Protein A does not induce allosteric structural changes in an IgG1 antibody during binding

Pabon, Juan P. Rincon; Kochert, Brent A.; Liu, Yan‐Hui; Richardson, Douglas D.; Weis, David D.

doi:10.1016/j.xphs.2021.02.027

Cited by 13 publications

(14 citation statements)

References 58 publications

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“… 539 The allosteric link between Fab and Fc was also recently tested directly by looking at the influence of protein A binding to the Fc, which noted no significant alterations in the Fab regions, at least within the context of an antibody-protein A interaction. 540 Overall, the HDX-MS studies to date on IgG provide strong evidence for a functional allosteric link between the Fc and Fab domains, which are likely context dependent. We expect that future HDX-MS studies looking at the interaction between antibodies, antigens, and immune complexes will elucidate such allosteric networks and examine their potential role in regulating the immune response.…”

Section: Current Uses Of Hdx-msmentioning

confidence: 94%

“…The strongest evidence of this Fab–Fc interaction was from the direct observation of distal effects within the CH2 and CH3 regions in a human IgG1 upon antigen binding at the Fab . The allosteric link between Fab and Fc was also recently tested directly by looking at the influence of protein A binding to the Fc, which noted no significant alterations in the Fab regions, at least within the context of an antibody-protein A interaction . Overall, the HDX-MS studies to date on IgG provide strong evidence for a functional allosteric link between the Fc and Fab domains, which are likely context dependent.…”

Section: Current Uses Of Hdx-msmentioning

confidence: 99%

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Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

et al. 2021

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Solution-phase hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) is a widespread tool for structural analysis across academia and the biopharmaceutical industry. By monitoring the exchangeability of backbone amide protons, HDX-MS can reveal information about higher-order structure and dynamics throughout a protein, can track protein folding pathways, map interaction sites, and assess conformational states of protein samples. The combination of the versatility of the hydrogen/deuterium exchange reaction with the sensitivity of mass spectrometry has enabled the study of extremely challenging protein systems, some of which cannot be suitably studied using other techniques. Improvements over the past three decades have continually increased throughput, robustness, and expanded the limits of what is feasible for HDX-MS investigations. To provide an overview for researchers seeking to utilize and derive the most from HDX-MS for protein structural analysis, we summarize the fundamental principles, basic methodology, strengths and weaknesses, and the established applications of HDX-MS while highlighting new developments and applications.

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Section: Current Uses Of Hdx-msmentioning

confidence: 94%

Section: Current Uses Of Hdx-msmentioning

confidence: 99%

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

et al. 2021

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“…An additional subset of LC-MS runs were collected in second laboratory using a custom HDX-MS setup coupled to an Agilent 6530 Q-TOF as described previously. , The flow rate through the protease column was 200 μL/min, corresponding to a digestion time of approximately 26 s. Peak areas were integrated with MassHunter Qualitative Analysis software. Peak area quantification included the summation of the monoisotopic peak areas across all observed charge states.…”

Section: Methodsmentioning

confidence: 99%

Rapid Assessment of Pepsin Column Activity for Reliable HDX-MS Studies

Vorauer

Wrigley

Pabon

et al. 2021

J. Am. Soc. Mass Spectrom.

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Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) is a widely used technique to probe protein structural dynamics, track conformational changes, and map protein–protein interactions. Most HDX-MS studies employ a bottom-up approach utilizing the acid active protease pepsin to digest the protein of interest, often utilizing immobilized protease in a column format. The extent of proteolytic cleavage will greatly influence data quality and presents a major source of variation in HDX-MS studies. Here, we present a simple cocktail of commonly available peptides that are substrates of pepsin and can serve as a rapid check of pepsin column activity. The peptide-based assay requires no system modifications and provides an immediate readout to check and benchmark pepsin activity across different HDX-MS platforms.

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“…HDX experiments were performed similarly as described elsewhere. 36 Briefly, automated HDX-MS experiments were carried out using a LEAP Technologies HDX PAL DHR robot (Carrboro, NC) with a three-valve configuration system coupled to a Bruker MaXis Q-ToF operating in positive-ion mode. HDX was initiated by diluting 9 μL of purified COQ8A (15 μM in 20 mM HEPES, 100 mM NaCl, at pH 7.2) with 81 μL of 20 mM HEPES, 100 mM NaCl, and 5% DMSO, at pD 7.2 in D 2 O (Cambridge Isotope Laboratories, Inc., Tewksbury, MA) with and without 8 mM 2-propylphenol.…”

mentioning

confidence: 99%

“…Differential HDX data were evaluated for significant differences by using a hybrid significance test. 38 Normalized HDX differences were clustered 36 to define thresholds for strong, moderate, and negligible HDX differences. In the Supporting Information is a volcano plot for the identification of statistically different peptides and the magnitude of the effects.…”

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confidence: 99%

2-Propylphenol Allosterically Modulates COQ8A to Enhance ATPase Activity

et al. 2022

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COQ8A is an atypical kinase-like protein that aids the biosynthesis of coenzyme Q, an essential cellular cofactor and antioxidant. COQ8A's mode of action remains unclear, in part due to the lack of small molecule tools to probe its function.Here, we blend NMR and hydrogen−deuterium exchange mass spectrometry to help determine how a small CoQ precursor mimetic, 2-propylphenol, modulates COQ8A activity. We identify a likely 2-propylphenol binding site and reveal that this compound modulates a conserved COQ8A domain to increase nucleotide affinity and ATPase activity. Our findings promise to aid further investigations into COQ8A's precise enzymatic function and the design of compounds capable of boosting endogenous CoQ production for therapeutic gain.

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Protein A does not induce allosteric structural changes in an IgG1 antibody during binding

Cited by 13 publications

References 58 publications

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

Rapid Assessment of Pepsin Column Activity for Reliable HDX-MS Studies

2-Propylphenol Allosterically Modulates COQ8A to Enhance ATPase Activity

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