints in the three fingers of some TFPDs.

Exogenous myristic acid acylates proteins in cultured rat hepatocytes

Fatty acid acylation is a functionally important modification of proteins. In the liver, however, acylated proteins remain largely unknown. This work was aimed at investigating fatty acid acylation of proteins in cultured rat hepatocytes. Incubation of these cells with [9,10-3H] myristic acid followed by two-dimensional electrophoresis separation of the delipidated cellular proteins and autoradiography evidenced the reproducible and selective incorporation of radioactivity from the precursor into 18 well-resolved proteins in the 10--120 kDa range and the 4--7 pH range. Radiolabeling of these proteins resulted from covalent linkage to the precursor [9,10-3H] myristic acid or to its elongation product, palmitic acid. The majority of the covalent linkages between the proteins and the fatty acids were broken by base hydrolysis, which indicated that the linkage was of thioester or ester-type. Only one of the studied proteins was attached to myristic acid via an amide linkage which resisted the basic treatment but was broken by acid hydrolysis. After incubation with [9,10-3H] palmitic acid, only two proteins previously detected with myristic acid were radiolabeled. Finally, the identified acylated proteins may be grouped into two classes: proteins involved in signal transduction (the alpha subunit of a heterotrimeric G protein and several small G proteins) and cytoskeletal proteins (cytokeratins, actin).

Interaction of riboflavin binding protein with riboflavin, quinacrine, chlorpromazine and daunomycin.

1. circular dichroism and fluorescence measurements were used to study the reversible unfolding of riboflavin-riboflavin binding protein complex. Both methods showed that the complex was unfolded according to the two-state model. 2. The results suggest that riboflavin was strongly bound in the hydrophobic cleft of the protein and that it could not be dislodged by TFE, MeOH or SDS without major unfolding of the unique tertiary structure of the protein. 3. In addition, it has been also shown that quinacrine, chlorpromazine and daunomycin did not form stereospecific complexes with riboflavin binding protein.

Unfolding processes of small globular proteins: The two-state vs multi-state model

1. The alcohol-induced unfolding of two homologous proteins, neurotoxin and cardiotoxin from Taiwan cobra (Naja naja atra) venom has been analysed. 2. It is postulated that the unfolding process for both proteins is a multi-state conformational transition. 3. It has been hypothesized that between the compact native state of the protein and its fully unfolded state there exists a quasi-continuous spectrum of conformational metastates of protein species. 4. The population distribution of these metastates is partially dependent on the nature of unfolding factors as well as the amino acid composition and sequence. 5. The sum of all transient conformational states and the protein species being in the folded and unfolded states respectively, can be detected by means of circular dichroism spectroscopy since the absorption of circularly polarized light is rapid relative to the rate of fluctuations of the protein structure.

Analysis of microdensitometric data in terms of probability of cleavage of DNA

Computer-aided analysis of autoradiographic films of DNA fragments is presented. The Powell least-squares procedure is used for optimization of parameters for components of complex densitometric curves. Since each densitometric spectrum may be divided for several non-overlapped blocks of bands, there is no upper limit on the number of parameters which must be optimized. Eight shapes for the component bands are utilized: symmetric and asymmetric Gauss and Cauchy functions, direct, symmetric and asymmetric product of Gauss function and inverse of Cauchy function, and log-normal function. The probability of DNA cleavage is calculated with correction for multiple cuts. The methods presented was applied to detailed analysis of densitometric spectra of a 21-bp DNA restriction fragment and allowed for direct correlation between structural microheterogeneity of DNA and the resulting cutting pattern. This method should facilitate the analysis of densitometric data from antibiotic-induced cleavage of DNA and footprinting experiments.

Computer-aided analysis of infrared, circular dichroism and absorption spectra

Marquardt and Powell optimization methods without constraints on the optimized spectral parameters were employed for decomposition of complex i.r., c.d. and absorption spectra into component bands. The procedure resolved experimental spectra into eight component bands and it can be easily adjusted for a larger set of component bands. The CPU time required for achievement of satisfactory convergence of parameters for eight component bands is rather large even when using mainframe computers and therefore division of spectra into a few non-overlapped parts is advisable. The program also can be used for calculation of absorption, c.d. and difference spectra from formatted raw spectral data.

Statistics of disulfide bond formation in proteins

Abstract A new set of statistical expressions describing the reformation of disulfide bonds from SH groups is proposed. The results of the statistical calculations of disulfide bond reformation are discussed in terms of protein folding.

CORGEN: A FORTRAN-77 generator of standard and non-standard DNA helices from the sequence

An analytical procedure CORGEN generates a variety of DNA double-stranded structures from user-supplied sequence using a nucleic acid database incorporated into a standard FORTRAN-77 program. Alternatively, the cylindrical polar coordinates of DNA components may be supplied from the external table. An algorithm that performs intercalation sites in DNA is described. This procedure can be used to generate complexes of antibiotics with DNA. Non-standard DNA structures can be built by alternating the global helical twist and global helical rise in the regular DNA helix. The procedures described can be used for computer generation of a variety of non-standard DNA structures which can be subjected to molecular mechanics and dynamics simulations.

Interaction of Rose Bengal with α-chymotrypsin and α-chymotrypsinogen-A

Abstract Absorption spectra of aqueous solutions of Rose Bengal—(α-chymotrypsin) and Rose Bengal—(α- chymotrypsinogen-A) measured at different pHs showed that both proteins formed the same complexes. The binding constants for the formation of RB/CHT and RB/CHTGA complexes were of the same order of magnitude. The binding isotherms showed that stoichiometry of RB complexes was 3:1 at pH 4.6 and 1:1 at pH 7.2 for both RB/CHT and RB/CHTGA, respectively. Equal binding between the dye and both proteins eliminated the possibility that RB could interact selectively with the active site of α-chymotrypsin. Charge-charge interaction between the carboxylic group of the dye and the positively charged protein surface was essential for the stability of the complexes. The binding reached its minimum when the pH of aqueous solution was equal to the pI of the protein. The complexes exhibited no CD at any pH and at any KCl concentration varied from 0.0 to 0.5 M, which showed that the complexes were achiral. At pH