Biomedical subjects

L O Essen

Publications and source records attributed to L O Essen.

23 records · Page 2Linked to original sources

PubMed1996

C2 domain conformational changes in phospholipase C-delta 1.

The structure of the PH-domain truncated core of rat phosphoinositide-specific phospholipase C-delta 1 has been determined at 2.4 A resolution and compared to the structure previously determined in a different crystal form. The stereochemical relationship between the EF, catalytic, and C2 domains is essentially identical. The Ca2+ analogue Sm3+ binds at two sites between the jaws of the C2 domain. Sm3+ binding ejects three lysine residues which bridge the gap between the jaws and occupy the Ca2+ site in the apoenzyme, triggering a conformational change in the jaws. The distal sections of the C2 jaws move apart, opening the mouth by 9 A and creating a gap large enough to bind a phospholipid headgroup.

Animals↗

PubMed1995

Crystals of an antibody Fv fragment against an integral membrane protein diffracting to 1.28 A resolution.

The Fv fragment of a monoclonal antibody, 7E2 (IgG1, kappa, murine), which is directed against the integral membrane protein cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans, was cloned and produced in Escherichia coli. Crystals suitable for high-resolution X-ray analysis were obtained by microdialysis under low salt conditions. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 51.51 A, b = 56.15 A, c = 99.86 A (1 A = 0.1 nm) and contain one Fv fragment per asymmetric unit. Using synchrotron radiation diffraction data were collected up to 1.28 A resolution. This high resolution is very unusual for a heterodimeric protein. The crystals should open the way for refining not only the atomic positions, but also for obtaining information about internal dynamics.

Amino Acid Sequence↗

PubMed1994

The de novo design of an antibody combining site. Crystallographic analysis of the VL domain confirms the structural model.

In a protein design approach the molecular model of an artificial antibody Fv fragment was generated with predicted complementarity to part of the known crystal structure of chicken egg-white cystatin. The model of the Fv fragment was based on the three-dimensional structure of the anti-lysozyme antibody HyHEL-10, which was modified by substituting amino acid side-chains in the complementarity-determining regions (CDRs) as well as the framework without altering the backbone. In the course of crystallization experiments with the bacterially produced Fv fragment crystals of the VL domain alone were obtained. These crystals diffracted X-rays to a resolution of 2.17 A and were shown to belong to the space group P2(1)2(1)2(1) with unit cell dimensions a = 46.89 A, b = 58.05 A, c = 83.22 A containing two VL monomers in the asymmetric unit. The crystal structure was solved by molecular replacement and refined to a crystallographic R-factor of 17.5%. The two VL monomers exhibit an asymmetric mode of association, which is different from other crystallized VL domains described before and shows the peculiar feature of an isopropanol precipitant molecule buried at the interface. Both VL structures reveal a high level of similarity to the predicted three-dimensional model. With the exception of two loop segments in the framework region that are involved in crystal packing contacts, the backbone structures of the two VL monomers in the crystal and the molecular model of the VL domain are practically identical. Although six amino acid residues had been replaced in the hypervariable regions, the CDR conformations remained conserved and only minor deviations in the orientation of some side-chains and peptide planes were detected. The crystallographic analysis of the VL domain modelled as part of a complex between an artificial Fv fragment and the small protein cystatin, deliberately chosen as antigen target, confirms the concept of distinct structural classes for CDR backbones and supports our strategy for the de novo design of an antibody combining site.

Amino Acid Sequence↗

PubMed1993

Single-step purification of a bacterially expressed antibody Fv fragment by immobilized metal affinity chromatography in the presence of betaine.

A procedure was developed for the rapid isolation of an antibody Fv fragment expressed in Escherichia coli via immobilized metal affinity chromatography. Metal affinity was mediated by fusing hexahistidine tails to both the VL and the VH domain and was thus independent of the antigen-binding specificity. Unexpectedly, it was not possible to isolate the Fv fragment with correct stoichiometric composition of the two variable domains under standard chromatographic conditions. Proper non-covalent association of VL and VH was, however, maintained when using glycine betaine as electrolyte, thus permitting purification of the intact Fv fragment to homogeneity in a single step.

Antibody Specificity↗

PubMed1992

Production and secretion in CHO cells of the extracellular domain of AMOG/beta 2, a type-II membrane protein.

A hybrid gene consisting of the sequences coding for the signal peptide and N terminus of a type-I membrane protein, the neural cell adhesion molecule (N-CAM), and the extracellular domain of the adhesion molecule on glia (AMOG/beta 2), a type-II membrane protein, was constructed. The sequence was inserted into a eukaryotic expression vector containing the human cytomegalovirus promoter and the glutamine synthetase selection marker, and used to transfect Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of soluble recombinant AMOG/beta 2 (reAMOG/beta 2), which was secreted into the culture medium as a heavily glycosylated 40-55-kDa protein. N-terminal sequence analysis revealed that the protein is not cleaved at the natural signal peptide cleavage site of N-CAM, but two amino acids (aa) further downstream. Treatment of reAMOG/beta 2 with N-glycosidase F (GlycoF) reduced the molecular mass to 27 kDa, corresponding to the calculated mass of the unglycosylated form. In contrast to AMOG/beta 2 isolated from mouse brain, which is sensitive to endoglycosidase H, the immunoaffinity-purified re-protein is more resistant to this treatment, indicating that the sugars attached to reAMOG/beta 2 are mainly of the complex type. Our results demonstrate the feasibility of secreting the extracellular domain of a type-II membrane protein, which is usually inserted into the membrane with the C terminus facing the extracellular side.

Adenosine Triphosphatases↗