Raman and CD spectroscopy of recombinant 68-kDa DNA human topoisomerase I and its complex with suicide DNA-substrate.

N-terminally truncated recombinant 68-kDa human topoisomerase (topo) I exhibits the same DNA-driving activities as the wild-type protein. In the present study, Raman and circular dichroism techniques were employed for detailed structural characterization of the 68-kDa human topo I and its transformations induced by the suicide sequence-specific oligonucleotide (solig) binding and cleavage. Spectroscopic data combined with statistical prediction techniques were employed to construct a model of the secondary structure distribution along the primary protein structure in solution. The 68-kDa topo I was found to consist of ca. 59% alpha-helix, 24% beta-strand and/or sheets, and 17% other structures. A secondary structure transition of the 68-kDa topo I was found to accompany solig binding and cleavage. Nearly 15% of the alpha-helix of 68-kDa topo I is transferred within the other structures when in the complex with its DNA substrate. Raman spectroscopy analysis also shows redistribution of the structural rotamers of the 68-kDa topo I disulfide bonds and significant changes in the H-bonding of the Tyr residues and in the microenvironment/conformation of the Trp side chains. No structural modifications of the DNA substrate were detected by spectroscopic techniques. The data presented provide the first direct experimental evidence of the human topo I conformational transition after the cleavage step in the reaction of binding and cleavage of DNA substrate by the enzyme. This evidence supports the model of the enzyme function requiring the protein conformational transition. The most probable location of the enzyme transformations was the core and the C-terminal conservative 68-kDa topo I structural domains. By contrast, the linker domain was found to have an extremely low potential for solig-induced structural transformations. The pattern of redistribution of protein secondary structures induced by solig binding and covalent suicide complex formation supports the model of an intramolecular bipartite mode of topo I/DNA interaction in the substrate binding and cleavage reaction.

The secondary structure and architecture of human elastin.

The presented work constitutes the first structural characterization of both insoluble human elastin and its solubilized form, kappa-elastin. Structural data were reached following the use of Fourier transform infrared, near infrared Fourier transform Raman and circular dichroism optical spectroscopic methods and their quantitative analysis permitted us to estimate approximately 10% alpha-helices, approximately 35% beta-strands and approximately 55% undefined conformations in the global secondary structure of insoluble human elastin in the solid state. Following the use of the LINK method, the probable local distribution of the secondary-structure elements along the sequence was determined and compared to that obtained for bovine elastin, the historical standard of elastin. This comparison led us to propose a globular architecture for the human elastomer and permitted us to delineate some elements of its structure-elasticity relationship.

Camptothecin-binding site in human serum albumin and protein transformations induced by drug binding.

Circular dichroism (CD) and Raman spectroscopy were employed in order to locate a camptothecin (CPT)-binding site within human serum albumin (HSA) and to identify protein structural transformations induced by CPT binding. A competitive binding of CPT and 3'-azido-3'-deoxythymidine (a ligand occupying IIIA structural sub-domain of the protein) to HSA does not show any competition and demonstrates that the ligands are located in the different binding sites, whereas a HSA-bound CPT may be replaced by warfarin, occupying IIA structural sub-domain of the protein. Raman and CD spectra of HSA and HSA/CPT complexes show that the CPT-binding does not induce changes of the global protein secondary structure. On the other hand, Raman spectra reveal pronounced CPT-induced local structural modifications of the HSA molecule, involving changes in configuration of the two disulfide bonds and transfer of a single Trp-residue to hydrophilic environment. These data suggest that CPT is bound in the region of interdomain connections within the IIA structural domain of HSA and it induces relative movement of the protein structural domains.

Bovine elastin and kappa-elastin secondary structure determination by optical spectroscopies.

Elastin is the macromolecular polymer of tropoelastin molecules responsible for the elastic properties of tissues. The understanding of its specific elasticity is uncertain because its structure is still unknown. Here, we report the first experimental quantitative determination of bovine elastin secondary structures as well as those of its corresponding soluble kappa-elastin. Using circular dichroism and Fourier transform infrared and near infrared Fourier transform Raman spectroscopic data, we estimated the secondary structure contents of elastin to be approximately 10% alpha-helices, approximately 45% beta-sheets, and approximately 45% undefined conformations. These values were very close to those we had previously determined for the free monomeric tropoelastin molecule, suggesting thus that elastin would be constituted of a closely packed assembly of globular beta structural class tropoelastin molecules cross-linked to form the elastic network (liquid drop model of elastin architecture). The presence of a strong hydration shell is demonstrated for elastin, and its possible contribution to elasticity is discussed.

Conformational changes of lipoxygenase (LOX) in modified environments. Contribution to the variation in specificity of soybean LOX type 1.

The addition of water-soluble cosolvents in the reaction medium of type 1 soybean lipoxygenase can modify the selectivity of the enzyme in the hydroperoxide synthesis reaction. This also results in changes in secondary reactions such as carbonyl compound formation. The possibility of a conformational change of the enzyme due to variations in its microenvironment was considered. Using enzyme immobilization and laser visible Raman spectroscopy, both indirect and direct observations of such a protein conformational rearrangement are described. Subtle modifications in the secondary and/or tertiary structures, for example in microenvironments of Tyr and Trp residues, in orientations of lateral side chains were evidenced, and their importance to enzyme specificity is discussed.

Characterization and conformational analysis by Raman spectroscopy of human airway lysozyme.

Human airway lysozyme, purified from pathological bronchial secretions, is characterized by a specific activity 3-fold higher than that of hen egg-white lysozyme. The amino acid composition of human airway lysozyme is identical to that of other human lysozymes. The laser Raman spectra of human airway lysozyme and hen egg-white lysozyme in phosphate buffer solution (pH 7.2) are recorded in the range 300-1900 cm-1 at 488 nm. Drastic intensity differences are observed between the spectra analyzed in the ranges characteristic of the peptide backbone (e.g., beta-sheet; C alpha-C, C alpha-N), and of the aromatic side-chain vibrations (tyrosine, tryptophan). The deconvolution of the Raman amide I band gives secondary structures of 38% and 39% alpha-helix, 25% and 20% beta-sheet, and 37% and 41% undefined structure for the human and hen lysozymes, respectively.