Structural studies of immunoglobulin molecules the Fv fragment of mouse myeloma protein M315

Thesis

The antibody is the main weapon in the body's defence against invading foreign materials. The different functions of the antibody molecule are performed by different parts of the structure. Some of these regions can be isolated in the form of enzymically-cleaved fragments. The Fv fragment, consisting of the variable regions of the light and heavy chains of the molecule, is the site of binding of the foreign antigen to which the antibody is specific.

Protein M315, a mouse myeloma protein, has an affinity for small molecules containing dinitrophenyl groups, comparable to that of a true antibody for its antigen. It is thus a particularly appropriate tool for examining antigen-antibody interactions.

The genetic mechanism of how the very large repertoire of antigenbinding specificities of antibodies arises in an organism has puzzled iiranunologists in the past. Recently, some progress on discovering this mechanism has been made, and some of the observations which have been reported and theories which have been proposed are reviewed.

The Fv fragment, which contains the antigen-binding site of the immunoglobulin, has been isolated from M315, and crystallized. The 6A* resolution data were measured on the four-circle diffractometer, and those to 2.7Å resolution by rotation photography. Phases were found by the method of isomorphous replacement with two heavy-atom compounds. The determination and refinement of the positions and extent of heavy-atom binding, are described. Electron density maps were calculated initially to 6Å and finally to 4.5Å resolution.

The 4.5Å resolution electron density map shows two complete Fv fragments in the asymmetric unit. The interactions between the V_H and V_L subunits are very close. However, the two molecules are oriented quite differently in the unit cell.

An initial examination of the binding of a small hapten, dinitroaniline, has been made. Crystals which were isomorphous with the native ones, 'were grown of Fv in the presence of the hapten. Preliminary indications are that the two crystallographically independent molecules may have different binding properties.

The directions in which the project may proceed, including the extension of the resolution and the assignment of atomic coordinates, are discussed.

Authors: Rose, D

• Publication date: 1981
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Subjects: Immunoglobulins; Myeloma proteins