Protein engineering and characterisation of a single Ig-binding domain of Protein L from Peptostreptococcus magnus

Beckingham, Jennifer Ann (1997) Protein engineering and characterisation of a single Ig-binding domain of Protein L from Peptostreptococcus magnus. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Protein L is displayed in the surface of approximately 10% Peptostreptococcus isolates, and has between 3 and 5 Ig-binding domain. The DNA of a single Ig-binding domain of Protein L from strain 3316 has been cloned into a pKK223-3 based vector and the protein product (Ppl) expressed and purified from E. coli. Ppl interacts with κ-chains with a high affinity in a non-antigenic manner. The interaction between Ppl and κ-chain was studied using the fluorescence properties of inserted tryptophan residues. The interaction was found to occur in two phases, an initial fast phase which formed initial complex with a Kd between 2-12μM followed by a second slow phase which led to an increase in affinity to give a final Kd of about 150nM. The second phase was rate limiting and is thought to involve a structural rearrangement of the proteins which allows further interactions to occur. The complex formed between Ppl and κ-chain is very stable and harsh conditions are required to promote the dissociation of the complex.

Chemical modification experiments were carried out to identify residues in Ppl that are involved in the interaction. The most notable effect was observed when tetranitromethane was used to modify tyrosine residues, the effect of which abolished the binding capacity of Ppl. Mutagenesis experiments were carried out to establish the role of tyr53 in the interaction by substituting it was a phenylalanine residue. The removal of the hydroxyl group reduced the affinity of the complex by 28 fold, with a difference in free energy of binding of 8.3 kJ mol^-1. The stopped-flow experiments showed that only the second phase of the interaction was affected by this mutation, which suggests it is involved in the formation of the high affinity complex measured at equilibrium. Conformational stability studies were carried out on the various mutant Ppl proteins, and it was observed that substitutions by amino acids with side chains that altered in size or charge caused a significant decrease in the stability of the domain, while more conservative alterations had a reduced effect on the stability of the domain.

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