No description available.

No description available.

The importance of tryptophan in the combining sites of anti-DNP antibodies is evaluated from a series of model studies. The thermodynamic parameters characterizing the formation of a DNP/tryptophan complex are determined. The contribution of this interaction to the affinity and specificity of anti-DNP antibodies is discussed. The accuracy of antibody combining site structures generated by model-building is examined, using available crystallographic data. The average error of alpha-carbon atom positions is estimated to be 1-2 Å. The binding of nitrophenyl compounds to the V_L dimer of the DNP-binding mouse myeloma protein 315 is investigated by ¹H n.m.r. It is concluded that any corformational changes are small, and that the physical basis for the DNP-binding specificity of the V_L dimer is the conservation of structural features which are important in determining the specificity of the intact protein 315. A model of the combining site of the V_L dimer is described. The proposed site is a large cavity bounded by the aromatic side-chains of the Trp-93_L and Tyr-34_L residues. The structure is able to explain the large upfield chemical shift changes of the ligand resonances observed on binding. The kinetic parameters and structural extent of the pH-dependent conformational change of the V_L dimer are investigated by fluorescence and ¹H n.m.r. The transition does not obey a reversible one-step mechanism, and is limited in extent. The involvement of two tyrosine residues in the hypervariable regions of protein 315 is investigated by specific nitration. Nitration of Tyr-34_L has no effect on the affinity of protein 315 or of the V_L dimer for several ligands. It is concluded that no hydrogen bond is formed between the phenolic group of Tyr-34_L and the 2-nitro group of the ligand. From measurements of the perturbation of the visible absorption spectrum of protein 315 nitrated at Tyr-33_H, it is concluded that this residue is in proximity to the side-chains, but not the nitrophenyl rings, of bound ligands. Several aspects of the interaction of a homogeneous mouse anti-DNP antibody, protein A3, with nitrophenyl and similar ligands are described. The findings are discussed in relation to the heterogeneity and cross-reactivity of antisera.

No description available.

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1968. / Bibliography: leaves [197]-208. / xiii, 208 l illus., tables

Polyreactive immunoglobulins (Ig) exhibit a capacity to recognise multiple, structurally dissimilar antigens through a single combining site. This characteristic differentiates these Igs from monoreactive Igs which bind to a single antigen, usually with high specificity and affinity. Chronic B lymphocytic leukaemia (B CLL) is a malignancy identified by the incessant accumulation, in the peripheral circulation, of B lymphocytes of a mature and resting morphology. B CLL malignant cells generally express both surface IgM and the pan T cell antigen CD5. Moreover, the IgM on the surface of these CD5 positive B CLL cells is frequently polyreactive. This thesis examines the structural diversity found in the combining sites of B CLL derived Igs in an attempt to elucidate the structural basis of polyreactive antigen binding displayed by a significant proportion of human Igs. The genes encoding the variable (V) domains of five B CLL derived IgM antibodies (Bel, Tre, Yar, Hod and Jak) were cloned and sequenced (Chapter Two). When the light chain V domain genes were aligned with the closest germline VL and JL coding DNA sequences it was determined that there was either a complete absence of somatic mutation (Tre, Yar and Jak) or a minimal number of mutations (Bel and Hod) present in the rearranged VL domain genes. A remarkable fidelity in the splicing of VL to JL genes was noted suggesting that the diversity, normally introduced through variability of splicing VL to JL, is reduced in Igs expressed by B CLL cells. Furthermore, the markedly reduced primary structural diversity was highlighted when two of the VL domain genes (Yar and Hod) were found to be different in sequence by only four nucleotides and two amino acids. The heavy chain V domain genes of the same five Igs were sequenced in another study (Brock, 1995), however, it was interesting to analyse the sequences of the VH domain genes and compare them with the VL domain genes. The naive or gerrnline nature of the B CLL antibodies was reflected in the VH genes by either an absence or a low frequency of mutations within these sequences compared with germline immunoglobulin gene sequences. No obvious conserved motif, which could be related to polyreactivity, was observed when the primary protein sequence was analysed for distribution of identical or similar amino acids. Thus, homology modelling was used to construct three-dimensional models of the Fv (VL-VH) portions of the five B CLL IgM molecules to examine the structures of the combining sites of these Igs (Chapter Three). Framework regions were constructed using X-ray coordinates taken from highly hon~ologous human variable domain structures. Complementarity determining regions (CDR) were predicted by grafting loops, taken from known Ig structures, onto the Fv framework models. The CDR templates were selected, where possible, to be of the same length and of high residue identity or similarity. If a single template CDR was not appropriate to model a particular CDR the loop was built from loop sterns of known conformation, followed by chain closure with a p-turn. Template models were refined using standard molecular mechanics simulations. The binding sites were either relatively flat or contained a deep cavity at the VL-VH domain interface. Further differences in topology were the result of some CDR loops protruding into the solvent. Examination of the electrostatic molecular surface did not reveal a common structural feature within the binding sites of the five polyreactive Fv. While two of the binding cavities were positively charged the other three structures displayed either negatively charged or predominantly hydrophobic combining sites. These findings suggested that a diversity of structural mechanisms are involved in polyreactive antigen binding. Rcsidues within CDRs which have aromatic side-chains and are partially exposed to solvent were distributed across large regions of the combining sites. It is possible that these aromatic residues are responsible for the conserved binding to mouse Igs observed (Chapter Two) for the B CLL derived polyreactive IgM molecules. Two Fv molecules (Be1 and Tre) were cloned as dicistronic constructs, into the bacterial expression vector pFLAG. The expression of the Fvs was fully characterised and unfortunately the VL and VH of Be1 and Tre Igs did not associate in an appropriate manner to yield large quantities of purified Fv (Chapter Four). Expression of correctly folded and stabilised fragments of human polyreactive immunoglobulins would enable the structural basis for the polyreactive binding phenomenon to be fully explored using protein crystallography.

ix, 179 leaves xxiv : ill. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1970

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Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

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