Interlaboratory Comparison of Hydrogen–Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb

Hudgens, Jeffrey W.; Gallagher, Elyssia S.; Karageorgos, Ioannis; Anderson, Kyle W.; Filliben, James J.; Huang, Richard Y.‐C.; Chen, Guodong; Bou-Assaf, George M.; Espada, Alfonso; Chalmers, Michael J.; Harguindey, Eduardo; Zhang, Hui Min; Walters, Benjamin T.; Zhang, Jennifer; Venable, John D.; Steckler, Caitlin; Park, Inhee; Brock, Ansgar; Lu, Xiaojun; Pandey, R.; Chandramohan, Arun; Anand, Ganesh S.; Nirudodhi, Sasidhar N.; Sperry, Justin B.; Rouse, Jason C.; Carroll, James A.; Rand, Kasper D.; Leurs, Ulrike; Weis, David D.; Al-Naqshabandi, Mohammed A.; Hageman, Tyler S.; Deredge, Daniel; Wintrode, Patrick L.; Papanastasiou, Malvina; Lambris, John D.; Li, Sheng; Urata, Sarah

doi:10.1021/acs.analchem.9b01100

Cited by 44 publications

(68 citation statements)

References 93 publications

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“…The introduction of cathepsin-L as a new tool to facilitate dual-protease techniques could be highly impactful for the study of important biological molecules beyond histones. Such cases may include protein classes known to be challenging for HX-MS, such as antibodies (81), or proteins which have been identified as cathepsin"s substrates (i.e. elastins, collagens and proteoglycans) (52).…”

Section: Fig 3 Peptide Maps Following Digestion With Cathepsin-l Omentioning

confidence: 99%

Chasing Tails: Cathepsin-L Improves Structural Analysis of Histones by HX-MS*[S]

Papanastasiou

Mullahoo

DeRuff

et al. 2019

Molecular & Cellular Proteomics

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The N-terminal regions (tails) of histone proteins are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications present on these regions contribute to transcriptional regulation. Considering their biological significance, relatively few structural details have been established for histone tails, mainly because of their inherently disordered nature. Although hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed in this context, presumably because of the poor N-terminal coverage provided by pepsin. Inspired from histone-clipping events, we profiled the activity of cathepsin-L under HX-MS quench conditions and characterized its specificity employing the four core histones (H2A, H2B, H3 and H4). Cathepsin-L demonstrated cleavage patterns that were substrate- and pH-dependent. Cathepsin-L generated overlapping N-terminal peptides about 20 amino acids long for H2A, H3, and H4 proving its suitability for the analysis of histone tails dynamics. We developed a comprehensive HX-MS method in combination with pepsin and obtained full sequence coverage for all histones. We employed our method to analyze histones H3 and H4. We observe rapid deuterium exchange of the N-terminal tails and cooperative unfolding (EX1 kinetics) in the histone-fold domains of histone monomers in-solution. Overall, this novel strategy opens new avenues for investigating the dynamic properties of histones that are not apparent from the crystal structures, providing insights into the structural basis of the histone code.

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Section: Fig 3 Peptide Maps Following Digestion With Cathepsin-l Omentioning

confidence: 99%

Chasing Tails: Cathepsin-L Improves Structural Analysis of Histones by HX-MS*[S]

Papanastasiou

Mullahoo

DeRuff

et al. 2019

Molecular & Cellular Proteomics

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“…In a recent publication, the authors performed HDX on both lipid-free and -bound forms of three ApoA-I variants (F71Y, L159R and L170P). Although these variants were studied in rHDL particles with a different size (11-12 nm for F71Y, L159R and L170P vs 8.4 nm for L75P and L174S) and lipid composition (DMPC vs POPC), and despite the fact that comparing HDX data is inherently complicated 28 , the present study describes common trends with the previous HDX data. Indeed, a general reduction in uptake when going from lipid-free to lipid-bound form was observed (Fig S2), as well as a decreased protection of the region spanning the h5 pair (L159R and L170P).…”

Section: Discussionmentioning

confidence: 55%

Structure dynamics of ApoA-I amyloidogenic variants in small HDL increase their ability to mediate cholesterol efflux

Lagerstedt

Nilsson

Lindvall

et al. 2020

Preprint

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24Specific mutations in Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) are 25 responsible for a late-onset systemic amyloidosis. Carriers do not exhibit increased 26 cardiovascular disease risk despite reduced levels of ApoA-I/ HDL-cholesterol. To explain 27 this paradox, we show that the HDL particle profile of L75P and L174S patients presents a 28 higher relative abundance of the 8.4 nm vs 9.6 nm particles, and that serum from patients, as 29 well as reconstituted 8.4 and 9.6 nm HDL particles (rHDL), possess increased capacity to 30 catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and 31 hydrogen-deuterium exchange revealed that the variants in 8.4 nm rHDL have altered 32 secondary structure composition and display a more flexible binding to lipids compared to 33 their native counterpart. The reduced HDL-cholesterol levels of patients carrying ApoA-I 34 amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles 35 and better cholesterol efflux due to altered, region-specific protein structure dynamics. 36 37 Keywords 38 Apolipoprotein A-I, amyloidosis, high-density lipoprotein, cholesterol efflux, cardiovascular 39 disease 40 41 Abbreviations 42 ABCA1, ATP binding cassette A1; ABCG1, ATP binding cassette G1, ApoA-I, apolipoprotein 43 A-I; ApoB, apolipoprotein B; CD, circular dichroism; CVD, cardiovascular disease; FC, 44 unesterified free cholesterol; HDL, high-density lipoprotein; HDX, hydrogen-deuterium 45 exchange; LCAT, lecithin-cholesteryl acyl transferase; PBS, phosphate buffer saline; POPC, 46 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; rHDL, reconstituted HDL; SRCD, 47 Synchrotron Radiation Circular Dichroism; TEV, tobacco etch virus; Tm, transition 48 temperature; WT, wild-type.49 3 Main 50 Amyloidoses are a broad group of diseases caused by protein instability and misfolding that 51 leads to pathological aggregation of proteins as amyloid deposits in tissues and organs 1 .52 These amyloid deposits are either localized, as in Alzheimer's and Parkinson's diseases, or 53 systemic, as is the case of transthyretin and light-chain amyloidoses. The different types of 54 amyloidoses commonly lead to severe and age-related damage of the tissues where 55 aggregation takes place. The understanding of the determinants leading to protein fibril 56 formation and amyloidosis development is thus key for finding ways to develop efficient 57 treatments for the affected individuals.58 Apolipoprotein A-I (ApoA-I), associated with hereditary systemic amyloidosis, has a key role 59 for the transportation of cholesterol and lipids in the circulation between peripheral tissues 60 and the liver 2 . Following its synthesis in the liver and intestines, ApoA-I mediates this 61 transportation by the formation of high-density lipoprotein (HDL) particles. These load 62 cholesterol and lipids from macrophages at the artery wall via a regulated efflux mechanism 63 catalyzed by the cellular ATP-binding cassette (ABC)A1 receptor. HDLs are then c...

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“…To directly investigate the dynamics of different regions of hMGL upon mutations, we have used the HDX-MS methodology that has been well established 40 , 41 . At the peptide length resolution these data provide insight into the changes of hMGL dynamics induced by mutations.…”

Section: Resultsmentioning

confidence: 99%

Conformational gating, dynamics and allostery in human monoacylglycerol lipase

Tyukhtenko

Rajarshi

et al. 2020

Sci Rep

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Inhibition of human Monoacylglycerol Lipase (hMGL) offers a novel approach for treating neurological diseases. The design of inhibitors, targeting active-inactive conformational transitions of the enzyme, can be aided by understanding the interplay between structure and dynamics. Here, we report the effects of mutations within the catalytic triad on structure, conformational gating and dynamics of hMGL by combining kinetics, NMR, and HDX-MS data with metadynamics simulations. We found that point mutations alter delicate conformational equilibria between active and inactive states. HDX-MS reveals regions of the hMGL that become substantially more dynamic upon substitution of catalytic acid Asp-239 by alanine. These regions, located far from the catalytic triad, include not only loops but also rigid α-helixes and β-strands, suggesting their involvement in allosteric regulation as channels for long-range signal transmission. The results identify the existence of a preorganized global communication network comprising of tertiary (residue-residue contacts) and quaternary (rigid-body contacts) networks that mediate robust, rapid intraprotein signal transmission. Catalytic Asp-239 controls hMGL allosteric communications and may be considered as an essential residue for the integration and transmission of information to enzymes’ remote regions, in addition to its well-known role to facilitate Ser-122 activation. Our findings may assist in the identification of new druggable sites in hMGL.

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Interlaboratory Comparison of Hydrogen–Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb

Cited by 44 publications

References 93 publications

Chasing Tails: Cathepsin-L Improves Structural Analysis of Histones by HX-MS*[S]

Chasing Tails: Cathepsin-L Improves Structural Analysis of Histones by HX-MS*[S]

Structure dynamics of ApoA-I amyloidogenic variants in small HDL increase their ability to mediate cholesterol efflux

Conformational gating, dynamics and allostery in human monoacylglycerol lipase

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