Genetic and fluorescence studies of affinity maturation in related antibodies.

Affinity maturation, the process by which an organism's response to infection becomes more specific and more effective over time, occurs after somatic hypermutation of antibody genes in B-cells. This increase in affinity might be a result of the evolution of either specific interactions between antigen and antibody over time (enthalpic factors) or antibody binding site rigidification (entropic factors) or both. Here, monoclonal antibodies, derived from antibodies elicited at different points in the murine immune response after inoculation with the same diketone hapten, have been characterized both genetically and functionally. Though this hapten has previously been shown to produce the catalytic aldolase antibody 38C2, antibodies described here are not catalytic and unlike 38C2, form no covalent enzyme-substrate complex. Thus, they provide a system in which to assess contributions to the evolution of binding affinity. The genes for these non-catalytic antibodies have been sequenced and analyzed both with regard to their relationships to germ line genes, to each other, and to two commercially available catalytic aldolase antibodies. Consequences of particular mutations for antigen binding behavior are discussed. The protein products of these genes have been expressed, purified, and binding properties measured by two complementary techniques: the hapten-induced quenching of the native antibody fluorescence and the changes in the anisotropy of Prodan (6-propionyl-2-(dimethylamino)naphthalene), a fluorescent hapten analogue. Differences in binding affinity are related back to differences in the lengths and amino acid sequences of the complementary determining region 3 (CDR3) binding loop. Taken together with our earlier results on binding site heterogeneity from tryptophan lifetime analysis [Mohan, G.S., Chiu, P.T., Southern, C.A., O'Hara, P.B., 2004. Steady-state and multifrequency phase fluorometry studies of binding site flexibility in related antibodies. J. Phys. Chem. A 108, 7871-7877], affinity appears to be modulated by a combination of entropic and enthalpic factors, and not dominated by one or the other. Because these antibodies are not related to the same germ line gene, however, these results do not provide evidence for the dominance of enthalpy or entropy in evolving binding affinity in this system.

Teaching structure: student use of software tools for understanding macromolecular structure in an undergraduate biochemistry course.

Because understanding the structure of biological macromolecules is critical to understanding their function, students of biochemistry should become familiar not only with viewing, but also with generating and manipulating structural representations. We report a strategy from a one-semester undergraduate biochemistry course to integrate use of structural representation tools into both laboratory and homework activities. First, early in the course we introduce the use of readily available open-source software for visualizing protein structure, coincident with modules on amino acid and peptide bond properties. Second, we use these same software tools in lectures and incorporate images and other structure representations in homework tasks. Third, we require a capstone project in which teams of students examine a protein-nucleic acid complex and then use the software tools to illustrate for their classmates the salient features of the structure, relating how the structure helps explain biological function. To ensure engagement with a range of software and database features, we generated a detailed template file that can be used to explore any structure, and that guides students through specific applications of many of the software tools. In presentations, students demonstrate that they are successfully interpreting structural information, and using representations to illustrate particular points relevant to function. Thus, over the semester students integrate information about structural features of biological macromolecules into the larger discussion of the chemical basis of function. Together these assignments provide an accessible introduction to structural representation tools, allowing students to add these methods to their biochemical toolboxes early in their scientific development.