Design of a calcium-binding protein with desired structure in a cell adhesion molecule

Ca2+, "a signal of life and death", controls numerous cellular processes through interactions with proteins. An effective approach to understanding the role of Ca2+ is the design of a Ca2+-binding protein with predicted structural and functional properties. To design de novo Ca2+-binding sites in proteins is challenging due to the high coordination numbers and the incorporation of charged ligand residues, in addition to Ca2+-induced conformational change. Here, we demonstrate the successful design of a Ca2+-binding site in the non-Ca2+-binding cell adhesion protein CD2. This designed protein, Ca.CD2, exhibits selectivity for Ca2+ versus other di- and monovalent cations. In addition, La3+ (Kd 5.0 microM) and Tb3+ (Kd 6.6 microM) bind to the designed protein somewhat more tightly than does Ca2+ (Kd 1.4 mM). More interestingly, Ca.CD2 retains the native ability to associate with the natural target molecule. The solution structure reveals that Ca.CD2 binds Ca2+ at the intended site with the designed arrangement, which validates our general strategy for designing de novo Ca2+-binding proteins. The structural information also provides a close view of structural determinants that are necessary for a functional protein to accommodate the metal-binding site. This first success in designing Ca2+-binding proteins with desired structural and functional properties opens a new avenue in unveiling key determinants to Ca2+ binding, the mechanism of Ca2+ signaling, and Ca2+-dependent cell adhesion, while avoiding the complexities of the global conformational changes and cooperativity in natural Ca2+-binding proteins. It also represents a major achievement toward designing functional proteins controlled by Ca2+ binding.     

Synthesis of Tetrafluoromethane by Graphite Fluorination with Elemental Fluorine

Formation of high-temperature inverse wave of the filtration combustion of graphite fixed bed in fluorine was studied. Scientific principles of the industrial process of the tetrafluoromethane synthesis from graphite and fluorine were developed.     

Al(III) complexes with the ampicillin,amoxicillin, and cephalexin anions

The interaction of Al(III) with ampicillin, amoxicillin, and cephalexin anions (L) was studied by the pH-metric titration in aqueous solutions at 20°C and ionic strength 0.1 (KNO₃). In weakly acidic medium, complexes with the composition Al(OH)L and Al(OH)₂L were formed. The diagrams of the distribution of the complex forms of Al(III) depending on the pH were plotted. The constants of formation of the complexes were determined.     

Stereoselective hydroxylation of an achiral cyclopentanecarboxylic acid derivative using engineered P450s BM-3

Substrate engineered, achiral carboxylic acid derivative was biohydroxylated with various mutants of cytochrome P450 BM-3 to give two out of the four possible diastereoisomers in high de and ee. The BM-3 mutants exhibit up to 9200 total turnovers for hydroxylation of the engineered substrate, which without the protecting group is not transformed by this enzyme.     

Structural Investigation of Guest-to-Host and Ligand-Ligand Interactions in Werner Clathrates of [Ni(4-Etpy)4(NCS)2]⋅ nG Type (G = Naphthalene Derivatives). The Crystal Structure of [Ni(4-Etpy)4(NCS)2]⋅1-Methylnaphthalene

The stoichiometry and spectral properties of [Ni(4-Etpy)4(NCS)2]nG clathrates have been studied where n = 2 for G = 1-BrN (N = naphthalene), n = 1 or 2 for G = 1-MeN, and n = 0.5 for 2-MeN and 2-BrN. The complexes under study show electronic absorption spectra typical of an octahedral environment of the Ni(II) central atom. The differences found in IR spectra for the (CN) and (Ni–-NNCS) vibrations are discussed. The crystal structure of [Ni(4-Etpy)4(NCS)2]1-MeN was determined by X-ray diffraction and refined to R = 0.0586. Discrete non-centrosymmetric [Ni(4-Etpy)4(NCS)2] molecules form layers of a host structure and the space between the layers is occupied by 1-MeN. The relationship between interatomic distances in the host complex of similar clathrates are discussed.     

Crystal structure of KAsUO<Subscript>6</Subscript>·<Subscript>3</Subscript>H<Subscript>2</Subscript>O at 100 and 293 K

The crystal structure of KAsUO₆·3H₂O was solved at 100 K and 293 K. KAsUO₆·3H₂O at T = 100 K: tetragonal, P4/ncc, a = 7.2037(6) Å, c = 17.811(2) Å; Z = 4, R1 = 0.0263, wR2 = 0.0546, for 618 independent reflections; at T = 293 K: tetragonal, P4/ncc, a = 7.1600(4) Å, c = 17.746(1) Å; Z = 4, R1 = 0.0263, wR2 = 0.0433 for 427 independent reflections. The results of X-ray analysis are compared with our previous data on heat capacity of this compound, and changes that take place in the structure at elevated temperature are considered.     

Biocompatible hydrogels based on chitosan,cellulose/starch,PVA and PEDOT:PSS with high flexibility and high mechanical strength

Fabricating mechanically strong hydrogels that can withstand the conditions in internal tissues is a challenging task. We have designed hydrogels based on multicomponent systems by combining chitosan, starch/cellulose, PVA, and PEDOT:PSS via one-pot synthesis. The starch-based hydrogels were homogeneous, while the cellulose-based hydrogels showed the presence of cellulose micro- and nanofibers. The cellulose-based hydrogels demonstrated a swelling ratio between 121 and 156%, while the starch-based hydrogels showed higher values, from 234 to 280%. Tensile tests indicated that the presence of starch in the hydrogels provided high flexibility (strain at break?>?300%), while combination with cellulose led to the formation of stiffer hydrogels (elastic moduli 3.9–6.6 MPa). The ultimate tensile strength for both types of hydrogels was similar (2.8–3.9 MPa). The adhesion and growth of human osteoblast-like SAOS-2 cells was higher on hydrogels with cellulose than on hydrogels with starch, and was higher on hydrogels with PEDOT:PSS than on hydrogels without this polymer. The metabolic activity of cells cultivated for 3 days in the hydrogel infusions indicated that no acutely toxic compounds were released. This is promising for further possible applications of these hydrogels in tissue engineering or in wound dressings.

Graphical abstract

     

Immunological detection of glycoproteins on blots based on labeling with digoxigenin

In the following, we describe the application of the DIG/antiDIG system for the (structural) analysis of glycoproteins on blots. Special emphasis is being placed on the variety of the different DIG applications in order to obtain structural information concerning the glycoprotein carbohydrate chains. Prominent among them is the use of lectins with well-known specificities for carbohydrate structures. This article first appeared in: Hounsell, E. F., ed. (1993),Methods in Molecular Biollogy, vol. 14: Glycoprotein Analysis in Biomedicine, Humana Press Inc., Totowa, NJ.     

Construction of 4D-QSAR Models for Use in the Design of Novel p38-MAPK Inhibitors

The p38-mitogen-activated protein kinase (p38-MAPK) plays a key role in lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) release during the inflammatory process, emerging as an attractive target for new anti-inflammatory agents. Four-dimensional quantitative structure-activity relationship (4D-QSAR) analysis [Hopfinger et al., J. Am. Chem. Soc., 119 (1997) 10509] was applied to a series of 33 (a training set of 28 and a test set of 5) pyridinyl-imidazole and pyrimidinyl-imidazole inhibitors of p38-MAPK, with IC50 ranging from 0.11 to 2100 nM [Liverton et al., J. Med. Chem., 42 (1999) 2180]. Five thousand conformations of each analogue were sampled from a molecular dynamics simulation (MDS) during 50 ps at a constant temperature of 303 K. Each conformation was placed in a 2 angstroms grid cell lattice for each of three trial alignments. 4D-QSAR models were constructed by genetic algorithm (GA) optimization and partial least squares (PLS) fitting, and evaluated by leave-one-out cross-validation technique. In the best models, with three to six terms, the adjusted cross-validated squared correlation coefficients, Q2adj, ranged from 0.67 to 0.85. Model D (Q2adj = 0.84) was identified as the most robust model from alignment 1, and it is representative of the other best models. This model encompasses new molecular regions as containing pharmacophore sites, such as the amino-benzyl moiety of pyrimidine analogs and the N1-substituent in the imidazole ring. These regions of the ligands should be further explored to identify better anti-inflammatory inhibitors of p38-MAPK.