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.e., from tau = 33 micros at 310 K to tau = 1.7 micros at 355 K). Thermodynamic analyses of gpW reveal probe dependent thermal denaturation, complex coupling between two denaturing agents, and differential scanning calorimetry (DSC) thermogram characteristic of folding over a negligible thermodynamic folding barrier. The free energy surface analysis of gpW folding kinetics also produces a marginal folding barrier of about thermal energy ( RT) at the denaturation midpoint. From these results we conclude that gpW folds in the downhill regime and is close to the global downhill limit. This protein seems to be poised toward downhill folding by a loosely packed hydrophobic core with low aromatic content, large stabilizing contributions from local interactions, and abundance of positive charges on the native surface. These special features, together with a complex functional role in bacteriophage lambda assembly, suggest that gpW has been engineered to fold downhill by natural selection.

For many decades, protein folding experimentalists have worked with no information about the timescales of relevant protein folding motions and without methods for estimating the height of folding barriers. Experiments in protein folding 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 experiment was circumscribed to apparent equilibrium constants and relative folding rates. Recent developments are changing this situation dramatically. The combination of fast-folding experiments with the development of analytical methods more closely connected to physical theory reveals that folding barriers in native conditions range from minimally high (~14 RT for the very slow folder AcP) to nonexisting. While slow-folding (i.e. 1 millisecond or longer) single domain proteins are expected to fold in a two-state fashion, microsecond-folding proteins should exhibit complex behavior arising from crossing marginal or negligible folding barriers. This realization opens a realm of exciting opportunities for experimentalists. The free energy surface of a protein with marginal (or no) barrier can be mapped using equilibrium experiments, which could resolve energetic from structural factors in folding. Kinetic experiments on these proteins provide the unique opportunity to measure folding dynamics directly. Furthermore, the complex distributions of time-dependent folding behaviors expected for these proteins might be accessible to single molecule measurements. Here, we discuss some of these recent developments in protein folding, emphasizing aspects that can serve as a guide for experimentalists interested in exploiting this new avenue of research.In folding to their biologically active 3D structures, proteins must coordinate the vast number of degrees of freedom of their polypeptide chains by forming complex networks of noncovalent interactions. Therefore, understanding protein folding involves determining the relations between the energetics of weak interactions and protein conformation, and the collective chain dynamics that govern the search in conformational space. In modern rate theory, these issues are resolved by mapping the potential energy of the molecule as a function of the relevant coordinates. The dynamics are then represented as diffusion on such an energy surface(1,2). For folding reactions, however, even the solvent-averaged free energy surface is hyper-dimensional due to the large number of relevant coordinates (i.e. thousands of atomic coordinates for a small protein)(3). Folding hypersurfaces should also be topographically complex due to frustration between the myriads of possible interactions (3,4). Moreover, molecular simulations(5-8) and NMR dynamics experiments(9) indicate that protein conformational motions span a wide range of timescales (i.e. from picoseconds to milliseconds). The implication is that measuri...

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

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. Detailed characterization of the IgG2 disulfide linkages often involve MS/MS approaches that include electrospray ionization or electron-transfer dissociation, and method optimization is often cumbersome due to the large size and heterogeneity of the disulfide-bonded peptides. As reported here, we developed a rapid LC-MALDI-TOF/TOF workflow that can both identify the IgG2 disulfide linkages and provide a semi-quantitative assessment of the distribution of the disulfide isoforms. We established signature disulfide-bonded IgG2 hinge peptides that correspond to the A, A/B, and B disulfide isoforms and can be applied to the fast classification of IgG2 isoforms in heterogeneous mixtures.

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. Our characterization of these ADCs demonstrated that the disulfide configuration affects the kinetics of disulfide bond reduction, but has minimal effect on the primary sites of reduction. The IgG2-A mAbs yielded ADCs with higher drug-to-antibody ratios (DARs) due to the easier reduction of its interchain disulfides. However, hinge-region cysteines were the primary conjugation sites for both IgG2-A and IgG2-B mAbs.