Vladimír Kopecký

Anharmonic effects in IR, Raman, and Raman optical activity spectra of alanine and proline zwitterions

The difference spectroscopy of the Raman optical activity (ROA) provides extended information about molecular structure. However, interpretation of the spectra is based on complex and often inaccurate simulations. Previously, the authors attempted to make the calculations more robust by including the solvent and exploring the role of molecular flexibility for alanine and proline zwitterions. In the current study, they analyze the IR, Raman, and ROA spectra of these molecules with the emphasis on the force field modeling. Vibrational harmonic frequencies obtained with 25 ab initio methods are compared to experimental band positions. The role of anharmonic terms in the potential and intensity tensors is also systematically explored using the vibrational self-consistent field, vibrational configuration interaction (VCI), and degeneracy-corrected perturbation calculations. The harmonic approach appeared satisfactory for most of the lower-wavelength (200-1800 cm(-1)) vibrations. Modern generalized gradient approximation and hybrid density functionals, such as the common B3LYP method, provided a very good statistical agreement with the experiment. Although the inclusion of the anharmonic corrections still did not lead to complete agreement between the simulations and the experiment, occasional enhancements were achieved across the entire region of wave numbers. Not only the transitional frequencies of the C-H stretching modes were significantly improved but also Raman and ROA spectral profiles including N-H and C-H lower-frequency bending modes were more realistic after application of the VCI correction. A limited Boltzmann averaging for the lowest-frequency modes that could not be included directly in the anharmonic calculus provided a realistic inhomogeneous band broadening. The anharmonic parts of the intensity tensors (second dipole and polarizability derivatives) were found less important for the entire spectral profiles than the force field anharmonicities (third and fourth energy derivatives), except for a few weak combination bands which were dominated by the anharmonic tensor contributions.

Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies

BackgroundFungal β-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-N-acetylhexosaminidase. The fungal β-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined.ResultsThe complete primary structure of the fungal β-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.ConclusionWhereas the intracellular bacterial β-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzymes strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys448 with Cys483 stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.

Soluble recombinant CD69 receptors optimized to have an exceptional physical and chemical stability display prolonged circulation and remain intact in the blood of mice

We investigated the soluble forms of the earliest activation antigen of human leukocyte CD69. This receptor is expressed at the cell surface as a type II homodimeric membrane protein. However, the elements necessary to prepare the soluble recombinant CD69 suitable for structural studies are a matter of controversy. We describe the physical, biochemical and in vivo characteristics of a highly stable soluble form of CD69 obtained by bacterial expression of an appropriate extracellular segment of this protein. Our construct has been derived from one used for CD69 crystallization by further optimization with regard to protein stability, solubility and easy crystallization under conditions promoting ligand binding. The resulting protein is stable at acidic pH and at temperatures of up to 65 °C, as revealed by long‐term stability tests and thermal denaturation experiments. Protein NMR and crystallography confirmed the expected protein fold, and revealed additional details of the protein characteristics in solution. The soluble CD69 refolded in a form of noncovalent dimers, as revealed by gel filtration, sedimentation velocity measurements, NMR and dynamic light scattering. The soluble CD69 proved to be remarkably stable in vivo when injected into the bloodstream of experimental mice. More than 70% of the most stable CD69 proteins is preserved intact in the blood 24 h after injection, whereas the less stable CD69 variants are rapidly taken up by the liver.

Structural analysis of natural killer cell receptor protein 1 (NKR- P1) extracellular domains suggests a conserved long loop region involved in ligand specificity

Receptor proteins at the cell surface regulate the ability of natural killer cells to recognize and kill a variety of aberrant target cells. The structural features determining the function of natural killer receptor proteins 1 (NKR-P1s) are largely unknown. In the present work, refined homology models are generated for the C-type lectin-like extracellular domains of rat NKR-P1A and NKR-P1B, mouse NKR-P1A, NKR-P1C, NKR-P1F, and NKR-P1G, and human NKR-P1 receptors. Experimental data on secondary structure, tertiary interactions, and thermal transitions are acquired for four of the proteins using Raman and infrared spectroscopy. The experimental and modeling results are in agreement with respect to the overall structures of the NKR-P1 receptor domains, while suggesting functionally significant local differences among species and isoforms. Two sequence regions that are conserved in all analyzed NKR-P1 receptors do not correspond to conserved structural elements as might be expected, but are represented by loop regions, one of which is arranged differently in the constructed models. This region displays high flexibility but is anchored by conserved sequences, suggesting that its position relative to the rest of the domain might be variable. This loop may contribute to ligand-binding specificity via a coupled conformational transition.

Phe475 and Glu446 but not Ser445 participate in ATP-binding to the α-subunit of Na+/K+-ATPase

Abstract The ATP-binding site of Na + /K + -ATPase is localized on the large cytoplasmic loop of the α-subunit between transmembrane helices H 4 and H 5 . Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser 445 , Glu 446 , and Phe 475 were proposed to be close to the binding pocket. Replacement of Phe 475 with Trp and Glu 446 with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser 445 with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser 445 is close to the binding site, although it does not participate in binding.

Molecular architecture of mouse activating NKR-P1 receptors

Receptors belonging to NKR-P1 family and their specific Clr ligands form an alternative missing self recognition system critical in immunity against tumors and viruses, elimination of tumor cells subjected to genotoxic stress, activation of T cell dependent immune response, and hypertension. The three-dimensional structure of the extracellular domain of the mouse natural killer (NK) cell receptor mNKR-P1Aex has been determined by X-ray diffraction. The core of the C-type lectin domain (CTLD) is homologous to the other CTLD receptors whereas one quarter of the domain forms an extended loop interacting tightly with a neighboring loop in the crystal. This domain swapping mechanism results in a compact interaction interface. A second dimerization interface resembles the known arrangement of other CTLD NK receptors. A functional dimeric form of the receptor is suggested, with the loop, evolutionarily conserved within this family, proposed to participate in interactions with ligands.

Vibrational spectroscopy and computer modeling of proteins: solving structure of α 1 -acid glycoprotein

This work introduces a new approach connecting vibrational spectroscopy with homology and energetic molecular modeling of proteins. Combination of both methods can compensate their disadvantages and result in realistic three-dimensional protein models. The approach is most powerful for membrane proteins or glycoproteins with high carbohydrate content where X-ray or NMR analysis is not always successful. Nevertheless, it can also serve as a tool of preliminary analysis of any protein with unknown structure. Power of the approach is demonstrated on human α1-acid glycoprotein. Its predicted structure pub- lished in (V. Kopecký Jr. et al., Biochem. Biophys. Res. Commun. 300 (2003), 41-46) is discussed in detail with respect to the approach and its general employment.

Membrane activity of the pentaene macrolide didehydroroflamycoin in model lipid bilayers

Didehydroroflamycoin (DDHR), a recently isolated member of the polyene macrolide family, was shown to have antibacterial and antifungal activity. However, its mechanism of action has not been investigated. Antibiotics from this family are amphiphilic; thus, they have membrane activity, their biological action is localized in the membrane, and the membrane composition and physical properties facilitate the recognition of a particular compound by the target organism. In this work, we use model lipid membranes comprised of giant unilamellar vesicles (GUVs) for a systematic study of the action of DDHR. In parallel, experiments are conducted using filipin III and amphotericin B, other members of the family, and the behavior observed for DDHR is described in the context of that of these two heavily studied compounds. The study shows that DDHR disrupts membranes via two different mechanisms and that the involvement of these mechanisms depends on the presence of cholesterol. The leakage assays performed in GUVs and the conductance measurements using black lipid membranes (BLM) reveal that the pores that develop in the absence of cholesterol are transient and their size is dependent on the DDHR concentration. In contrast, cholesterol promotes the formation of more defined structures that are temporally stable.

Structural and dynamic changes of the serum response element and the core domain of serum response factor induced by their association

Transcriptional activity of serum response factor (SRF) is dependent on its binding to the CC(A/T)(6)GG box (CArG box) of serum response element (SRE). By Raman spectroscopy, we carried out a comparative analysis, in solution, of the complexes obtained from the association of core-SRF with 20-mer SREs bearing wild-type and mutated c-fos CArG boxes. In case of association with the wild type c-fos CArG box, the complex does not bring out the expected Raman signature of a stable bending of the targeted SRE but keeps a bend-linear conformer oligonucleotide interconversion. The linear conformer population is larger than that of free oligonucleotide. In the core-SRF moiety of the wild-type complex a large spectral change associated with the CO-groups from Asp and/or Glu residues shows that their ionization states and the strength of their interactions decrease as compared to those of mutated non-specific complexes. Structural constraints evidenced on the free core-SRF are released in the wild-type complex and environmental heterogeneities appear in the vicinity of Tyr residues, due to higher water molecule access. The H-bonding configuration of one Tyr OH-group, in average, changes with a net transfer from H-bond acceptor character to a combined donor and acceptor character. A charge repartition distributed on both core-SRF and targeted SRE stabilizes the specific complex, allowing the two partners to experience a variety of conformations.

Antimicrobial Peptide from the Eusocial Bee Halictus sexcinctus Interacting with Model Membranes

Halictine-1 (Hal-1)—a linear antibacterial dodecapeptide isolated from the venom of the eusocial bee Halictus sexcinctus—has been subjected to a detailed spectroscopic study including circular dichroism, fluorescence, and vibrational spectroscopy. We investigated Hal-1 ability to adopt an amphipathic α-helical structure upon interaction with model lipid-based bacterial membranes (phosphatidylcholine/phosphatidylglycerol-based large unilamellar vesicles and sodium dodecylsulfate micelles) and helix inducing components (trifluoroethanol). It was found that Hal-1 responds sensitively to the composition of the membrane model and to the peptide/lipid ratio. The amphipathic nature of the helical Hal-1 seems to favour flat charged surfaces of the model lipid particles over the nondirectional interaction with trifluoroethanol. Increasing fraction of polyproline II type conformation was detected at low peptide/lipid ratios.