TY  - JOUR
AB  - Imaging in the shortwave infrared (SWIR) region offers fast, high-resolution visualization of in vivo targets in a multiplexed manner. These methods require bright, bathochromically shifted fluorescent dyes with sufficient emission at SWIR wavelengths-ideally above 1500 nm for high-resolution deep tissue imaging. Polymethine dyes are a privileged class of contrast agents due to their excellent absorption and high degree of modularity. In this work, we push flavylium and chromenylium dyes further into the SWIR region through polymethine chain extension. This panel of nonamethine dyes boasts absorbances as red as 1149 nm and tail emission beyond 1500 nm. These dyes are the brightest organic fluorophores at their respective bandgaps to date, with εmax ∼ 105 M-1 cm-1 and ΦF up to 0.5%. Using two nonamethine dyes, Chrom9 and JuloFlav9, we performed two-color all-SWIR multiplexed imaging (Excitation at 1060 and 1150 nm; Emission collection at >1500 nm), enhancing the depths and resolutions able to be obtained in multicolor SWIR imaging with small molecule contrast agents. Finally, we combine the nonamenthine dyes with other SWIR-emissive fluorophores and demonstrate five-color awake imaging in an unrestrained mouse, simultaneously pushing the multiplexing, resolution, and speed limits of in vivo optical imaging.
AU  - Spearman, A.L.*
AU  - Lin, E.Y.*
AU  - Mobley, E.B.*
AU  - Chmyrov, A.
AU  - Arus, B.A.
AU  - Turner, D.W.*
AU  - Garcia, C.A.*
AU  - Bui, K.*
AU  - Rowlands, C.J.*
AU  - Bruns, O.T.
AU  - Sletten, E.M.*
C1  - 74417
C2  - 57437
CY  - 1155 16th St, Nw, Washington, Dc 20036 Usa
TI  - High-resolution multicolor shortwave infrared dynamic in vivo imaging with chromenylium nonamethine dyes.
JO  - J. Am. Chem. Soc.
PB  - Amer Chemical Soc
PY  - 2025
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.
AU  - Daniilidis, M.*
AU  - Sperl, L.E.*
AU  - Müller, B.S.*
AU  - Babl, A.*
AU  - Hagn, F.
C1  - 70752
C2  - 55730
CY  - 1155 16th St, Nw, Washington, Dc 20036 Usa
SP  - 15403-15410
TI  - Efficient segmental isotope labeling of integral membrane proteins for high-resolution NMR studies.
JO  - J. Am. Chem. Soc.
VL  - 146
IS  - 22
PB  - Amer Chemical Soc
PY  - 2024
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Deposition of amyloid plaques in the brains of Alzheimer's disease (AD) patients is a hallmark of the disease. AD plaques consist primarily of the beta-amyloid (Aβ) peptide but can contain other factors such as lipids, proteoglycans, and chaperones. So far, it is unclear how the cellular environment modulates fibril polymorphism and how differences in fibril structure affect cell viability. The small heat-shock protein (sHSP) alpha-B-Crystallin (αBC) is abundant in brains of AD patients, and colocalizes with Aβ amyloid plaques. Using solid-state NMR spectroscopy, we show that the Aβ40 fibril seed structure is not replicated in the presence of the sHSP. αBC prevents the generation of a compact fibril structure and leads to the formation of a new polymorph with a dynamic N-terminus. We find that the N-terminal fuzzy coat and the stability of the C-terminal residues in the Aβ40 fibril core affect the chemical and thermodynamic stability of the fibrils and influence their seeding capacity. We believe that our results yield a better understanding of how sHSP, such as αBC, that are part of the cellular environment, can affect fibril structures related to cell degeneration in amyloid diseases.
AU  - Rodina, N.
AU  - Hornung, S.*
AU  - Sarkar, R.
AU  - Suladze, S.*
AU  - Peters, C.*
AU  - Schmid, P.W.N.*
AU  - Niu, Z.*
AU  - Haslbeck, M.*
AU  - Buchner, J.*
AU  - Kapurniotu, A.*
AU  - Reif, B.
C1  - 71063
C2  - 55922
CY  - 1155 16th St, Nw, Washington, Dc 20036 Usa
SP  - 19077–19087
TI  - Modulation of Alzheimer's disease Aβ40 fibril polymorphism by the small heat shock protein αB-crystallin.
JO  - J. Am. Chem. Soc.
VL  - 146
IS  - 28
PB  - Amer Chemical Soc
PY  - 2024
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - The deposition of islet amyloid polypeptide (hIAPP) fibrils is a hallmark of β-cell death in type II diabetes. In this study, we employ state-of-the-art MAS solid-state spectroscopy to investigate the previously elusive N-terminal region of hIAPP fibrils, uncovering both rigidity and heterogeneity. Comparative analysis between wild-type hIAPP and a disulfide-deficient variant (hIAPPC2S,C7S) unveils shared fibril core structures yet strikingly distinct dynamics in the N-terminus. Specifically, the variant fibrils exhibit extended β-strand conformations, facilitating surface nucleation. Moreover, our findings illuminate the pivotal roles of specific residues in modulating secondary nucleation rates. These results deepen our understanding of hIAPP fibril assembly and provide critical insights into the molecular mechanisms underpinning type II diabetes, holding promise for future therapeutic strategies.
AU  - Suladze, S.
AU  - Sustay Martinez, C.*
AU  - Rodriguez Camargo, D.C.
AU  - Engler, J.
AU  - Rodina, N.
AU  - Sarkar, R.
AU  - Zacharias, M.*
AU  - Reif, B.
C1  - 70657
C2  - 55699
CY  - 1155 16th St, Nw, Washington, Dc 20036 Usa
SP  - 13783-13796
TI  - Structural insights into seeding mechanisms of hIAPP fibril formation.
JO  - J. Am. Chem. Soc.
VL  - 146
IS  - 20
PB  - Amer Chemical Soc
PY  - 2024
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.
AU  - Koller, T.O.*
AU  - Scheid, U.*
AU  - Kösel, T.*
AU  - Herrmann, J.*
AU  - Krug, D.*
AU  - Boshoff, H.I.M.*
AU  - Beckert, B.*
AU  - Evans, J.C.*
AU  - Schlemmer, J.*
AU  - Sloan, B.*
AU  - Weiner, D.M.*
AU  - Via, L.E.*
AU  - Moosa, A.*
AU  - Ioerger, T.R.*
AU  - Gräf, M.*
AU  - Zinshteyn, B.*
AU  - Abdelshahid, M.*
AU  - Nguyen, F.*
AU  - Arenz, S.*
AU  - Gille, F.*
AU  - Siebke, M.
AU  - Seedorf, T.*
AU  - Plettenburg, O.
AU  - Green, R.*
AU  - Warnke, A.-L.
AU  - Ullrich, J.*
AU  - Warrass, R.*
AU  - Barry, C.E.*
AU  - Warner, D.F.*
AU  - Mizrahi, V.*
AU  - Kirschning, A.*
AU  - Wilson, D.N.*
AU  - Müller, R.*
C1  - 67185
C2  - 54235
CY  - 1155 16th St, Nw, Washington, Dc 20036 Usa
SP  - 851-863
TI  - The
       myxobacterial antibiotic myxovalargin: Biosynthesis, structural 
      revision, total synthesis and molecular characterization of ribosomal 
      inhibition.
JO  - J. Am. Chem. Soc.
VL  - 145
IS  - 2
PB  - Amer Chemical Soc
PY  - 2023
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Recently, proton-detected magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy has become an attractive tool to study the structure and dynamics of insoluble proteins at atomic resolution. The sensitivity of the employed multidimensional experiments can be systematically improved when both transversal components of the magnetization are transferred simultaneously after an evolution period. The method of preservation of equivalent pathways has been explored in solution-state NMR; however, it does not find widespread application due to relaxation issues connected with increased molecular size. We present here for the first time heteronuclear transverse mixing sequences for correlation experiments at moderate and fast MAS frequencies. Optimal control allows to boost the signal-to-noise ratio (SNR) beyond the expected factor of 2 for each indirect dimension. In addition to the carbon-detected sensitivity-enhanced 2D NCA experiment, we present a novel proton-detected, doubly sensitivity-enhanced 3D hCANH pulse sequence for which we observe a 3-fold improvement in SNR compared to the conventional experimental implementation. The sensitivity gain turned out to be essential to unambiguously characterize a minor fibril polymorph of a human lambda-III immunoglobulin light chain protein that escaped detection so far.
AU  - Blahut, J.*
AU  - Brandl, M.J.*
AU  - Pradhan, T.*
AU  - Reif, B.
AU  - Tošner, Z.*
C1  - 66162
C2  - 53100
SP  - 17336-17340
TI  - Sensitivity-enhanced multidimensional solid-state NMR spectroscopy by optimal-control-based transverse mixing sequences.
JO  - J. Am. Chem. Soc.
VL  - 144
IS  - 38
PY  - 2022
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - The archetype inhibitors of ferroptosis, ferrostatin-1 and liproxstatin-1, were identified via high-throughput screening of compound libraries for cytoprotective activity. These compounds have been shown to inhibit ferroptosis by suppressing propagation of lipid peroxidation, the radical chain reaction that drives cell death. Herein, we present the first rational design and optimization of ferroptosis inhibitors targeting this mechanism of action. Engaging the most potent radical-trapping antioxidant (RTA) scaffold known (phenoxazine, PNX), and its less reactive chalcogen cousin (phenothiazine, PTZ), we explored structure-reactivity-potency relationships to elucidate the intrinsic and extrinsic limitations of this approach. The results delineate the roles of inherent RTA activity, H-bonding interactions with phospholipid headgroups, and lipid solubility in determining activity/potency. We show that modifications which increase inherent RTA activity beyond that of the parent compounds do not substantially improve RTA kinetics in phospholipids or potency in cells, while modifications that decrease intrinsic RTA activity lead to corresponding erosions to both. The apparent "plateau"of RTA activity in phospholipid bilayers (kinh ∼2 × 105 M-1 s-1) and cell potency (EC50 ∼4 nM) may be the result of diffusion-controlled reactivity between the RTA and lipid-peroxyl radicals and/or the potential limitations on RTA turnover/regeneration by endogenous reductants. The metabolic stability of selected derivatives was assessed to identify a candidate for in vivo experimentation as a proof-of-concept. This PNX-derivative demonstrated stability in mouse liver microsomes comparable to liproxstatin-1 and was successfully used to suppress acute renal failure in mice brought on by tissue-specific inactivation of the ferroptosis regulator GPX4.
AU  - Farmer, L.A.*
AU  - Wu, Z.*
AU  - Poon, J.*
AU  - Zilka, O.*
AU  - Lorenz, S.
AU  - Hühn, S.
AU  - Proneth, B.
AU  - Conrad, M.
AU  - Pratt, D.A.*
C1  - 65911
C2  - 52559
SP  - 14706-14721
TI  - Intrinsic and extrinsic limitations to the design and optimization of inhibitors of lipid peroxidation and associated cell death.
JO  - J. Am. Chem. Soc.
VL  - 144
IS  - 32
PY  - 2022
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - The hepatitis B virus (HBV) is the leading cause of persistent liver infections. Its DNA-based genome is synthesized through reverse transcription of an RNA template inside the assembled capsid shell. In addition to the structured assembly domain, the capsid protein harbors a C-terminal extension that mediates both the enclosure of RNA during capsid assembly and the nuclear entry of the capsid during infection. The arginine-rich motifs within this extension, though common to many viruses, have largely escaped atomic-scale investigation. Here, we leverage solution and solid-state nuclear magnetic resonance spectroscopy at ambient and cryogenic temperatures, under dynamic nuclear polarization signal enhancement, to investigate the organization of the genome within the capsid. Transient interactions with phosphate groups of the RNA backbone confine the arginine-rich motifs to the interior capsid space. While no secondary structure is induced in the C-terminal extension, interactions with RNA counteract the formation of a disulfide bond, which covalently tethers this peptide arm onto the inner capsid surface. Electrostatic and covalent contributions thus compete in the spatial regulation of capsid architecture. This disulfide switch represents a coupling mechanism between the structured assembly domain of the capsid and the enclosed nucleic acids. In particular, it enables the redox-dependent regulation of the exposure of the C-terminal extension on the capsid surface, which is required for nuclear uptake of the capsid. Phylogenetic analysis of capsid proteins from hepadnaviruses points toward a function of this switch in the persistence of HBV infections.
AU  - Harati Taji, Z.
AU  - Bielytskyi, P.
AU  - Shein, M.
AU  - Sani, M.A.*
AU  - Seitz, S.*
AU  - Schütz, A.K.
C1  - 65023
C2  - 52140
SP  - 8536-8550
TI  - Transient RNA interactions leave a covalent imprint on a viral capsid protein.
JO  - J. Am. Chem. Soc.
VL  - 144
IS  - 19
PY  - 2022
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Optical imaging within the shortwave infrared (SWIR, 1000-2000 nm) region of the electromagnetic spectrum has enabled high-resolution and high-contrast imaging in mice, non-invasively. Polymethine dyes, with their narrow absorption spectra and high absorption coefficients, are optimal probes for fast and multiplexed SWIR imaging. Here, we expand upon the multiplexing capabilities in SWIR imaging by obtaining brighter polymethine dyes with varied excitation wavelengths spaced throughout the near-infrared (700-1000 nm) region. Building on the flavylium polymethine dye scaffold, we explored derivatives with functional group substitution at the 2-position, deemed chromenylium polymethine dyes. The reported dyes have reduced nonradiative rates and enhanced emissive properties, enabling non-invasive imaging in mice in a single color at 300 fps and in three colors at 100 fps. Combined with polymethine dyes containing a red-shifted julolidine flavylium heterocycle and indocyanine green, distinct channels with well-separated excitation wavelengths provide non-invasive video-rate in vivo imaging in four colors.
AU  - Cosco, E.
AU  - Arus, B.A.
AU  - Spearman, A.L.*
AU  - Atallah, T.L.*
AU  - Lim, I.*
AU  - Leland, O.S.*
AU  - Caram, J.R.*
AU  - Bischof, T.S.
AU  - Bruns, O.T.
AU  - Sletten, E.M.*
C1  - 61951
C2  - 50528
CY  - 1155 16th St, Nw, Washington, Dc 20036 Usa
SP  - 6836-6846
TI  - Bright chromenylium polymethine dyes enable fast, four-color in vivo imaging with shortwave infrared detection.
JO  - J. Am. Chem. Soc.
VL  - 143
IS  - 18
PB  - Amer Chemical Soc
PY  - 2021
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - The human ATPase p97, also known as valosin containing protein or Cdc48, is a highly abundant AAA+ engine that fuels diverse energy-consuming processes in the human cell. p97 represents a potential target for cancer therapy and its malfunction causes a degenerative disease. Here, we monitor the enzymatic activity of p97 in real time via an NMR-based approach that allows us to follow the steps that couple ATP turnover to mechanical work. Our data identify a transient reaction intermediate, the elusive ADP.P-i nucleotide state, which has been postulated for many ATPases but has so far escaped direct detection. In p97, this species is crucial for the regulation of adenosine triphosphate turnover in the first nucleotide-binding domain. We further demonstrate how the enzymatic cycle is detuned by disease-associated gain-of-function mutations. The high-resolution insight obtained into conformational transitions in both protein and nucleotide bridges the gap between static enzyme structures and the dynamics of substrate conversion. Our approach relies on the close integration of solution- and solid-state NMR methods and is generally applicable to shed light on the mechanochemical operating modes of large molecular engines.
AU  - Rydzek, S.*
AU  - Shein, M.*
AU  - Bielytskyi, P.*
AU  - Schütz, A.K.
C1  - 59905
C2  - 49108
CY  - 1155 16th St, Nw, Washington, Dc 20036 Usa
SP  - 14472-14480
TI  - Observation of a transient reaction intermediate illuminates the mechanochemical cycle of the AAA-ATPase p97.
JO  - J. Am. Chem. Soc.
VL  - 142
IS  - 34
PB  - Amer Chemical Soc
PY  - 2020
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Tissue is translucent to shortwave infrared (SWIR) light, rendering optical imaging superior in this region. However, the widespread use of optical SWIR imaging has been limited, in part, by the lack of bright, biocompatible contrast agents that absorb and emit light above 1000 nm. J-Aggregation offers a means to transform stable, near-infrared (NJR) fluorophores into red-shifted SWIR contrast agents. Here we demonstrate that J-aggregates of NIR fluorophore IR-140 can be prepared inside hollow mesoporous silica nanoparticles (HMSNs) to result in nanomaterials that absorb and emit SWIR light. The J-aggregates inside PEGylated HMSNs are stable for multiple weeks in buffer and enable high resolution imaging in vivo with 980 nm excitation.
AU  - Chen, W.*
AU  - Cheng, C.A.*
AU  - Cosco, E.
AU  - Ramakrishnan, A.
AU  - Lingg, J.G.P.*
AU  - Bruns, O.T.
AU  - Zink, J.I.*
AU  - Sletten, E.M.*
C1  - 56708
C2  - 47200
CY  - 1155 16th St, Nw, Washington, Dc 20036 Usa
SP  - 12475-12480
TI  - Shortwave infrared imaging with J-aggregates stabilized in hollow mesoporous silica nanoparticles.
JO  - J. Am. Chem. Soc.
VL  - 141
IS  - 32
PB  - Amer Chemical Soc
PY  - 2019
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - sn2-15-Hydroperoxy-eicasotetraenoyl-phosphatidylethanolamines (sn2-15-HpETE-PE) generated by mammalian 15-lipoxygenase/phosphatidylethanolamine binding protein-1 (15-LO/PEBP1) complex is a death signal in a recently identified type of programmed cell demise, ferroptosis. How the enzymatic complex selects sn2-ETE-PE as the substrate among 1 of similar to 100 total oxidizable membrane PUFA phospholipids is a central, yet unresolved question. To unearth the highly selective and specific mechanisms of catalytic competence, we used a combination of redox lipidomics, mutational and computational structural analysis to show they stem from (i) reactivity toward readily accessible hexagonally organized membrane sn2-ETE-PEs, (ii) relative preponderance of sn2-ETE-PE species vs other sn2-ETE-PLs, and (iii) allosteric modification of the enzyme in the complex with PEBP1. This emphasizes the role of enzymatic vs random stochastic free radical reactions in ferroptotic death signaling.
AU  - Amoscato, A.A.*
AU  - Mikulska-Ruminska, K.*
AU  - Rosenbaum, J.C.*
AU  - Mao, G.*
AU  - Zhao, J.*
AU  - Conrad, M.
AU  - Kellum, J.A.*
AU  - Wenzel, S.E.*
AU  - VanDemark, A.P.*
AU  - Bahar, I.*
AU  - Kagan, V.E.*
AU  - Baylr, H.*
C1  - 55112
C2  - 46085
CY  - 1155 16th St, Nw, Washington, Dc 20036 Usa
SP  - 17835-17839
TI  - Empowerment of 15-lipoxygenase catalytic competence in selective oxidation of membrane ETE-PE to ferroptotic death signals, HpETE-PE.
JO  - J. Am. Chem. Soc.
VL  - 140
IS  - 51
PB  - Amer Chemical Soc
PY  - 2018
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Ferroptosis is a regulated form of necrotic cell death implicated in carcinogenesis and neurodegeneration that is driven by phospholipid peroxidation. Lipid-derived electrophiles (LDEs) generated during this process can covalently modify proteins ("carbonylation") and affect their functions. Here we report the development of a quantitative chemoproteomic method to profile carbonylations in ferroptosis by an aniline-derived probe. Using the method, we established a global portrait of protein carbonylations in ferroptosis with >400 endogenously modified proteins and for the first time, identified >20 residue sites with endogenous LDE modifications in ferroptotic cells. Specifically, we discovered and validated a novel cysteine site of modification on voltage-dependent anion-selective channel protein 2 (VDAC2) that might play an important role in sensitizing LDE signals and mediating ferroptosis. Our results will contribute to the understanding of ferroptotic signaling and pathogenesis and provide potential biomarkers for ferroptosis detection.
AU  - Chen, Y.*
AU  - Liu, Y.*
AU  - Lan, T.H.*
AU  - Qin, W.*
AU  - Zhu, Y.*
AU  - Qin, K.*
AU  - Gao, J.*
AU  - Wang, H.*
AU  - Hou, X.*
AU  - Chen, N.*
AU  - Friedmann Angeli, J.P.F.
AU  - Conrad, M.
AU  - Wang, C.*
C1  - 53296
C2  - 44575
SP  - 4712-4720
TI  - Quantitative profiling of protein carbonylations in ferroptosis by an aniline-derived probe.
JO  - J. Am. Chem. Soc.
VL  - 140
IS  - 13
PY  - 2018
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - We introduce a selective and cell-permeable calcium sensor for photoacoustics (CaSPA), a versatile imaging technique that allows for fast volumetric mapping of photoabsorbing molecules with deep tissue penetration. To optimize for Ca2+-dependent photoacoustic signal changes, we synthesized a selective metallochromic sensor with high extinction coefficient, low quantum yield, and high photobleaching resistance. Micromolar concentrations of Ca2+ lead to a robust blueshift of the absorbance of CaSPA, which translated into an accompanying decrease of the peak photoacoustic signal. The acetoxymethyl esterified sensor variant was readily taken up by cells without toxic effects and thus allowed us for the first time to perform live imaging of Ca2+ fluxes in genetically unmodified cells and heart organoids as well as in zebrafish larval brain via combined fluorescence and photoacoustic imaging.
AU  - Roberts, S.
AU  - Seeger, M.
AU  - Jiang, Y.
AU  - Mishra, A.
AU  - Sigmund, F.
AU  - Stelzl, A.
AU  - Lauri, A.
AU  - Symvoulidis, P.
AU  - Rolbieski, H.*
AU  - Preller, M.*
AU  - Dean-Ben, X.L.
AU  - Razansky, D.
AU  - Orschmann, T.*
AU  - Desbordes, S.C.
AU  - Vetschera, P.*
AU  - Bach, T.*
AU  - Ntziachristos, V.
AU  - Westmeyer, G.G.
C1  - 52065
C2  - 43681
CY  - Washington
SP  - 2718-2721
TI  - Calcium sensor for photoacoustic imaging.
JO  - J. Am. Chem. Soc.
VL  - 140
IS  - 8
PB  - Amer Chemical Soc
PY  - 2018
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - The orchestrated recognition of phosphoinositides and concomitant intracellular release of Ca2+ is pivotal to almost every aspect of cellular processes, including membrane homeostasis, cell division and growth, vesicle trafficking, as well as secretion. Although Ca2+ is known to directly impact phosphoinositide clustering, little is known about the molecular basis for this or its significance in cellular signaling. Here, we study the direct interaction of Ca2+ with phosphatidylinositol sphosphate (PI(4,5)P-2), the main lipid marker of the plasma membrane. Electrokinetic potential measurements of PI(4,5)P-2 containing liposomes reveal that Ca2+ as well as Mg2+ reduce the zeta potential of liposomes to nearly background levels of pure phosphatidylcholine membranes. Strikingly, lipid recognition by the default PI(4,5)P-2 lipid sensor, phospholipase C delta 1 pleckstrin homology domain (PLC delta 1-PH), is completely inhibited in the presence of Ca2+, while Mg2+ has no effect with 100 nm liposomes and modest effect with giant unilamellar vesicles. Consistent with biochemical data, vibrational sum frequency spectroscopy and atomistic molecular dynamics simulations reveal how Ca2+ binding to the PI(4,5)P-2 headgroup and carbonyl regions leads to confined lipid headgroup tilting and conformational rearrangements. We rationalize these findings by the ability of calcium to block a highly specific interaction between PLC delta 1-PH and PI(4,5)P-2, encoded within the conformational properties of the lipid itself. Our studies demonstrate the possibility that switchable phosphoinositide conformational states can serve as lipid recognition and controlled cell signaling mechanisms.
AU  - Bilkova, E.
AU  - Pleskot, R.*
AU  - Rissanen, S.*
AU  - Sun, S.*
AU  - Czogalla, A.
AU  - Cwiklik, L.*
AU  - Róg, T.*
AU  - Vattulainen, I.*
AU  - Cremer, P.S.*
AU  - Jungwirth, P.*
AU  - Coskun, Ü.
C1  - 50864
C2  - 42762
CY  - Washington
SP  - 4019-4024
TI  - Calcium directly regulates phosphatidylinositol 4,5-Bbsphosphate headgroup conformation and recognition.
JO  - J. Am. Chem. Soc.
VL  - 139
IS  - 11
PB  - Amer Chemical Soc
PY  - 2017
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Solid-state NMR is becoming a viable alternative for obtaining information about structures and dynamics of large biomolecular complexes, including ones that are not accessible to other high-resolution biophysical techniques. In this context, methods for probing protein-protein interfaces at atomic resolution are highly desirable. Solvent paramagnetic relaxation enhancements (sPREs) proved to be a powerful method for probing protein-protein interfaces in large complexes in solution but have not been employed toward this goal in the solid state. We demonstrate that 1 H and 15 N relaxation-based sPREs provide a powerful tool for characterizing intermolecular interactions in large assemblies in the solid state. We present approaches for measuring sPREs in practically the entire range of magic angle spinning frequencies used for biomolecular studies and discuss their benefits and limitations. We validate the approach on crystalline GB1, with our experimental results in good agreement with theoretical predictions. Finally, we use sPREs to characterize protein-protein interfaces in the GB1 complex with immunoglobulin G (IgG). Our results suggest the potential existence of an additional binding site and provide new insights into GB1:IgG complex structure that amend and revise the current model available from studies with IgG fragments. We demonstrate sPREs as a practical, widely applicable, robust, and very sensitive technique for determining intermolecular interaction interfaces in large biomolecular complexes in the solid state.
AU  - Öster, C.*
AU  - Kosol, S.*
AU  - Hartlmüller, C.
AU  - Lamley, J.M.*
AU  - Iuga, D.*
AU  - Oss, A.*
AU  - Org, M.L.*
AU  - Vanatalu, K.*
AU  - Samoson, A.*
AU  - Madl, T.
AU  - Lewandowski, J.R.*
C1  - 51911
C2  - 43596
CY  - Washington
SP  - 12165-12174
TI  - Characterization of protein-protein interfaces in large complexes by solid-state NMR solvent paramagnetic relaxation enhancements.
JO  - J. Am. Chem. Soc.
VL  - 139
IS  - 35
PB  - Amer Chemical Soc
PY  - 2017
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Integrated experimental approaches play an increasingly important role in structural biology, taking advantage of the complementary information provided by different techniques. In particular, the combination of NMR data with X-ray diffraction patterns may provide accurate and precise information about local conformations not available from average-resolution X-ray structures alone. Here, we refined the structure of a ternary protein-protein-RNA complex comprising three domains, Sxl and Unr, bound to a single-stranded region derived in the msl2 mRNA. The joint X-ray and NMR refinement reveals that - despite the poor quality of the fit found for the original structural model - the NMR data can be largely accommodated within the uncertainty in the atom positioning (structural noise) from the primary X-ray data and that the overall domain arrangements and binding interfaces are preserved on passing from the crystalline state to the solution. The refinement highlights local conformational differences, which provide additional information on specific features of the structure. For example, conformational dynamics and heterogeneity observed at the interface between the CSD1 and the Sxl protein components in the ternary complex are revealed by the combination of NMR and crystallographic data. The joint refinement protocol offers unique opportunities to detect structural differences arising from various experimental conditions and reveals static or dynamic differences in the conformation of the biomolecule between the solution and the crystals.
AU  - Carlon, A.*
AU  - Ravera, E.*
AU  - Hennig, J.
AU  - Parigi, G.*
AU  - Sattler, M.
AU  - Luchinat, C.*
C1  - 47963
C2  - 39825
CY  - Washington
SP  - 1601-1610
TI  - Improved accuracy from joint X-ray and NMR refinement of a protein-RNA complex structure.
JO  - J. Am. Chem. Soc.
VL  - 138
IS  - 5
PB  - Amer Chemical Soc
PY  - 2016
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - We introduce a labeling scheme for magic angle spinning (MAS) solid-state NMR that is based on deuteration in combination with dilution of the carbon spin system. The labeling strategy achieves spectral editing by simplification of the HinnodataalphaCinnodataalpha and aliphatic side chain spectral region. A reduction in both proton and carbon spin density in combination with fast spinning (≥50 kHz) is essential to retrieve artifact-free 13C-R1 relaxation data for aliphatic carbons. We obtain good agreement between the NMR experimental data and order parameters extracted from a molecular dynamics (MD) trajectory, which indicates that carbon based relaxation parameters can yield complementary information on protein backbone as well as side chain dynamics. (Graph Presented).
AU  - Asami, S.*
AU  - Porter, J.R.*
AU  - Lange, O.F.*
AU  - Reif, B.
C1  - 43225
C2  - 36329
CY  - Washington
SP  - 1094-1100
TI  - Access to Cα backbone dynamics of biological solids by 13C T1 relaxation and molecular dynamics simulation.
JO  - J. Am. Chem. Soc.
VL  - 137
IS  - 3
PB  - Amer Chemical Soc
PY  - 2015
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Multidomain proteins containing intrinsically disordered linkers exhibit large-scale dynamic modes that play key roles in a multitude of molecular recognition and signaling processes. Here, we determine the conformational space sampled by the multidomain splicing factor U2AF65 using complementary nuclear magnetic resonance spectroscopy and small-angle scattering data. Available degrees of conformational freedom are initially stochastically sampled and experimental data then used to delineate the potential energy landscape in terms of statistical probability. The spatial distribution of U2AF65 conformations is found to be highly anisotropic, comprising significantly populated interdomain contacts that appear to be electrostatic in origin. This hypothesis is supported by the reduction of signature PREs reporting on expected interfaces with increasing salt concentration. The described spatial distribution reveals the complete spectrum of the unbound forms of U2AF65 that coexist with the small percentage of a preformed RNA-bound domain arrangement required for polypyrimidine-tract recognition by conformational selection. More generally, the proposed approach to describing conformational equilibria of multidomain proteins can be further combined with other experimental data that are sensitive to domain dynamics.
AU  - Huang, J.*
AU  - Warner, L.R.
AU  - Sanchez, C.
AU  - Gabel, F.*
AU  - Madl, T.
AU  - Mackereth, C.D.
AU  - Sattler, M.
AU  - Blackledge, M.J.*
C1  - 31590
C2  - 34574
CY  - Washington
SP  - 7068-7076
TI  - Transient electrostatic interactions dominate the conformational equilibrium sampled by multidomain splicing factor U2AF65: A combined NMR and SAXS study.
JO  - J. Am. Chem. Soc.
VL  - 136
IS  - 19
PB  - Amer Chemical Soc
PY  - 2014
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - In vitro protein-folding studies using chemical denaturants such as urea are indispensible in elucidating the forces and mechanisms determining the stability, structure, and dynamics of water-soluble proteins. By contrast, a-helical membrane-associated proteins largely evade such approaches because they are resilient to extensive unfolding We have used of the alpha-helical membrane-associated protein Mistic as well as dissection of the effects of urea on the structure and dynamics optical and NMR spectroscopy to provide an atomistic-level its interactions with detergent and solvent molecules. In the presence of the zwitterionic detergent lauryl dimethylamine oxide, increasing concentrations of urea result in a complex sequence of conformational changes that go beyond simple two-state unfolding. Exploiting this finding, we report the first high-resolution structural models of the urea denaturation process of an alpha-helical membrane-associated protein and its completely unfolded state, which contains almost no regular secondary structure but nevertheless retains a topology close to that of the folded state.
AU  - Jacso, T.
AU  - Bardiaux, B.*
AU  - Broecker, J.*
AU  - Fiedler, S.*
AU  - Baerwinkel, T.
AU  - Mainz, A.*
AU  - Fink, U.*
AU  - Vargas, C.*
AU  - Oschkinat, H.*
AU  - Keller, S.*
AU  - Reif, B.
C1  - 29135
C2  - 32897
SP  - 18884-18891
TI  - The mechanism of denaturation and the unfolded state of the α-helical membrane-associated protein Mistic.
JO  - J. Am. Chem. Soc.
VL  - 135
IS  - 50
PB  - Amer. Chemical Soc.
PY  - 2013
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - The design of liposome-nanoparticle hybrids offers a rich toolbox for the fabrication of multifunctional modalities. A self-assembled liposome-gold nanorod hybrid vesicular system that consists of lipid-bilayer-associated gold nanorods designed to allow deep tissue detection, therapy, and monitoring in living animals using multispectral optoacoustic tomography has been fabricated and characterized in vitro and in vivo.
AU  - Lozano, N.*
AU  - Al-Jamal, W.T.*
AU  - Taruttis, A.
AU  - Bézière, N.
AU  - Burton, N.C.
AU  - Van den Bossche, J.*
AU  - Mazza, M.*
AU  - Herzog, E.
AU  - Ntziachristos, V.
AU  - Kostarelos, K.*
C1  - 8563
C2  - 30180
SP  - 13256-13258
TI  - Liposome-gold nanorod hybrids for high-resolution visualization deep in tissues.
JO  - J. Am. Chem. Soc.
VL  - 134
IS  - 32
PB  - Amer Chemical Soc.
PY  - 2012
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - The 41-amino acid peptide corticotropin releasing factor (CRF) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the corticotropin releasing factor receptor 1 (CRF(1)R), a typical member of the class-B G-protein-coupled receptors (GPCRs) and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRF with the transmembrane domain of its cognate receptor, we developed a high-throughput conjugation approach that mimics the natural activation mechanism of class-B GPCRs. An acetylene-tagged peptide library was synthesized and conjugated to an azide-modified high-affinity carrier peptide derived from the CRF C-terminus using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstituted potent agonists and were tested in situ for activation of the CRF(1) receptor in a cell-based assay. By use of this approach we (i) defined the minimal sequence motif that is required for full receptor activation, (ii) identified the critical functional groups and structure-activity relationships, (iii) developed an optimized, highly modified peptide probe with high potency (EC(50) = 4 nM) that is specific for the activation domain of the receptor, and (iv) probed the behavioral role of CRF receptors in living mice. The membrane recruitment by a high-affinity carrier enhanced the potency of the tethered peptides by >4 orders of magnitude and thus allowed the testing of very weak initial fragments that otherwise would have been inactive on their own. As no chromatography purification of the test peptides was necessary, a substantial increase in screening throughput was achieved. Importantly, the peptide conjugates can be used to probe the endogenous receptor in its native environment in vivo.
AU  - Devigny, C.*
AU  - Perez-Balderas, F.*
AU  - Hoogeland, B.*
AU  - Cuboni, S.*
AU  - Wachtel, R.*
AU  - Mauch, C.P.*
AU  - Webb, K.J.
AU  - Deussing, J.M.
AU  - Hausch, F.*
C1  - 6510
C2  - 28836
CY  - Washington, DC
SP  - 8927-2933
TI  - Biomimetic screening of class-B G protein-coupled receptors.
JO  - J. Am. Chem. Soc.
VL  - 133
IS  - 23
PB  - American Chemical Society
PY  - 2011
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Magic-angle spinning (MAS) solid-state NMR becomes an increasingly important tool for the determination of structures of membrane proteins and amyloid fibrils. Extensive deuteration of the protein allows multidimensional experiments with exceptionally high sensitivity and resolution to be obtained. Here we present an experimental strategy to measure highly unambiguous spatial correlations for distances up to 13 Å. Two complementary three-dimensional experiments, or alternatively a four-dimensional experiment, yield highly unambiguous cross-peak assignments, which rely on four encoded chemical shift dimensions. Correlations to residual aliphatic protons are accessible via synchronous evolution of the (15)N and (13)C chemical shifts, which encode valuable amide-methyl distance restraints. On average, we obtain six restraints per residue. Importantly, 50% of all restraints correspond to long-range distances between residues i and j with |i - j| > 5, which are of particular importance in structure calculations. Using ARIA, we calculate a high-resolution structure for the microcrystalline 7.2 kDa α-spectrin SH3 domain with a backbone precision of ∼1.1 Å.
AU  - Linser, R.*
AU  - Bardiaux, B.*
AU  - Higman, V.*
AU  - Fink, U.*
AU  - Reif, B.
C1  - 6550
C2  - 28862
SP  - 5905-5912
TI  - Structure calculation from unambiguous long-range amide and methyl 1H-1H distance restraints for a microcrystalline protein with MAS solid-state NMR spectroscopy.
JO  - J. Am. Chem. Soc.
VL  - 133
IS  - 15
PB  - Amer Chemical Soc
PY  - 2011
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - Biological magic angle spinning (MAS) solid-state nuclear magnetic resonance spectroscopy has developed rapidly over the past two decades. For the structure determination of a protein by solid-state NMR, routinely C-13,C-13 distance restraints as well as dihedral restraints are employed. In protonated samples, this is achieved by growing the bacterium on a medium which contains [1,3]-C-13 glycerol or [2]-C-13 glycerol to dilute the C-13 spin system. Labeling schemes, which rely on heteronuclei, are insensitive both for detection and in terms of quantification of distances, since they are relying on low-gamma nuclei. Proton detection can in principle provide a gain in sensitivity by a factor of 8 and 31, compared to the C-13 or N-15 detected version of the experiment. We report here a new labeling scheme, which enables H-1-detection of aliphatic resonances with high resolution in MAS solid-state NMR spectroscopy. We prepared microcrystals of the SH3 domain of chicken a-spectrin with 5% protonation at nonexchangeable sites and obtained line widths on the order of 25 Hz for aliphatic H-1 resonances. We show further that C-13 resolved 3D-H-1,H-1 correlation experiments yield access to long-range proton-proton distances in the protein.
AU  - Asami, S.*
AU  - Schmieder, P.*
AU  - Reif, B.
C1  - 5686
C2  - 28002
CY  - Washington
SP  - 15133-15135
TI  - High resolution 1H-detected solid-state NMR spectroscopy of protein aliphatic resonances: Access to tertiary structure information.
JO  - J. Am. Chem. Soc.
VL  - 132
IS  - 43
PB  - Amer Chemical Soc
PY  - 2010
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - The measurement of (13)C directed-detected paramagnetic relaxation enhancements (PREs) on spin-labeled proteins combines the efficacy of PREs for the detection of long-range distance information with the favorable sensitivity and resolution of (13)C direct-detected experiments. The (13)C PREs provide long-range distance restraints to map binding interfaces in proteins and protein complexes and are especially useful for studies of high-molecular weight perdeuterated molecules.
AU  - Madl, T.
AU  - Felli, I.C.*
AU  - Bertini, I.*
AU  - Sattler, M.
C1  - 5659
C2  - 27928
SP  - 7285-7287
TI  - Structural analysis of protein interfaces from ¹³C direct-detected paramagnetic relaxation enhancements.
JO  - J. Am. Chem. Soc.
VL  - 132
IS  - 21
PB  - American Chemical Society
PY  - 2010
SN  - 0002-7863
ER  - 

TY  - JOUR
AU  - George, G.N.*
AU  - Pickering, I.J.*
AU  - Harris, H.H.*
AU  - Gailer, J.
AU  - Klein, D.
AU  - Lichtmannegger, J.
AU  - Summer, K.H.
C1  - 10110
C2  - 20857
SP  - 1704-1705
TI  - Tetrahiomolybdate Causes Formation of Hepatic Copper-Molybdenum Clusters in an Animal Model of Wilson's Disease.
JO  - J. Am. Chem. Soc.
VL  - 125
PY  - 2003
SN  - 0002-7863
ER  - 

TY  - JOUR
AU  - Leufgen, K.*
AU  - Mutter, M.*
AU  - Vogel, H.*
AU  - Szymczak, W.
C1  - 22331
C2  - 21173
SP  - 8911-8915
TI  - Orientation Modulation of a Synthetic Polypeptide in Self-Assembled Monolayers : a TOF-SIMS  Study.
JO  - J. Am. Chem. Soc.
VL  - 125
PY  - 2003
SN  - 0002-7863
ER  - 

TY  - JOUR
AB  - The volume changes, ΔV, which accompany the selective binding of ions in ion-exchange reactions at 25° between aqueous electrolyte mixtures and polyelectrolyte gels were measured in an attempt to establish the range of applicability of the Katchalsky and the Rice-Harris theories. The ΔV for the exchange of singly charged ions in lightly cross-linked polystyrene type strong-acid or strong-base ion-exchanger gels were small and positive or negative depending on the nature of the ions. Large, positive volume changes were observed with multiply charged cations. In general, ΔV became more positive with increased gel cross linking and with the strength of binding as measured by the decrease in the standard free energy, ΔG°, in the ion-exchange reaction. Four types of ion-binding processes were distinguished: (a) field binding; (b) site binding via ion-pair formation; (c) structure-enforced binding; and (d) charge-transfer binding. Multiply charged cations (i.e., Ca2+, Ba2+, La3+, Th4+) were selectively bound as ion pairs in all gels. Field binding could be inferred only for the selective uptake of singly charged cations in the lightly cross-linked gel.
AU  - Boyd, G.E.*
AU  - Bunzl, K.W.
C1  - 41133
C2  - 38085
SP  - 2054-2058
TI  - Selective binding of ions by polyelectrolyte gels. The volume change criterion.
JO  - J. Am. Chem. Soc.
VL  - 96
IS  - 7
PY  - 1974
SN  - 0002-7863
ER  -