Specificity of immobilized porcine pepsin in H/D exchange compatible conditions

Hamuro, Yoshitomo; Coales, Stephen J.; Molnar, Kathleen S.; Tuske, Steve; Morrow, Jeffrey A.

doi:10.1002/rcm.3467

Cited by 147 publications

(142 citation statements)

References 19 publications

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“…S7 in the supplemental material). For example, an L369P mutation between the clade C and B isolates likely disrupted a potential pepsin cleavage site (79) in the clade B isolates and yielded differential coverage of the CD4 bind-ing loop across clades. Similarly, peptides spanning V1 and V4, the most densely glycosylated regions of gp120, were not observed for any isolates.…”

Section: Resultsmentioning

confidence: 99%

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Davenport

Guttman

Guo

et al. 2013

J Virol

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dThe HIV-1 envelope glycoprotein (Env) mediates viral entry into host cells and is the sole target of neutralizing antibodies. Much of the sequence diversity in the HIV-1 genome is concentrated within Env, particularly within its gp120 surface subunit. While dramatic functional diversity exists among HIV-1 Env isolates-observable even in the context of monomeric gp120 proteins as differences in antigenicity and immunogenicity-we have little understanding of the structural features that distinguish Env isolates and lead to isolate-specific functional differences, as crystal structures of truncated gp120 "core" proteins from diverse isolates reveal a high level of structural conservation. Because gp120 proteins are used as prospective vaccine immunogens, it is critical to understand the structural factors that influence their reactivity with antibodies. Here, we studied four full-length, glycosylated gp120 monomers from diverse HIV-1 isolates by using small-angle X-ray scattering (SAXS) to probe the overall subunit morphology and hydrogen/deuterium-exchange with mass spectrometry (HDX-MS) to characterize the local structural order of each gp120. We observed that while the overall subunit architecture was similar among isolates by SAXS, dramatic isolate-specific differences in the conformational stability of gp120 were evident by HDX-MS. These differences persisted even with the CD4 receptor bound. Furthermore, surface plasmon resonance (SPR) and enzyme-linked immunosorbance assays (ELISAs) showed that disorder was associated with poorer recognition by antibodies targeting conserved conformational epitopes. These data provide additional insight into the structural determinants of gp120 antigenicity and suggest that conformational dynamics should be considered in the selection and design of optimized Env immunogens.

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Section: Resultsmentioning

confidence: 99%

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Davenport

Guttman

Guo

et al. 2013

J Virol

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“…Digestion of the mAbs, under conditions of slow exchange, was achieved using an immobilised pepsin column. Cleavage of peptide bonds by pepsin is somewhat nonspecific, 20 and consequently the peptide's mass alone could not be used for definitive identification. Therefore, identification of the peptides, produced by pepsin digestion of the proteins, was achieved using a combination of accurate mass (<1 ppm) from the FT-ICR measurements and MS/MS product ion spectra of selected ions using QToF mass spectrometry.…”

Section: Resultsmentioning

confidence: 99%

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Burkitt¹,

Domann²,

O’Connor³

2010

Protein Science

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Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247-253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the C H 2 and C H 3 domains.

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“…Spatial resolution (the size of the average HDX-characterized fragment) is experimentally related to the size of the pepsin-generated fragments (17), as well as to the detection limits imposed by the mass spectrometric analysis. Pepsin digestion of unliganded and CD4-bound gp120 resulted in 38 gp120 peptic fragments, which overlapped in 16 independent regions and covered 98% of the gp120 sequence ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Local Conformational Stability of HIV-1 gp120 in Unliganded and CD4-Bound States as Defined by Amide Hydrogen/Deuterium Exchange

Kong

Huang

Coales³

et al. 2010

J Virol

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The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states, meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrometry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydrogens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange correlated well with alterations in binding surface (P value ‫؍‬ 0.0004). For the structurally variable gp120, however, reductions in flexibility extended outside the binding surface, and regions of expected high structural diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse conformations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for understanding how local conformational stability influences entropic change, conformational diversity, and structural rearrangements in the gp120-CD4 binding reaction.

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Specificity of immobilized porcine pepsin in H/D exchange compatible conditions

Cited by 147 publications

References 19 publications

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Local Conformational Stability of HIV-1 gp120 in Unliganded and CD4-Bound States as Defined by Amide Hydrogen/Deuterium Exchange

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