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The dynamics of the backbone and side-chains of protein are routinely studied by interpreting experimentally determined 15 N spin relaxation rates. R 1 ( 15 N), the longitudinal relaxation rate, reports on fast motions and encodes, together with the transverse relaxation R 2 , structural information about the shape of the molecule and the orientation of the amide bond vectors in the internal diffusion frame. Determining error-free 15 N longitudinal relaxation rates remains a challenge for small, disordered, and medium-sized proteins. Here, we show that mono-exponential fitting is sufficient, with no statistical preference for bi-exponential fitting up to 800 MHz. A detailed comparison of the TROSY and HSQC techniques at medium and high fields showed no statistically significant differences. The least error-prone DD/CSA interference removal technique is the selective inversion of amide signals while avoiding water resonance. The exchange of amide with solvent deuterons appears to affect the rate R 1 of solvent-exposed amides in all fields tested and in each DD/CSA interference removal technique in a statistically significant manner. In summary, the most accurate R 1 ( 15 N) rates in proteins are achieved by selective amide inversion, without the addition of D 2 O. Importantly, at high magnetic fields stronger than 800 MHz, when non-mono-exponential decay is involved, it is advisable to consider elimination of the shortest delays (typically up to 0.32 s) or bi-exponential fitting., (© 2024. The Author(s).)

The unique photophysical properties of single-walled carbon nanotubes (SWCNTs) exhibit great potential for bioimaging applications. This led to extensive exploration of photosensitization methods to improve their faint shortwave infrared (SWIR) photoluminescence. Here, we report the mechanisms of SWCNT-assisted J-aggregation of cyanine dyes and the associated photoluminescence enhancement of SWCNTs in the SWIR spectral region. Surprisingly, we found that excitation energy transfer between the cyanine dyes and SWCNTs makes a negligible contribution to the overall photoluminescence enhancement. Instead, the shielding of SWCNTs from the surrounding water molecules through hydrogen bond-assisted macromolecular reorganization of ionic surfactants triggered by counterions and the physisorption of the dye molecules on the side walls of SWCNTs play a primary role in the photoluminescence enhancement of SWCNTs. We observed 2 orders of magnitude photoluminescence enhancement of SWCNTs by optimizing these factors. Our findings suggest that the proper shielding of SWCNTs is the critical factor for their photoluminescence enhancement, which has important implications for their application as imaging agents in biological settings., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

A series of 10 cyclic, biaryl analogs of enkephalin, with Tyr or Phe residues at positions 1 and 4, were synthesized according to the Miyaura borylation and Suzuki coupling methodology. Biaryl bridges formed by side chains of the two aromatic amino acid residues are of the meta-meta, meta-para, para-meta, and para-para configuration. Conformational properties of the peptides were studied by CD and NMR. CD studies allowed only to compare conformations of individual peptides while NMR investigations followed by XPLOR calculations provided detailed information on their conformation. Reliability of the XPLOR calculations was confirmed by quantum chemical ones performed for one of the analogs. No intramolecular hydrogen bonds were found in all the peptides. They are folded and adopt the type IV β-turn conformation. Due to a large steric strain, the aromatic carbon atoms forming the biaryl bond are distinctly pyramidalized. Seven of the peptides were tested in vitro for their affinity for the µ-opioid receptor., (© 2024. The Author(s).)

DNA damage is a serious threat to all living organisms and may be induced by environmental stressors. Previous studies have revealed that the tardigrade (Ramazzotius varieornatus) DNA damage suppressor protein Dsup has protective effects in human cells and tobacco. However, whether Dsup provides radiation damage protection more widely in crops is unclear. To explore the effects of Dsup in other crops, stable Dsup overexpression lines through Agrobacterium-mediated transformation were generated and their agronomic traits were deeply investigated. In this study, the overexpression of Dsup not only enhanced the DNA damage resistance at the seeds and seedlings stages, they also exhibited grain size enlargement and starch granule structure and cell size alteration by the scanning electron microscopy observation. Notably, the RNA-seq revealed that the Dsup plants increased radiation-related and abiotic stress-related gene expression in comparison to wild types, suggesting that Dsup is capable to coordinate normal growth and abiotic stress resistance in rice. Immunoprecipitation enrichment with liquid chromatography-tandem mass spectrometry (IP-LC-MS) assays uncovered 21 proteins preferably interacting with Dsup in plants, suggesting that Dsup binds to transcription and translation related proteins to regulate the homeostasis between DNA protection and plant development. In conclusion, our data provide a detailed agronomic analysis of Dsup plants and potential mechanisms of Dsup function in crops. Our findings provide novel insights for the breeding of crop radiation resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

ASH1L is a histone methyltransferase that regulates gene expression through methylation of histone H3 on lysine K36. While the catalytic SET domain of ASH1L has low intrinsic activity, several studies found that it can be vastly enhanced by the interaction with MRG15 protein and proposed allosteric mechanism of releasing its autoinhibited conformation. Here, we found that full-length MRG15, but not the MRG domain alone, can enhance the activity of the ASH1L SET domain. In addition, we showed that catalytic activity of MRG15-ASH1L depends on nucleosome binding mediated by MRG15 chromodomain. We found that in solution MRG15 binds to ASH1L, but has no impact on the conformation of the SET domain autoinhibitory loop or the S-adenosylmethionine cofactor binding site. Moreover, MRG15 binding did not impair the potency of small molecule inhibitors of ASH1L. These findings suggest that MRG15 functions as an adapter that enhances ASH1L catalytic activity by recruiting nucleosome substrate., Competing Interests: Declaration of interests T.C. and J.G. received prior research support from Kura Oncology Inc. for an unrelated project, served as consultants for Kura Oncology, and have equity ownership in the company. The remaining authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Metal ions present in cellular microenvironment have been implicated as drivers of aggregation of amyloid forming proteins. Zinc (Zn 2+ ) ions have been reported to directly interact with α-synuclein (AS), a causative agent of Parkinson's disease and other neurodegenerative diseases, and promote its aggregation. AS is a small intrinsically disordered protein (IDP) i.e., understanding molecular factors that drive its misfolding and aggregation has been challenging since methods used routinely to study protein structure are not effective for IDPs. Here, we report the atomic details of Zn 2+ binding to AS at physiologically relevant conditions using proton-less NMR techniques that can be applied to highly dynamic systems like IDPs. We also examined how human serum albumin (HSA), the most abundant protein in human blood, binds to AS and whether Zn 2+ and/or ionic strength affect this. We conclude that Zn 2+ enhances the anti-aggregation chaperoning role of HSA that relies on protecting the hydrophobic N-terminal and NAC regions of AS, rather than polar negatively charged C-terminus. This suggested a previously undocumented role of Zn 2+ in HSA function and AS aggregation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Efficient determination of protein ligandability, or the propensity to bind small-molecules, would greatly facilitate drug development for novel targets. Ligandability is currently assessed using computational methods that typically consider the static structural properties of putative binding sites or by experimental fragment screening. Here, we evaluate ligandability of conserved BTB domains from the cancer-relevant proteins LRF, KAISO, and MIZ1. Using fragment screening, we discover that MIZ1 binds multiple ligands. However, no ligands are uncovered for the structurally related KAISO or LRF. To understand the principles governing ligand-binding by BTB domains, we perform comprehensive NMR-based dynamics studies and find that only the MIZ1 BTB domain exhibits backbone µs-ms time scale motions. Interestingly, residues with elevated dynamics correspond to the binding site of fragment hits and recently defined HUWE1 interaction site. Our data argue that examining protein dynamics using NMR can contribute to identification of cryptic binding sites, and may support prediction of the ligandability of novel challenging targets., (© 2022. The Author(s).)

Chromatin marks are recognized by distinct binding modules, many of which are embedded in multidomain proteins. How the different functionalities of such complex chromatin modulators are regulated is often unclear. Here, we delineated the interplay of the H3 amino terminus- and K9me-binding activities of the multidomain hUHRF1 protein. We show that the phosphoinositide PI5P interacts simultaneously with two distant flexible linker regions connecting distinct domains of hUHRF1. The binding is dependent on both, the polar head group, and the acyl part of the phospholipid and induces a conformational rearrangement juxtaposing the H3 amino terminus and K9me3 recognition modules of the protein. In consequence, the two features of the H3 tail are bound in a multivalent, synergistic manner. Our work highlights a previously unidentified molecular function for PI5P outside of the context of lipid mono- or bilayers and establishes a molecular paradigm for the allosteric regulation of complex, multidomain chromatin modulators by small cellular molecules.

Multidomain kinases use many ways to integrate and process diverse stimuli. Here, we investigated the mechanism by which the protein tyrosine kinase 2-beta (PYK2) functions as a sensor and effector of cellular calcium influx. We show that the linker between the PYK2 kinase and FAT domains (KFL) encompasses an unusual calmodulin (CaM) binding element. PYK2 KFL is disordered and engages CaM through an ensemble of transient binding events. Calcium increases the association by promoting structural changes in CaM that expose auxiliary interaction opportunities. KFL also forms fuzzy dimers, and dimerization is enhanced by CaM binding. As a monomer, however, KFL associates with the PYK2 FERM-kinase fragment. Thus, we identify a mechanism whereby calcium influx can promote PYK2 self-association, and hence kinase-activating trans-autophosphorylation. Collectively, our findings describe a flexible protein module that expands the paradigms for CaM binding and self-association, and their use for controlling kinase activity., (© 2022. The Author(s).)

Human serum albumin (HSA) is the main zinc(II) carrier in blood plasma. The HSA site with the strongest affinity for zinc(II), multi-metal binding site A, is disrupted by the presence of fatty acids (FAs). Therefore, the FA concentration in the blood influences zinc distribution, which may affect both normal physiological processes and a range of diseases. Based on the current knowledge of HSA's structure and its coordination chemistry with zinc(II), we investigated zinc interactions and the effect of various FAs, including lipoic acid (LA), on the protein structure, stability, and zinc(II) binding. We combined NMR experiments and isothermal titration calorimetry to examine zinc(II) binding to HSA at a sub-atomic level in a quantitative manner as well as the effect of FAs. Free HSA results indicate the existence of one high-affinity zinc(II) binding site and multiple low-affinity sites. Upon the binding of FAs to HSA, we observed a range of behaviors in terms of zinc(II) affinity, depending on the type of FA. With FAs that disrupt zinc binding, the addition of LA restores HSA's affinity for zinc ions to the levels seen with free defatted HSA, indicating the possible mechanism of LA, which is effective in the treatment of diabetes and cardiovascular diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor JL declared a past co-authorship with the author LJ., (Copyright © 2022 Al-Harthi, Chandra and Jaremko.)

The etiological role of NSD2 enzymatic activity in solid tumors is unclear. Here we show that NSD2, via H3K36me2 catalysis, cooperates with oncogenic KRAS signaling to drive lung adenocarcinoma (LUAD) pathogenesis. In vivo expression of NSD2 E1099K , a hyperactive variant detected in individuals with LUAD, rapidly accelerates malignant tumor progression while decreasing survival in KRAS-driven LUAD mouse models. Pathologic H3K36me2 generation by NSD2 amplifies transcriptional output of KRAS and several complementary oncogenic gene expression programs. We establish a versatile in vivo CRISPRi-based system to test gene functions in LUAD and find that NSD2 loss strongly attenuates tumor progression. NSD2 knockdown also blocks neoplastic growth of PDXs (patient-dervived xenografts) from primary LUAD. Finally, a treatment regimen combining NSD2 depletion with MEK1/2 inhibition causes nearly complete regression of LUAD tumors. Our work identifies NSD2 as a bona fide LUAD therapeutic target and suggests a pivotal epigenetic role of the NSD2-H3K36me2 axis in sustaining oncogenic signaling., Competing Interests: Declaration of interests O.G. is a co-scientific founder, consultant, and stockholder of EpiCypher, Inc. and K36 Therapeutics, Inc. P.K.M. is a scientific co-founder, consultant, and stockholder of Amplified Medicines, Inc. and Ikena Oncology, Inc., (Copyright © 2021 Elsevier Inc. All rights reserved.)