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.     

Zur Kenntnis der Halogensauerstoffverbindungm

Zusammenfassung Es wurde die Kinetik der Bromatbildungsreaktion nach 3 Br₃+6 OH=8 Br+BrO₃+3 H₂O, beziehungsweise 3 Br₂+6 OH=5 Br+BrO₃+3 H₂O untersucht und gefunden, daß die Geschwindigkeit der Bromatbildung mit den Konzentrationen von Brom und Hydroxylion steigt und mit zunehmender Bromionkonzentration abnimmt. Die Werte der Potenzexponenten der Konzentrationen variieren mit der Geschwindigkeit. Neutralsalze verzögern.Es wurde ferner für die rasche Reaktion das Zeitgesetz –d[Br₃]/d=k ₁[OH]/[Br]³[Br₃]², beziehungsweise –d[Br₂]/d=k₁[OH]/[Br][Br₂]² und für die langsame Reaktion das Zeitgesetz –d[Br₃]/d=k ₂[OH]⁴/[Br]⁷[Br₃]³, beziehungsweise –d[Br₂]/d=k₂[OH]⁴/[Br]⁴[Br₂]³ wahrscheinlich gemacht. Der Temperaturkoeffizient der in einer Monophosphat-Biphosphatlösung gemessenen langsamen Reaktion ist von der Größenordnung 17.Aus den Geschwindigkeitskoeffizienten der raschen Reaktion und dem der Reaktion HBrO+OH BrO₃ läßt sich das Brom-Hypobromitgleichgewicht und aus den Koeffizienten der langsamen Reaktion und dem der Reaktion BrO₃+Br+H^. Br₂ das Brom-Bromatgleichgewicht berechnen.Vgl. die vorhergehende Arbeit.     

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.     

Synthesis and biological activity of fluorinated 2-amino-4-aryl-3,4-dihydro[1,3,5]triazino[1,2-a]benzimidazoles

The heterocyclic nucleus s-triazino[1,2-a]benzimidazole has been reported to exhibit antibacterial activity. In this study, seven new 3,4-dihydro[1,3,5]triazino[1,2-a]benzimidazole derivatives were prepared via cyclocondensation between 2-guanidinobenzimidazole and fluorine substituted (including trifluoromethyl) benzaldehydes. The structures of all the compounds were confirmed by ¹H, ¹³C NMR and IR spectral data. Spectral data also suggested the existence of various tautomeric forms of the fluorine-containing s-triazino[1,2-a]benzimidazole compounds. The synthesized compounds were also screened for antibacterial and bovine dihydrofolate reductase (DHFR) inhibitory activities. The compound 3g substituted with a 3′,5′-bis(trifluoromethyl)phenyl moiety demonstrated the best antibacterial activity in the series. None of the tested compounds significantly inhibited bovine DHFR.     

Synthese neuer Aminofluorsilane,sowie Alkoxylierung von Bis (trimethylsilyl)-amino-fluorsilanen

The reaction of aminofluorsilanes of the type (R=H,F) (Me ₃Si)₂N?SiF₂R with two moles of ammonia, or of a mono- or dialkylamine, yields the corresponding amino-compounds, e.g. (Me ₃Si)₂N?Si(F)R?NH₂, (Me ₃Si)₂N?Si(F)R?NHR′ and (Me ₃Si)₂N?Si(F)R?NR₂′ (R′=Me, Et). Analogous products are obtained by reaction of the aminofluorosilanes with lithium salts of amines with bulky organic substituents in a 1 : 1 molar ratio. Alkoxy- and aryloxyaminofluorosilanes are prepared by the reaction of sodium alcoholates and sodium phenolate with (Me ₃Si)₂N?Si(F₂)R (R=H, C₂H₃, C₂H₅, C₆H₅). The i.r.-, mass-,¹H- and¹⁹F-NMR spectra of the above compounds are reported.     

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.