Phillip A. Patten

The immunological evolution of catalysis.

The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 104, primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, kcat/Km, from 1.7 × 102 M−1 min−1 to 1.4 × 104 M−1 min−1. The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that none of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry or limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

An Analysis of T Cell Receptor–Ligand Interaction Using a Transgenic Antigen Model for T Cell Tolerance and T Cell Receptor Mutagenesis

Publisher Summary This chapter presents an analysis of T cell receptor-ligand interaction using a transgenic antigen model for T-cell tolerance and T-cell receptor mutagenesis. It presents an analysis of the molecular basis of T-cell receptor (TCR) recognition of foreign peptide-major histocompatibility complexes (MHC). The chapter presents a few experiments that succeeded in transferring Staphylococcus aureus enterotoxin B (SEB) reactivity. T-cell receptor sequences, particularly V regions, are very similar to immunoglobulins, and the consensus of a number of experts is that they probably fold and pair in much the same way. However, the preponderance of their sequence diversity lies in the V(D)J junctional or CDR3-equivalent region, exceeding the diversity of immunoglobulin CDR3s by many orders of magnitude in the absence of somatic mutation.

Directed evolution studies with combinatorial libraries of T4 lysozyme mutants

SummaryGene duplication with divergence to new functions has been an important mechanism in protein evolution. However, the questions of how many new functions can arise from a particular ancestral gene and how many mutational steps are typically required to generate new functions have been difficult to approach experimentally. We have addressed these questions using T4 lysozyme as a model system by synthesizing two combinatorial libraries of >107 mutant T4 lysozyme genes: one library with an average of 14 missense mutations spread throughout the gene and one library in which 13 active site residues have been simultaneously randomized. These libraries were placed under selection inlacZ orpheA deficient strains ofE. coli to investigate whether they sample sufficient diversity to contain mutants with acquired β-galactosidase or prephenate dehydratase activities. Although neither selection yielded T4 lysozyme mutants with these new activities, a novelE. coli locus was cloned that weakly complements these mutants, allowing them to form 1 mm colonies in 4–6 weeks. This growth rate corresponds to a turnover number of approximately 1000 or 25 min− for thelacZ orpheA complementation systems, respectively, thus defining the limits of evolved enzymatic activity detectable in these selections. Thus, the strong selective pressure uncovered an unexpected solution to the biochemical blocks, a frequently observed phenomenon in selection experiments. The characterization of this locus will allow its elimination from futureE. coli complementation schemes.

The Genes of the Murine T Cell Receptor

T cells recognize antigen in a way fundamentally different from B cells. It has been known now for a decade (1–3) that the T cell receptor sees the antigen, not alone, but in association the major histocompatibility complex (MHC, H-2 in the mouse). As a general rule cytotoxic T (Tc) cells recognize antigen in association with Class I MHC molecules and helper T (Th) cells do so with Class II structures (4,5).