20 research outputs found
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H/D Exchange and Mass Spectrometry in the Studies of Protein Conformation and Dynamics: Is There a Need for a Top-Down Approach?
Hydrogen/deuterium exchange (HDX) combined with mass spectrometry (MS) detection has matured in recent years to become a powerful tool in structural biology and biophysics. Several limitations of this technique can and will be addressed by tapping into ever expanding arsenal of methods to manipulate ions in the gas phase offered by mass spectrometry
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Existence of a Noncanonical State of Iron-Bound Transferrin at Endosomal pH Revealed by Hydrogen Exchange and Mass Spectrometry
Transferrin is an enigmatic metalloprotein, which exhibits a profound conformational change upon binding of ferric ion and a synergistic anion (oxalate or carbonate). While the apo- and holo-forms of the protein have well-defined and stable conformations termed “open” and “closed,” certain aspects of transferrin behavior imply the existence of alternative protein states. In this work hydrogen/deuterium exchange was used in combination with mass spectrometry to map solvent-accessible surfaces of the iron-bound and iron-free forms of the N-lobe of human serum transferrin at both neutral and endosomal pH. While the deuterium uptake is significantly decelerated in the iron-bound state of the protein (compared to the apo-form) at neutral pH, the changes are distributed very unevenly across the protein sequence. Protein segments exhibiting most noticeable gain in protection map onto the inter-domain cleft region housing the iron-binding site. At the same time, protection levels of segments located in the bulk of the protein are largely unaffected by the presence of the metal. These observations are fully consistent with the notion of a metal-induced switch from the open to the closed conformation with solvent-inaccessible inter-domain cleft. However, differences in the exchange behavior between the apo- and holo-forms of transferrin become much less noticeable at endosomal pH, including the segments located in the inter-domain cleft region. Intriguingly, a significant patch in the cleft region becomes slightly less protected in the presence of the metal, suggesting that the holo-protein exists in the open conformation under these slightly acidic conditions. The existence of a non-canonical state of holo-transferrin was postulated several years ago; however, this work provides for the first time conclusive evidence that such alternative states are indeed populated in solution
Conformation and Dynamics of Biopharmaceuticals: Transition of Mass Spectrometry-Based Tools from Academe to Industry
Mass spectrometry plays a very visible role in biopharmaceutical industry, although its use in development, characterization, and quality control of protein drugs is mostly limited to the analysis of covalent structure (amino acid sequence and post-translational modifications). Despite the centrality of protein conformation to biological activity, stability, and safety of biopharmaceutical products, the expanding arsenal of mass spectrometry-based methods that are currently available to probe higher order structure and conformational dynamics of biopolymers did not, until recently, enjoy much attention in the industry. This is beginning to change as a result of recent work demonstrating the utility of these experimental tools for various aspects of biopharmaceutical product development and manufacturing. In this work, we use a paradigmatic protein drug interferon β-1a as an example to illustrate the utility of mass spectrometry as a powerful tool not only to assess the integrity of higher order structure of a protein drug, but also to predict consequences of its degradation at a variety of levels
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Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches
Unlike small molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both the higher order structure and conformational dynamics of a protein, induced by an array of extrinsic factors, is of central importance in producing, purifying, and formulating a commercial biopharmaceutical with consistent therapeutic properties. In this study we demonstrate that two complementary mass spectrometry-based approaches (analysis of ionic charge state distribution and hydrogen/deuterium exchange) can be a potent tool in monitoring conformational changes in protein biopharmaceuticals. The utility of these approaches is demonstrated by detecting and characterizing conformational changes in the biopharmaceutical product interferon β-1a (IFN-β1a). The protein degradation process was modeled by inducing a single chemical modification of IFN-β1a (alkylation of its only free cysteine residue with N-ethylmaleimide), which causes significant reduction in its antiviral activity. Analysis of IFN-β1a ionic charge state distributions unequivocally reveals a significant decrease of conformational stability in the degraded protein, while hydrogen/deuterium exchange measurements provide a clear indication that the higher order structure is affected well beyond the covalent modification site. Importantly, neither technique required that the location or indeed the nature of the chemical modification be known prior to or elucidated in the process of the analysis. In contrast, application of the standard armamentarium of biophysical tools, which are commonly employed for quality control of protein pharmaceuticals, met with very limited success in detection and characterization of conformational changes in the modified IFN-β1a. This work highlights the role mass spectrometry can and should play in the biopharmaceutical industry beyond the presently assigned task of primary structure analysis
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Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin
Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated Fe(C)hTF. The new bridge sequence of this construct, designated Fe(C)TEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe
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Structural and Functional Consequences of the Substitution of Glycine 65 with Arginine in the N-Lobe of Human Transferrin
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Evolution reversed: The ability to bind iron restored to the N-lobe of the murine inhibitor of carbonic anhydrase by strategic mutagenesis
The murine inhibitor of carbonic anhydrase (mICA) is a member of the superfamily related to the bilobal iron transport protein transferrin (TF), which binds a ferric ion within a cleft in each lobe. Although the gene encoding ICA in humans is classified as a pseudogene, an apparently functional ICA gene has been annotated in mice, rats, cows, pigs, and dogs. All ICAs lack one (or more) of the amino acid ligands in each lobe essential for high-affinity coordination of iron and the requisite synergistic anion, carbonate. The reason why ICA family members have lost the ability to bind iron is potentially related to acquiring a new function(s), one of which is inhibition of certain carbonic anhydrase (CA) isoforms. A recombinant mutant of the mICA (W124R/S188Y) was created with the goal of restoring the ligands required for both anion (Arg124) and iron (Tyr188) binding in the N-lobe. Absorption and fluorescence spectra definitively show that the mutant binds ferric iron in the N-lobe. Electrospray ionization mass spectrometry confirms the presence of both ferric iron and carbonate. At the putative endosomal pH of 5.6, iron is released by two slow processes indicative of high-affinity coordination. Induction of specific iron binding implies that (1) the structure of mICA resembles those of other TF family members and (2) the N-lobe can adopt a conformation in which the cleft closes when iron binds. Because the conformational change in the N-lobe indicated by metal binding does not impact the inhibitory activity of mICA, inhibition of CA was tentatively assigned to the C-lobe. Proof of this assignment is provided by limited trypsin proteolysis of porcine ICA