Hydrogen exchange-mass spectrometry (HX-MS) is a technique that allows for a sensitive characterization of a protein through exchanging an amide hydrogen on the protein’s backbone with a deuterium. Sections of the protein that have slower rates of exchange indicate strong hydrogen bonding or burial within the core of the protein, while faster rates could imply no hydrogen bonding or more solvent exposure. By determining the rate of exchange through increases in mass in different sections of the protein, conclusions about higher-order structure can be established. However, there is yet to be a solid understanding on the interrelationship between protein structure, protein function, and HX-MS. Hence, the long-term goal is to establish this important interrelationship to aid in product quality evaluations for protein therapies. In this present work we investigated the relationship between structure and function to provide data that can determine how these attributes relate to HX-MS data. The antigen binding domain of the single-chain F5 camelid (VHH) antibody has previously been determined to have strong binding to the ricin toxin A subunit (RTA). Using the F5 VHH and RTA as a model system, single amino acid changes were introduced into the VHH antibody outside of the paratope, since binding occurs in this region. Through E. coli protein expression and purification, the wild-type protein and proteins that contained the V75A, V75I, and C50AC97A mutations were expressed. The mutations were compared to the wild-type using an antigen binding assay to determine if there was a decrease in binding affinity when introduced to RTA. Along with using ELISA, this project incorporated the use of SDS-PAGE and mass spectrometry for confirmation of the desired proteins through protein expression and purification. Data from the ELISAs showed that there was a significant decrease in binding affinity of the VHH with the C50AC97A and V75I mutations when compared to the wild-type. The V75A mutation did not show significant changes in binding affinity when compared to the wild-type.

Determination of the binding affinity of point-mutated antibody fragments against ricin toxin subunit A

ACS Spring 2022

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