Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination.

Human antibodies of the IgG2 subclass exhibit complex inter-chain disulfide bonding patterns that result in three structures, namely A, A/B, and B. In therapeutic applications, the distribution of disulfide isoforms is a critical product quality attribute because each configuration affects higher order structure, stability, isoelectric point, and antigen binding. The current standard for quantification of IgG2 disulfide isoform distribution is based on chromatographic or electrophoretic techniques that require additional characterization using mass spectrometry (MS)-based methods to confirm disulfide linkages.

Influence of disulfide bond isoforms on drug conjugation sites in cysteine-linked IgG2 antibody-drug conjugates.

Cysteine-linked antibody-drug conjugates (ADCs) produced from IgG2 monoclonal antibodies (mAbs) are more heterogeneous than ADCs generated from IgG1 mAbs, as IgG2 ADCs are composed of a wider distribution of molecules, typically containing 0 - 12 drug-linkers per antibody. The three disulfide isoforms (A, A/B, and B) of IgG2 antibodies confer differences in solvent accessibilities of the interchain disulfides and contribute to the structural heterogeneity of cysteine-linked ADCs. ADCs derived from either IgG2-A or IgG2-B mAbs were compared to better understand the role of disulfide isoforms on attachment sites and distribution of conjugated species.

Expanding the realm of ultrafast protein folding: gpW, a midsize natural single-domain with alpha+beta topology that folds downhill.

All ultrafast folding proteins known to date are either very small in size (less than 45 residues), have an alpha-helix bundle topology, or have been artificially engineered. In fact, many of them share two or even all three features. Here we show that gpW, a natural 62-residue alpha+beta protein expected to fold slowly in a two-state fashion, folds in microseconds (i.

Structure nor stability of the transmembrane spanning 6/7 domain of presenilin I correlates with pathogenicity.

Since its cloning in 1995, missense point mutations in presenilin I (PS-I) have been shown to be responsible for greater than 70% of the cases of early onset familial Alzheimer's disease (EOFAD), which can affect individuals as early as age 18. PS-I is known to be a component of gamma-secretase, the enzyme responsible for cleavage of the amyloid precursor protein (APP) into 42 amino acid peptides that aggregate to form the plaques surrounding neurons of Alzheimer's patients. It has recently been hypothesized that wild-type (wt) PS-I contains an autoinhibitory module that prevents gamma-secretase cleavage of the APP, while pathogenic PS-I point mutants lack a structure necessary for this inhibition.

Dynamics, energetics, and structure in protein folding.

For many decades, protein folding experimentalists have worked with no information about the time scales of relevant protein folding motions and without methods for estimating the height of folding barriers. Protein folding experiments have been interpreted using chemical models in which the folding process is characterized as a series of equilibria between two or more distinct states that interconvert with activated kinetics. Accordingly, the information to be extracted from experiments was circumscribed to apparent equilibrium constants and relative folding rates.