Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed ßα protein.

The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring ßα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable ßα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.

Deamidation and isomerization liability analysis of 131 clinical-stage antibodies.

Contemporary in vivo and in vitro discovery platform technologies greatly increase the odds of identifying high-affinity monoclonal antibodies (mAbs) towards essentially any desired biologically relevant epitope. Lagging discovery throughput is the ability to select for highly developable mAbs with drug-like properties early in the process. Upstream consideration of developability metrics should reduce the frequency of failures in later development stages. As the field moves towards incorporating biophysical screening assays in parallel to discovery processes, similar approaches should also be used to ensure robust chemical stability. Optimization of chemical stability in the early stages of discovery has the potential to reduce complications in formulation development and improve the potential for successful liquid formulations. However, at present, our knowledge of the chemical stability characteristics of clinical-stage therapeutic mAbs is fragmented and lacks comprehensive comparative assessment. To address this knowledge gap, we produced 131 mAbs with amino acid sequences corresponding to the variable regions of clinical-stage mAbs, subjected these to low and high pH stresses and identified the resulting modifications at amino acid-level resolution via tryptic peptide mapping. Among this large set of mAbs, relatively high frequencies of asparagine deamidation events were observed in CDRs H2 and L1, while CDRs H3, H2 and L1 contained relatively high frequencies of instances of aspartate isomerization.

Database-Centric Method for Automated High-Throughput Deconvolution and Analysis of Kinetic Antibody Screening Data.

The state-of-the-art industrial drug discovery approach is the empirical interrogation of a library of drug candidates against a target molecule. The advantage of high-throughput kinetic measurements over equilibrium assessments is the ability to measure each of the kinetic components of binding affinity. Although high-throughput capabilities have improved with advances in instrument hardware, three bottlenecks in data processing remain: (1) intrinsic molecular properties that lead to poor biophysical quality in vitro are not accounted for in commercially available analysis models, (2) processing data through a user interface is time-consuming and not amenable to parallelized data collection, and (3) a commercial solution that includes historical kinetic data in the analysis of kinetic competition data does not exist. Herein, we describe a generally applicable method for the automated analysis, storage, and retrieval of kinetic binding data. This analysis can deconvolve poor quality data on-the-fly and store and organize historical data in a queryable format for use in future analyses. Such database-centric strategies afford greater insight into the molecular mechanisms of kinetic competition, allowing for the rapid identification of allosteric effectors and the presentation of kinetic competition data in absolute terms of percent bound to antigen on the biosensor.

Rapid assessment of oxidation via middle-down LCMS correlates with methionine side-chain solvent-accessible surface area for 121 clinical stage monoclonal antibodies.

Susceptibility of methionine to oxidation is an important concern for chemical stability during the development of a monoclonal antibody (mAb) therapeutic. To minimize downstream risks, leading candidates are usually screened under forced oxidation conditions to identify oxidation-labile molecules. Here we report results of forced oxidation on a large set of in-house expressed and purified mAbs with variable region sequences corresponding to 121 clinical stage mAbs. These mAb samples were treated with 0.1% H2O2 for 24 hours before enzymatic cleavage below the hinge, followed by reduction of inter-chain disulfide bonds for the detection of the light chain, Fab portion of heavy chain (Fd) and Fc by liquid chromatography-mass spectrometry. This high-throughput, middle-down approach allows detection of oxidation site(s) at the resolution of 3 distinct segments. The experimental oxidation data correlates well with theoretical predictions based on the solvent-accessible surface area of the methionine side-chains within these segments. These results validate the use of upstream computational modeling to predict mAb oxidation susceptibility at the sequence level.

Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY.

The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. Here, we report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structure of the excited state ensemble. This prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. Using these results, we then predict incisive single molecule FRET experiments as a means of model validation. This study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments.

Understanding ForteBio's Sensors for High-Throughput Kinetic and Epitope Screening for Purified Antibodies and Yeast Culture Supernatant.

Real-time and label-free antibody screening systems are becoming more popular because of the increasing output of purified antibodies and antibody supernatant from many antibody discovery platforms. However, the properties of the biosensor can greatly affect the kinetic and epitope binning results generated by these label-free screening systems. ForteBio human-specific ProA, anti-human IgG quantitation (AHQ), anti-human Fc capture (AHC) sensors, and custom biotinylated-anti-human Fc capture (b-AHFc) sensors were evaluated in terms of loading ability, regeneration, kinetic characterization, and epitope binning with both purified IgG and IgG supernatant. AHC sensors proved unreliable for kinetic or binning assays at times, whereas AHQ sensors showed poor loading and regeneration abilities. ProA sensors worked well with both purified IgG and IgG supernatant. However, the interaction between ProA sensors and the Fab region of the IgG with VH3 germline limited the application of ProA sensors, especially in the epitope binning experiment. In an attempt to generate a biosensor type that would be compatible with a variety of germlines and sample types, we found that the custom b-AHFc sensors appeared to be robust working with both purified IgG and IgG supernatant, with little evidence of sensor-related artifacts.

Clusters of isoleucine, leucine, and valine side chains define cores of stability in high-energy states of globular proteins: Sequence determinants of structure and stability.

Measurements of protection against exchange of main chain amide hydrogens (NH) with solvent hydrogens in globular proteins have provided remarkable insights into the structures of rare high-energy states that populate their folding free-energy surfaces. Lacking, however, has been a unifying theory that rationalizes these high-energy states in terms of the structures and sequences of their resident proteins. The Branched Aliphatic Side Chain (BASiC) hypothesis has been developed to explain the observed patterns of protection in a pair of TIM barrel proteins. This hypothesis supposes that the side chains of isoleucine, leucine, and valine (ILV) residues often form large hydrophobic clusters that very effectively impede the penetration of water to their underlying hydrogen bond networks and, thereby, enhance the protection against solvent exchange. The linkage between the secondary and tertiary structures enables these ILV clusters to serve as cores of stability in high-energy partially folded states. Statistically significant correlations between the locations of large ILV clusters in native conformations and strong protection against exchange for a variety of motifs reported in the literature support the generality of the BASiC hypothesis. The results also illustrate the necessity to elaborate this simple hypothesis to account for the roles of adjacent hydrocarbon moieties in defining stability cores of partially folded states along folding reaction coordinates.

Modulation of frustration in folding by sequence permutation.

Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an off-pathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N- and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray-scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.

Microsecond barrier-limited chain collapse observed by time-resolved FRET and SAXS.

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59-heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~27 µs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency>90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.

Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam.

Small-angle X-ray scattering (SAXS) is a well established technique to probe the nanoscale structure and interactions in soft matter. It allows one to study the structure of native particles in near physiological environments and to analyze structural changes in response to variations in external conditions. The combination of microfluidics and SAXS provides a powerful tool to investigate dynamic processes on a molecular level with sub-millisecond time resolution. Reaction kinetics in the sub-millisecond time range has been achieved using continuous-flow mixers manufactured using micromachining techniques. The time resolution of these devices has previously been limited, in part, by the X-ray beam sizes delivered by typical SAXS beamlines. These limitations can be overcome using optics to focus X-rays to the micrometer size range providing that beam divergence and photon flux suitable for performing SAXS experiments can be maintained. Such micro-SAXS in combination with microfluidic devices would be an attractive probe for time-resolved studies. Here, the development of a high-duty-cycle scanning microsecond-time-resolution SAXS capability, built around the Kirkpatrick-Baez mirror-based microbeam system at the Biophysics Collaborative Access Team (BioCAT) beamline 18ID at the Advanced Photon Source, Argonne National Laboratory, is reported. A detailed description of the microbeam small-angle-scattering instrument, the turbulent flow mixer, as well as the data acquisition and control and analysis software is provided. Results are presented where this apparatus was used to study the folding of cytochrome c. Future prospects for this technique are discussed.

Minireview: structural insights into early folding events using continuous-flow time-resolved small-angle X-ray scattering.

Small-angle X-ray scattering (SAXS) is a powerful method for obtaining quantitative structural information on the size and shape of proteins, and it is increasingly used in kinetic studies of folding and association reactions. In this minireview, we discuss recent developments in using SAXS to obtain structural information on the unfolded ensemble and early folding intermediates of proteins using continuous-flow mixing devices. Interfacing of these micromachined devices to SAXS beamlines has allowed access to the microsecond time regime. The experimental constraints in implementation of turbulence and laminar flow-based mixers with SAXS detection and a comparison of the two approaches are presented. Current improvements and future prospects of microsecond time-resolved SAXS and the synergy with ab initio structure prediction and molecular dynamics simulations are discussed.