المؤلفون: Tran, Quang Hieu¹, Eder, Oliver Maximilian¹, Winkler, Andreas^1,2 andreas.winkler@tugraz.at

المصدر: Journal of Biological Chemistry. May2024, Vol. 300 Issue 5, p1-12. 12p.

مصطلحات موضوعية: *PHYTOCHROMES, *HYDROGEN-deuterium exchange, *ALLOSTERIC regulation, *MASS spectrometry, *CYCLASES, *GLUTAMATE receptors

مستخلص: Sensor-effector proteins integrate information from different stimuli and transform this into cellular responses. Some sensory domains, like red-light responsive bacteriophytochromes, show remarkable modularity regulating a variety of effectors. One effector domain is the GGDEF diguanylate cyclase catalyzing the formation of the bacterial second messenger cyclic-dimericguanosine monophosphate. While critical signal integration elements have been described for different phytochromes, a generalized understanding of signal processing and communication over large distances, roughly 100 Å in phytochrome diguanylate cyclases, is missing. Here we show that dynamicsdriven allostery is key to understanding signal integration on a molecular level.We generated protein variants stabilized in their far-red-absorbing Pfr state and demonstrated by analysis of conformational dynamics using hydrogen-deuterium exchange coupled to mass spectrometry that single amino acid replacements are accompanied by altered dynamics of functional elements throughout the protein. We show that the conformational dynamics correlate with the enzymatic activity of these variants, explaining also the increased activity of a nonphotochromic variant. In addition, we demonstrate the functional importance of mixed Pfr/intermediate state dimers using a fast-reverting variant that still enables wild-type-like foldchanges of enzymatic stimulation by red light. This supports the functional role of single protomer activation in phytochromes, a property that might correlate with the non-canonical mixed Pfr/intermediate-state spectra observed for many phytochrome systems. We anticipate our results to stimulate research in the direction of dynamics-driven allosteric regulation of different bacteriophytochrome-based sensor-effectors. This will eventually impact design strategies for the creation of novel sensoreffector systems for enriching the optogenetic toolbox. [ABSTRACT FROM AUTHOR]

المؤلفون: Cajka, Tomas, Hricko, Jiri, Rakusanova, Stanislava, Brejchova, Kristyna, Novakova, Michaela, Rudl Kulhava, Lucie, Hola, Veronika, Paucova, Michaela, Kuda, Ondrej, Fiehn, Oliver

المصدر: International Journal of Molecular Sciences. 25(5)

مصطلحات موضوعية: hydrogen/deuterium exchange, liquid chromatography, mass spectrometry, metabolomics, unknown identification, Deuterium, Chromatography, Liquid, Metabolomics, Hydrogen Deuterium Exchange-Mass Spectrometry, Hydrophobic and Hydrophilic Interactions

الوصف: Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/51s9q33hTest

المؤلفون: Anderson, Jake W.¹, Vaisar, David¹, Jones, David N.², Pegram, Laurel M.¹, Vigers, Guy P.³, Huifen Chen⁴, Moffat, John G.⁴, Ahn, Natalie G.¹ natalie.ahn@colorado.edu

المصدر: eLife. 3/27/2024, p1-27. 27p.

مصطلحات موضوعية: *HYDROGEN-deuterium exchange, *MOLECULAR docking, *SIGNAL recognition particle receptor, *STACKING interactions, *DISPERSION (Chemistry), *G protein coupled receptors

مستخلص: Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named 'L' and 'R,' where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix aF segments surrounding the activation loop, as well as helix aL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix aC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors. [ABSTRACT FROM AUTHOR]

المؤلفون: Ruofan Chen¹, Glauninger, Hendrik², Kahan, Darren N.², Shangguan, Julia², Sachleben, Joseph R.³, Riback, Joshua A.^2,4,5, Drummond, D. Allan^2,6, Sosnick, Tobin R.^1,2,6 trsosnic@uchicago.edu

المصدر: Proceedings of the National Academy of Sciences of the United States of America. 3/26/2024, Vol. 121 Issue 13, p1-9. 26p.

مصطلحات موضوعية: *CONDENSATION, *HYDROGEN-deuterium exchange, *THERMAL stresses, *EUKARYOTIC cells, *FREE surfaces, *EXCHANGE

مستخلص: Eukaryotic cells form condensates to sense and adapt to their environment [S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen, Nat. Rev. Mol. Cell Biol. 18, 285-298 (2017), H. Yoo, C. Triandafillou, D. A. Drummond, J. Biol. Chem. 294, 7151-7159 (2019)]. Poly(A)-binding protein (Pab1), a canonical stress granule marker, condenses upon heat shock or starvation, promoting adaptation [J. A. Riback et al., Cell 168, 1028-1040. e19 (2017)]. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. We apply hydrogen-deuterium exchange/mass spectrometry to investigate the mechanism of Pab1's condensation. Pab1's four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe populations allows us to identify which regions contribute to the condensate's interaction network. Our data yield a picture of Pab1's stress-triggered condensation, which we term sequential activation (Fig. 1A), wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. Subsequent association is dictated more by the underlying free energy surface than specific interactions, an effect we refer to as thermodynamic specificity. Our study represents an advance for elucidating the interactions that drive condensation. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple stresses, a potentially general mechanism for stress-responsive proteins. [ABSTRACT FROM AUTHOR]

المؤلفون: Vankova, Pavla¹ (AUTHOR), Pacheco‐Garcia, Juan Luis² (AUTHOR), Loginov, Dmitry S.³ (AUTHOR), Gómez‐Mulas, Atanasio² (AUTHOR), Kádek, Alan³ (AUTHOR), Martín‐Garcia, José Manuel⁴ (AUTHOR), Salido, Eduardo⁵ (AUTHOR), Man, Petr³ (AUTHOR) pman@biomed.cas.cz, Pey, Angel L.⁶ (AUTHOR) angelpey@ugr.es

المصدر: FEBS Letters. Feb2024, Vol. 598 Issue 4, p485-499. 15p.

مصطلحات موضوعية: *STRUCTURAL dynamics, *HYDROGEN-deuterium exchange, *PATHOGENESIS, *PROTEIN stability

مستخلص: Primary hyperoxaluria type I (PH1) is caused by deficient alanine:glyoxylate aminotransferase (AGT) activity. PH1‐causing mutations in AGT lead to protein mistargeting and aggregation. Here, we use hydrogen‐deuterium exchange (HDX) to characterize the wild‐type (WT), the LM (a polymorphism frequent in PH1 patients) and the LM G170R (the most common mutation in PH1) variants of AGT. We provide the first experimental analysis of AGT structural dynamics, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants only show local destabilization. Enzymatic transamination of the pyridoxal 5‐phosphate cofactor bound to AGT hardly affects stability. Our study, thus, supports that AGT misfolding is not caused by dramatic effects on structural dynamics. [ABSTRACT FROM AUTHOR]

المؤلفون: Kant, Ravi¹ (AUTHOR) ravi.kant@wustl.edu, Mishra, Nawneet² (AUTHOR) nmishra@wustl.edu, Gross, Michael L.¹ (AUTHOR) mgross@wustl.edu

المصدر: International Journal of Molecular Sciences. Dec2023, Vol. 24 Issue 24, p17342. 10p.

مصطلحات موضوعية: *HYDROGEN-deuterium exchange, *MASS spectrometry, *ENERGY policy, *ANTIGENS, *HIGH energy forming, *DEUTERIUM

مستخلص: We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody–protein interactions by using hydrogen–deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody. [ABSTRACT FROM AUTHOR]

المؤلفون: Huang, Li-Kun¹ (AUTHOR), Huang, Yi-Cyuan² (AUTHOR), Chen, Pin-Chuan¹ (AUTHOR), Lee, Ching-Hung¹ (AUTHOR), Lin, Shih-Ming³ (AUTHOR), Hsu, Yuan-Hao Howard² (AUTHOR) howardhsu@thu.edu.tw, Pan, Rong-Long¹ (AUTHOR) rlpan@life.nthu.edu.tw

المصدر: Journal of Membrane Biology. Dec2023, Vol. 256 Issue 4-6, p443-458. 16p.

مصطلحات موضوعية: *HYDROGEN-deuterium exchange, *MASS spectrometry, *DEUTERIUM oxide, *DEUTERIUM, *HYDROGEN atom, *MUNG bean, *STRUCTURAL dynamics

مستخلص: Vigna radiata H+–translocating pyrophosphatases (VrH+-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They transport H+ into the lumen by hydrolyzing pyrophosphate, which is a by-product of many essential anabolic reactions. Although the crystal structure of H+-PPases has been elucidated, the H+ translocation mechanism of H+-PPases in the solution state remains unclear. In this study, we used hydrogen–deuterium exchange (HDX) coupled with mass spectrometry (MS) to investigate the dynamics of H+-PPases between the previously proposed R state (resting state, Apo form), I state (intermediate state, bound to a substrate analog), and T state (transient state, bound to inorganic phosphate). When hydrogen was replaced by proteins in deuterium oxide solution, the backbone hydrogen atoms, which were exchanged with deuterium, were identified through MS. Accordingly, we used deuterium uptake to examine the structural dynamics and conformational changes of H+-PPases in solution. In the highly conserved substrate binding and proton exit regions, HDX-MS revealed the existence of a compact conformation with deuterium exchange when H+-PPases were bound with a substrate analog and product. Thus, a novel working model was developed to elucidate the in situ catalytic mechanism of pyrophosphate hydrolysis and proton transport. In this model, a proton is released in the I state, and the TM5 inner wall serves as a proton piston. [ABSTRACT FROM AUTHOR]

المؤلفون: Lusiany, Tina¹ (AUTHOR) hendra@biken.osaka-u.ac.jp, Terada, Tohru² (AUTHOR) tterada@g.ecc.u-tokyo.ac.jp, Kishikawa, Jun-ichi³ (AUTHOR) kishijun@kit.ac.jp, Hirose, Mika³ (AUTHOR) mhirose@protein.osaka-u.ac.jp, Chen, David Virya^4,5 (AUTHOR) david@biken.osaka-u.ac.jp, Sugihara, Fuminori⁶ (AUTHOR) fsugihara@mail.biken.or.jp, Ismanto, Hendra Saputra¹ (AUTHOR) slli@biken.osaka-u.ac.jp, van Eerden, Floris J.⁴ (AUTHOR) fvaneerden@ifrec.osaka-u.ac.jp, Li, Songling^1,4 (AUTHOR), Kato, Takayuki³ (AUTHOR) tkato@protein.osaka-u.ac.jp, Arase, Hisashi^7,8 (AUTHOR) arase@biken.osaka-u.ac.jp, Yoshiharu, Matsuura⁵ (AUTHOR), Okada, Masato⁹ (AUTHOR) okadam@biken.osaka-u.ac.jp, Standley, Daron M.^1,4 (AUTHOR) standley@biken.osaka-u.ac.jp

المصدر: Viruses (1999-4915). Dec2023, Vol. 15 Issue 12, p2421. 12p.

مصطلحات موضوعية: *SARS-CoV-2, *HYDROGEN-deuterium exchange, *ANGIOTENSIN converting enzyme, *MOLECULAR dynamics, *PROTEINS, *IMMUNOGLOBULINS

مستخلص: The entry of SARS-CoV-2 into host cells is mediated by the interaction between the spike receptor-binding domain (RBD) and host angiotensin-converting enzyme 2 (ACE2). Certain human antibodies, which target the spike N-terminal domain (NTD) at a distant epitope from the host cell binding surface, have been found to augment ACE2 binding and enhance SARS-CoV-2 infection. Notably, these antibodies exert their effect independently of the antibody fragment crystallizable (Fc) region, distinguishing their mode of action from previously described antibody-dependent infection-enhancing (ADE) mechanisms. Building upon previous hypotheses and experimental evidence, we propose that these NTD-targeting infection-enhancing antibodies (NIEAs) achieve their effect through the crosslinking of neighboring spike proteins. In this study, we present refined structural models of NIEA fragment antigen-binding region (Fab)–NTD complexes, supported by molecular dynamics simulations and hydrogen–deuterium exchange mass spectrometry (HDX-MS). Furthermore, we provide direct evidence confirming the crosslinking of spike NTDs by NIEAs. Collectively, our findings advance our understanding of the molecular mechanisms underlying NIEAs and their impact on SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

المؤلفون: Zhang, Qian^1,2 (AUTHOR), Xia, Yaoyu^1,2 (AUTHOR), Song, Wei² (AUTHOR), Chen, Chunguang¹ (AUTHOR), Wang, Hao‐Yang² (AUTHOR) haoyangwang@sioc.ac.cn

المصدر: Rapid Communications in Mass Spectrometry: RCM. Sep2023 Supplement 1, Vol. 37, p1-13. 13p.

مصطلحات موضوعية: *ELECTROSPRAY ionization mass spectrometry, *HYDROGEN-deuterium exchange, *DEUTERIUM, *EXCHANGE reactions, *MASS spectrometry, *ORGANIC compounds, *ISOTOPIC analysis

مستخلص: Rationale: Deuterium‐labeled organic compounds, reagents, and drugs are widely used in many scientific research fields. Isotopic purity as the feature parameter of deuterated compounds is of great importance. In this article, we used electrospray ionization with high‐resolution mass spectrometry (ESI‐HRMS) to study isotopic purity of deuterium‐labeled organic compounds based on assigning and distinguishing the corresponding H/D (hydrogen–deuterium) isotopolog ions of deuterated compounds. Methods: We systematically considered the specific requirements of accuracy and resolution of ESI‐HRMS when measuring isotopic purity and demonstrated some actual cases using ESI‐HRMS and ultraperformance liquid chromatography (UPLC)‐HRMS. Meanwhile, ESI‐HRMS/MS of deuterated compounds was performed to obtain more information on deuterium‐labeled position characterization. Results: Two isotopic purity calculation methods based on the relative abundance in ESI‐HRMS and UPLC‐HRMS of H/D isotopolog ions (D0–Dn) were compared, which gave consistent isotopic purity values and were in good agreement with the certified isotopic purity values. We further studied and monitored the H/D exchange reaction of ethyl 3‐(4‐bromophenyl)‐3‐oxopropanoate (EBPO) by evaluating the dynamic isotopic purity changes in EBPO‐D2 in the H/D exchange reaction in situ. Conclusion: The isotopic purity characterization methods using ESI‐HRMS discussed in this article have some outstanding advantages: rapid, highly sensitive, very low sample consumption (even below nanogram level), and deuterated solvent‐free. Thus, this low‐impact analytical method requires less time and is cost effective and might have good application potential for in‐situ isotopic purity analysis and for monitoring the H/D exchange reactions. [ABSTRACT FROM AUTHOR]

المؤلفون: Singh, Sunil¹ (AUTHOR), Machida, Satoru¹ (AUTHOR), Tulsian, Nikhil Kumar^1,2 (AUTHOR), Choong, Yeu Khai¹ (AUTHOR), Ng, Joel¹ (AUTHOR), Shankar, Srihari¹ (AUTHOR), Liu, Yaochen¹ (AUTHOR), Chandiramani, Krisha Vashdev¹ (AUTHOR), Shi, Jian¹ (AUTHOR), Sivaraman, J¹ (AUTHOR) dbsjayar@nus.edu.sg

المصدر: Advanced Science. Sep2023, Vol. 10 Issue 27, p1-14. 14p.

مصطلحات موضوعية: *UBIQUITIN ligases, *HYDROGEN-deuterium exchange, *DIMERIZATION, *UBIQUITINATION

مستخلص: HACE1 is an ankyrin repeat (AKR) containing HECT‐type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well‐known tumor suppressor, its structure and mode of ubiquitination are not understood. The authors present the cryo‐EM structures of human HACE1 along with in vitro functional studies that provide insights into how the enzymatic activity of HACE1 is regulated. HACE1 comprises of an N‐terminal AKR domain, a middle (MID) domain, and a C‐terminal HECT domain. Its unique G‐shaped architecture interacts as a homodimer, with monomers arranged in an antiparallel manner. In this dimeric arrangement, HACE1 ubiquitination activity is hampered, as the N‐terminal helix of one monomer restricts access to the C‐terminal domain of the other. The in vitro ubiquitination assays, hydrogen‐deuterium exchange mass spectrometry (HDX–MS) analysis, mutagenesis, and in silico modeling suggest that the HACE1 MID domain plays a crucial role along with the AKRs in RAC1 substrate recognition. [ABSTRACT FROM AUTHOR]