David D. Weis

Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis.

Proteins that undergo cooperative unfolding events display EX1 kinetic signatures in hydrogen exchange mass spectra. The hallmark bimodal isotope pattern observed for EX1 kinetics is distinct from the binomial isotope pattern for uncorrelated exchange (EX2), the normal exchange regime for folded proteins. Detection and characterization of EX1 kinetics is simple when the cooperative unit is large enough that the isotopic envelopes in the bimodal pattern are resolved in the m/z scale but become complicated in cases where the unit is small or there is a mixture of EX1 and EX2 kinetics. Here we describe a data interpretation method involving peak width analysis that makes characterization of EX1 kinetics simple and rapid. The theoretical basis for EX1 and EX2 isotopic signatures and the effects each have on peak width are described. Modeling of EX2 widening and analysis of empirical data for proteins and peptides containing purely EX2 kinetics showed that the amount of widening attributable to stochastic forward- and back exchange in a typical experiment is small and can be quantified. Proteins and peptides with both obvious and less obvious EX1 kinetics were analyzed with the peak width method. Such analyses provide the half-life for the cooperative unfolding event and the relative number of residues involved. Automated analysis of peak width was performed with custom Excel macros and the DEX software package. Peak width analysis is robust, capable of automation, and provides quick interpretation of the key information contained in EX1 kinetic events.

Correlating excipient effects on conformational and storage stability of an IgG1 monoclonal antibody with local dynamics as measured by hydrogen/deuterium-exchange mass spectrometry.

The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability (T(m) values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAbs conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (C(H)2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the C(H)2 domain (notably heavy chain 241-251) and the mAbs overall physical stability.

Mapping unstructured regions and synergistic folding in intrinsically disordered proteins with amide H/D exchange mass spectrometry.

Mapping the structured and disordered regions and identifying disorder-to-order transitions are essential to understanding intrinsically disordered proteins (IDPs). One technique that can provide such information is H/D exchange coupled with mass spectrometry (H/D-MS). To explore the feasibility of H/D-MS for mapping disordered and ordered regions in IDPs, we undertook a systematic evaluation of an unstructured protein, a molten globular protein, and the well-folded complex of the two proteins. Most segments of the unstructured protein, ACTR (activator of thyroid and retinoid receptors, NCOA3_HUMAN, residues 1018-1088), exchange at rates consistent with its assignment as an unstructured protein, but there is slight protection in regions that become helical in the ACTR-CBP complex. The molten globular protein, CBP (the nuclear coactivator binding domain of the CREB binding protein, CBP_MOUSE, residues 2059-2117), is moderately protected from exchange, and the protection is nearly uniform across the length of the protein. The uniformity arises because of rapid interconversion between an ensemble of folded conformers and an ensemble of unstructured conformers. Rapid interconversion causes the H/D exchange kinetics to be dominated by exchange by molecules in unstructured conformations. For the folded ACTR-CBP complex, the exchange data provide a qualitatively accurate description of the complex. Our results provide a useful framework to use in the interpretation of H/D-MS data of intrinsically disordered proteins.

Effects of Salts from the Hofmeister Series on the Conformational Stability, Aggregation Propensity, and Local Flexibility of an IgG1 Monoclonal Antibody

This work examines the effect of three anions from the Hofmeister series (sulfate, chloride, and thiocyanate) on the conformational stability and aggregation rate of an IgG1 monoclonal antibody (mAb) and corresponding changes in the mAbs backbone flexibility (at pH 6 and 25 °C). Compared to a 0.1 M NaCl control, thiocyanate (0.5 M) decreased the melting temperatures (Tm) for three observed conformational transitions within the mAb by 6-9 °C, as measured by differential scanning calorimetry. Thiocyanate also accelerated the rate of monomer loss at 40 °C over 12 months, as monitored by size exclusion chromatography. Backbone flexibility, as measured via H/D exchange mass spectrometry, increased in two segments in the CH2 domain with more subtle changes across several additional regions. Chloride (0.5 M) caused slight increases in the Tm values, small changes in aggregation rate, and minimal yet consistent decreases in flexibility across various domains with larger effects noted within the VL, CH1, and CH3 domains. In contrast, 0.5 M sulfate increased Tm values, had small stabilizing influences on aggregate formation over time, yet substantially increased the flexibility of two specific regions in the CH1 and VL domains. While thiocyanate-induced conformational destabilization of the mAb correlated with increased local flexibility of specific regions in the CH2 domain (especially residues 241-251 in the heavy chain), the stabilizing anion sulfate did not affect these CH2 regions.

Infrared spectroscopic signatures of (NH4)2SO4 aerosols

Ammonium sulfate particles in air with average diameters ranging from 0.1 to 0.5-μm have been generated by atomizing aqueous solutions of (NH4)2SO4 of various concentrations at ambient temperatures and pressures. The infrared spectra from 4000 to 600 cm−1 of the resulting aerosols have been investigated. This spectral region has allowed us to study the four infrared-active vibrational modes of this salt: ν3(NH4+), ν4(NH4+), ν3(SO42−), and ν4(SO42−). The frequencies of these modes are similar to published results obtained from infrared studies of the single crystal but are displaced to higher wavenumbers. Depending on relative humidity, the aerosol particles are crystalline or supersaturated aqueous droplets. These phase identifications are possible because liquid water absorption features are found in the droplets but not in the crystals. Extensive Mie theory calculations have been performed for spheres of diameters ranging from 0.1-μm to 2.0-μm to explore frequency shifts and the relative contributions to extinction of scattering and absorption with particle size. We show that, for the smaller particles, the molecular cross section in the ν3(SO42−) region can be used to determine the number of (NH4)2SO4 molecules in an aerosol sample. The (small) frequency shifts in this region provide information on the aerosol particle size. A Mie theory calculation of extinction for a model polydisperse aerosol, believed to approximate that of an experimental aerosol, gives reasonable agreement with the observed spectrum. While calculated band centers of the four modes are within 1% of those observed, values of extinction can differ by as much as 50%. We discuss possible reasons for the discrepancies. Spectroscopic changes observed for an aerosol as the particles settle are discussed in terms of kinetic models and Mie theory. We discuss the potential of spectroscopic signatures of tropospheric (NH4)2SO4 aerosols for the characterization of their size, morphology, phase, and composition. Finally, we propose a field experiment to measure sulfate aerosol in the arctic troposphere.

Structural basis for activation of calcineurin by calmodulin

The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.0 and 7.4. Segments covering the YTE mutation sites and the FcRn binding epitopes showed either subtle or no observable differences in local flexibility. Surprisingly, several adjacent segments in the CH2 and distant segments in the VH, CH1, and VL domains had significantly increased flexibility in the YTE mutant. Most notable among the observed differences is increased flexibility of the 244–254 segment of the CH2 domain, where increased flexibility has been shown previously to correlate with decreased conformational stability and increased aggregation propensity in other IgG1 mAbs (e.g., presence of destabilizing additives as well as upon de-glycosylation or methionine oxidation). DSC analysis showed decreases in both thermal onset (Tonset) and unfolding (Tm1) temperatures of 7°C and 6.7°C, respectively, for the CH2 domain of the YTE mutant. In addition, mAb-E aggregated faster than mAb-A under accelerated stability conditions as measured by SEC analysis. Hence, the relatively lower physical stability of the YTE mutant correlates with increased local flexibility of the 244–254 segment, providing a site-directed mutant example that this segment of the CH2 domain is an aggregation hot spot in IgG1 mAbs.

Partial cooperative unfolding in proteins as observed by hydrogen exchange mass spectrometry

Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, exist and in many cases are important for protein function. The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days. In this review, we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples. Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of intermolecular and intramolecular interactions in the global control of kinase function. Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signalling proteins. HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both the classification of and greater understanding of the prevalence of protein motion.

Hydrogen-Deuterium Exchange Mass Spectrometry as an Emerging Analytical Tool for Stabilization and Formulation Development of Therapeutic Monoclonal Antibodies

The dynamic nature of the structure of monoclonal antibodies (mAbs) can be probed at a resolution of 5-20 residues using hydrogen-deuterium exchange mass spectrometry (H/D-MS). Recent studies using H/D-MS have shown that distinct regions of IgG1 mAbs experience significant changes in backbone dynamics in response to specific physicochemical alterations, varying solution conditions, or exposure to different environmental stresses. Tracking such changes in local dynamics may therefore serve as a key analytical tool, not only to monitor stability changes, but also to design improved, and more stable formulations of therapeutic mAbs in pharmaceutical dosage forms. This review article describes the H/D-MS method as applied to the analysis of formulations containing mAbs and summarizes recent studies monitoring changes in mAb local dynamics in response to chemical modifications, physical degradation, and presence of stabilizing and destabilizing excipients. Furthermore, the nature of the local dynamics of a highly conserved peptide segment in the CH 2 domain of IgG1 mAbs is reviewed, and the results are correlated with decreased pharmaceutical stability, supporting the identification of a common aggregation hotspot sequence in the Fc region of human IgG1 mAbs. In addition, unresolved challenges (and opportunities) in applying H/D-MS technology for stabilization and formulation development of mAbs are discussed.

Mapping Residual Structure in Intrinsically Disordered Proteins at Residue Resolution Using Millisecond Hydrogen/Deuterium Exchange and Residue Averaging

AbstractMeasurement of residual structure in intrinsically disordered proteins can provide insights into the mechanisms by which such proteins undergo coupled binding and folding. The present work describes an approach to measure residual structure in disordered proteins using millisecond hydrogen/deuterium (H/D) exchange in a conventional bottom-up peptide-based workflow. We used the exchange mid-point, relative to a totally deuterated control, to quantify the rate of H/D exchange in each peptide. A weighted residue-by-residue average of these midpoints was used to map the extent of residual structure at near single-residue resolution. We validated this approach both by simulating a disordered protein and experimentally using the p300 binding domain of ACTR, a model disordered protein already well-characterized by other approaches. Secondary structure elements mapped in the present work are in good agreement with prior nuclear magnetic resonance measurements. The new approach was somewhat limited by a loss of spatial resolution and subject to artifacts because of heterogeneities in intrinsic exchange. Approaches to correct these limitations are discussed. Graphical Abstractᅟ