Structure and Function of the Bacterial Protein Toxin Phenomycin.

Phenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime toward mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell-free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of phenomycin, however, have remained unanswered. In this paper, we have used morphological profiling to show that direct inhibition of translation underlies the toxicity of phenomycin in cells. We have performed studies of the cellular uptake mechanism of phenomycin, showing that endosomal escape is the toxicity-limiting step, and we have solved a solution phase high-resolution structure of the protein using NMR spectroscopy. Through bioinformatic as well as functional comparisons between phenomycin and two homologs, we have identified a peptide segment, which constitutes one of two loops in the structure that is critical for the toxicity of phenomycin.

Disorder induced polymorphic transitions in the high hydrogen density compound Sr(BH4)2(NH3BH3)2.

The new compound Sr(BH4)2(NH3BH3)2 has been synthesized and characterized with in situ powder X-ray diffraction and fast (28 or 60 kHz) magic angle spinning 1H, 11B and 15N NMR and structurally optimized with density functional theory calculations. This investigation reveals complex structural rearrangements for this compound as a function of temperature. A room temperature orthorhombic polymorph, α-Sr(BH4)2(NH3BH3)2, with the space group symmetry Pbca, has been determined with a layered structure of alternating ammonia borane and Sr(BH4)2, partially stabilized by dihydrogen bonding. Surprisingly the crystal symmetry is lowered upon heating, as evidenced both by in situ synchrotron powder X-ray diffraction and 11B MAS NMR data, resulting in an intermediate polymorph, ß'-Sr(BH4)2(NH3BH3)2, present from ∼65 to 115 °C. ß-Sr(BH4)2(NH3BH3)2, a sub structure of the ß'-polymorph showing higher symmetry with the space group symmetry Aba2, forms upon further heating. Ab initio molecular dynamics simulations show that the ammonia borane molecule can dynamically alternate between a bidentate and a tridentate coordination to Sr at finite temperature. The dynamic properties of the ammonia borane molecule in the solid state are suggested to cause the observed structural complexity. Based on simultaneous thermogravimetric analysis, differential scanning calorimetry and mass spectrometry, the decomposition of the compound was investigated showing a stabilization of ammonia borane in the structure relative to other metal borohydride ammonia boranes and neat ammonia borane.

Critical Influence of Cosolutes and Surfaces on the Assembly of Serpin-Derived Amyloid Fibrils.

Many proteins and peptides self-associate into highly ordered and structurally similar amyloid cross-ß aggregates. This fibrillation is critically dependent on properties of the protein and the surrounding environment that alter kinetic and thermodynamic equilibria. Here, we report on dominating surface and solution effects on the fibrillogenic behavior and amyloid assembly of the C-36 peptide, a circulating bioactive peptide from the α1-antitrypsin serine protease inhibitor. C-36 converts from an unstructured peptide to mature amyloid twisted-ribbon fibrils over a few hours when incubated on polystyrene plates under physiological conditions through a pathway dominated by surface-enhanced nucleation. In contrast, in plates with nonbinding surfaces, slow bulk nucleation takes precedence over surface catalysis and leads to fibrillar polymorphism. Fibrillation is strongly ion-sensitive, underlining the interplay between hydrophilic and hydrophobic forces in molecular self-assembly. The addition of exogenous surfaces in the form of silica glass beads and polyanionic heparin molecules potently seeds the amyloid conversion process. In particular, heparin acts as an interacting template that rapidly forces ß-sheet aggregation of C-36 to distinct amyloid species within minutes and leads to a more homogeneous fibril population according to solid-state NMR analysis. Heparin's template effect highlights its role in amyloid seeding and homogeneous self-assembly, which applies both in vitro and in vivo, where glycosaminoglycans are strongly associated with amyloid deposits. Our study illustrates the versatile thermodynamic landscape of amyloid formation and highlights how different experimental conditions direct C-36 into distinct macromolecular structures.

Epoxy Matrices Modified by Green Additives for Recyclable Materials.

Epoxy-based thermosets are one of the most popular matrix materials in many industries, and significant environmental benefits can be obtained by developing a recyclable variant of this widely utilized material. Incorporation of a bio-based disulfide additive within a commercial epoxy system leads to a cross-linked material that can be fractionated under mild and environmentally benign conditions. The material has been analyzed by FTIR and solid-state NMR. Furthermore, modified epoxy matrices with low additive concentrations are demonstrated to have similar mechanical and thermal properties compared to commercially available benchmarks. Thus, additive formulation and fractionation based on green chemistry principles have been demonstrated, and a recyclable epoxy matrix has been developed.

Synthesis of ent-BE-43547A1 reveals a potent hypoxia-selective anticancer agent and uncovers the biosynthetic origin of the APD-CLD natural products.

Tumour hypoxia is speculated to be a key driver of therapeutic resistance and metastatic dissemination. Consequently, the discovery of new potent agents that selectively target the hypoxic cell population may reveal new and untapped antitumour mechanisms. Here we demonstrate that the BE-43547 subclass of the APD-CLD (amidopentadienoate-containing cyclolipodepsipeptides) natural products possesses highly hypoxia-selective growth-inhibitory activity against pancreatic cancer cells. To enable this discovery, we have developed the first synthesis of the BE-43547-macrocyclic scaffold in 16 steps (longest linear sequence), which also allowed access to the full panel of relative stereoisomers and ultimately to the assignment of stereochemical configuration. Discrepancies between the spectroscopic signatures of the synthetic compounds with that originally reported for the BE-43547 members stimulated us to re-isolate the natural product from a BE-43547-producing microorganism during which we elucidated the biosynthetic gene clusters for the BE-43547 family as well as for all other known APD-CLDs. Our studies underline the exciting possibilities for the further development of the anticancer activities of these natural products.

Handling the influence of chemical shift in amplitude-modulated heteronuclear dipolar recoupling solid-state NMR.

We present a theoretical analysis of the influence of chemical shifts on amplitude-modulated heteronuclear dipolar recoupling experiments in solid-state NMR spectroscopy. The method is demonstrated using the Rotor Echo Short Pulse IRrAdiaTION mediated Cross-Polarization ((RESPIRATION)CP) experiment as an example. By going into the pulse sequence rf interaction frame and employing a quintuple-mode operator-based Floquet approach, we describe how chemical shift offset and anisotropic chemical shift affect the efficiency of heteronuclear polarization transfer. In this description, it becomes transparent that the main attribute leading to non-ideal performance is a fictitious field along the rf field axis, which is generated from second-order cross terms arising mainly between chemical shift tensors and themselves. This insight is useful for the development of improved recoupling experiments. We discuss the validity of this approach and present quaternion calculations to determine the effective resonance conditions in a combined rf field and chemical shift offset interaction frame transformation. Based on this, we derive a broad-banded version of the (RESPIRATION)CP experiment. The new sequence is experimentally verified using SNNFGAILSS amyloid fibrils where simultaneous (15)N → (13)CO and (15)N → (13)Cα coherence transfer is demonstrated on high-field NMR instrumentation, requiring great offset stability.

Structural Investigation of Zn(II) Insertion in Bayerite, an Aluminum Hydroxide.

Bayerite was treated under hydrothermal conditions (120, 130, 140, and 150 °C) to prepare a series of layered double hydroxides (LDHs) with an ideal composition of ZnAl4(OH)12(SO4)0.5·nH2O (ZnAl4-LDHs). These products were investigated by both bulk techniques (powder X-ray diffraction (PXRD), transmission electron microscopy, and elemental analysis) and atomic-level techniques ((1)H and (27)Al solid-state NMR, IR, and Raman spectroscopy) to gain a detailed insight into the structure of ZnAl4-LDHs and sample composition. Four structural models (one stoichiometric and three different defect models) were investigated by Rietveld refinement of the PXRD data. These were assessed using the information obtained from other characterization techniques, which favored the ideal (nondefect) structural model for ZnAl4-LDH, as, for example, (27)Al magic-angle spinning NMR showed that excess Al was present as amorphous bayerite (Al(OH)3) and pseudoboehmite (AlOOH). Moreover, no evidence of cation mixing, that is, partial substitution of Zn(II) onto any of four Al sites, was observed. Altogether this study highlights the challenges involved to synthesize pure ZnAl4-LDHs and the necessity to use complementary techniques such as PXRD, elemental analysis, and solid-state NMR for the characterization of the local and extended structure of ZnAl4-LDHs.

In situ high-resolution structure of the baseplate antenna complex in Chlorobaculum tepidum.

Photosynthetic antenna systems enable organisms harvesting light and transfer the energy to the photosynthetic reaction centre, where the conversion to chemical energy takes place. One of the most complex antenna systems, the chlorosome, found in the photosynthetic green sulfur bacterium Chlorobaculum (Cba.) tepidum contains a baseplate, which is a scaffolding super-structure, formed by the protein CsmA and bacteriochlorophyll a. Here we present the first high-resolution structure of the CsmA baseplate using intact fully functional, light-harvesting organelles from Cba. tepidum, following a hybrid approach combining five complementary methods: solid-state NMR spectroscopy, cryo-electron microscopy, isotropic and anisotropic circular dichroism and linear dichroism. The structure calculation was facilitated through development of new software, GASyCS for efficient geometry optimization of highly symmetric oligomeric structures. We show that the baseplate is composed of rods of repeated dimers of the strongly amphipathic CsmA with pigments sandwiched within the dimer at the hydrophobic side of the helix.

A solid state NMR study of layered double hydroxides intercalated with para-amino salicylate, a tuberculosis drug.

Para-amino salicylate (PAS), a tuberculosis drug, was intercalated in three different layered double hydroxides (MgAl, ZnAl, and CaAl-LDH) and the samples were studied by multi-nuclear ((1)H, (13)C, and (27)Al) solid state NMR (SSNMR) spectroscopy in combination with powder X-ray diffraction (PXRD), elemental analysis and IR-spectroscopy to gain insight into the bulk and atomic level structure of these LDHs especially with a view to the purity of the LDH-PAS materials and the concentration of impurities. The intercalations of PAS in MgAl-, ZnAl-, and CaAl-LDH's were confirmed by (13)C SSNMR and PXRD. Moreover, (13)C MAS NMR and infrared spectroscopy show that PAS did not decompose during synthesis. Large amounts (20-41%) of amorphous aluminum impurities were detected in the structure using (27)Al single pulse and 3QMAS NMR spectra, which in combination with (1)H single and double quantum experiments also showed that the M(II):Al ratio was higher than predicted from the bulk metal composition of MgAl-PAS and ZnAl-PAS. Moreover, the first high-resolution (1)H SSNMR spectra of a CaAl LDH is reported and assigned using (1)H single and double quantum experiments in combination with (27)Al{(1)H} HETCOR.

How Glycosaminoglycans Promote Fibrillation of Salmon Calcitonin.

Glycosaminoglycans (GAGs) bind all known amyloid plaques and help store protein hormones in (acidic) granular vesicles, but the molecular mechanisms underlying these important effects are unclear. Here we investigate GAG interactions with the peptide hormone salmon calcitonin (sCT). GAGs induce fast sCT fibrillation at acidic pH and only bind monomeric sCT at acidic pH, inducing sCT helicity. Increasing GAG sulfation expands the pH range for binding. Heparin, the most highly sulfated GAG, binds sCT in the pH interval 3-7. Small angle x-ray scattering indicates that sCT monomers densely decorate and pack single heparin chains, possibly via hydrophobic patches on helical sCT. sCT fibrillates without GAGs, but heparin binding accelerates the process by decreasing the otherwise long fibrillation lag times at low pH and accelerates fibril growth rates at neutral pH. sCT·heparin complexes form ß-sheet-rich heparin-covered fibrils. Solid-state NMR reveals that heparin does not alter the sCT fibrillary core around Lys(11) but makes changes to Val(8) on the exterior side of the ß-strand, possibly through contacts to Lys(18) Thus GAGs significantly modulate sCT fibrillation in a pH-dependent manner by interacting with both monomeric and aggregated sCT.

Near-complete 1H, 13C, 15N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T.

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D (1)H-(15)N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain (1)H, (13)C and (15)N resonances for unfolded FAS1-4 A546T at 25 °C.

A unified heteronuclear decoupling strategy for magic-angle-spinning solid-state NMR spectroscopy.

A unified strategy of two-pulse based heteronuclear decoupling for solid-state magic-angle spinning nuclear magnetic resonance is presented. The analysis presented here shows that different decoupling sequences like two-pulse phase-modulation (TPPM), X-inverse-X (XiX), and finite pulse refocused continuous wave (rCW(A)) are basically specific solutions of a more generalized decoupling scheme which incorporates the concept of time-modulation along with phase-modulation. A plethora of other good decoupling conditions apart from the standard, TPPM, XiX, and rCW(A) decoupling conditions are available from the unified decoupling approach. The importance of combined time- and phase-modulation in order to achieve the best decoupling conditions is delineated. The consequences of different indirect dipolar interactions arising from cross terms comprising of heteronuclear and homonuclear dipolar coupling terms and also those between heteronuclear dipolar coupling and chemical-shift anisotropy terms are presented in order to unfold the effects of anisotropic interactions under different decoupling conditions. Extensive numerical simulation results are corroborated with experiments on standard amino acids.

Accurate measurements of ¹³C-¹³C distances in uniformly ¹³C-labeled proteins using multi-dimensional four-oscillating field solid-state NMR spectroscopy.

Application of sets of (13)C-(13)C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important (13)C-(13)C distances in uniformly (13)C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl ((13)C') and aliphatic ((13)C(aliphatic)) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly (13)C,(15)N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of (13)C'-(13)C(aliphatic) distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform (13)C,(15)N-labeling on the FGAIL fragment.

A hexameric peptide barrel as building block of amyloid-ß protofibrils.

Oligomeric and protofibrillar aggregates formed by the amyloid-ß peptide (Aß) are believed to be involved in the pathology of Alzheimer's disease. Central to Alzheimer pathology is also the fact that the longer Aß42 peptide is more prone to aggregation than the more prevalent Aß40 . Detailed structural studies of Aß oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aß that forms stable protofibrils and here we use solid-state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aß protomers into hexameric barrel-like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C-terminal hydrophobic regions of Aß, and hairpin loops extend from the core. The model accounts for why Aß42 forms oligomers and protofibrils more easily than Aß40 .

Automated robust and accurate assignment of protein resonances for solid state NMR.

The process of resonance assignment represents a time-consuming and potentially error-prone bottleneck in structural studies of proteins by solid-state NMR (ssNMR). Software for the automation of this process is therefore of high interest. Procedures developed through the last decades for solution-state NMR are not directly applicable for ssNMR due to the inherently lower data quality caused by lower sensitivity and broader lines, leading to overlap between peaks. Recently, the first efforts towards procedures specifically aimed for ssNMR have been realized (Schmidt et al. in J Biomol NMR 56(3):243-254, 2013). Here we present a robust automatic method, which can accurately assign protein resonances using peak lists from a small set of simple 2D and 3D ssNMR experiments, applicable in cases with low sensitivity. The method is demonstrated on three uniformly (13)C, (15)N labeled biomolecules with different challenges on the assignments. In particular, for the immunoglobulin binding domain B1 of streptococcal protein G automatic assignment shows 100% accuracy for the backbone resonances and 91.8% when including all side chain carbons. It is demonstrated, by using a procedure for generating artificial spectra with increasing line widths, that our method, GAMES_ASSIGN can handle a significant amount of overlapping peaks in the assignment. The impact of including different ssNMR experiments is evaluated as well.

Preparation of uniformly (13)C,(15)N-labeled recombinant human amylin for solid-state NMR investigation.

A number of diseases are caused by the formation of amyloid fibrils. Detailed understanding of structural features of amyloid fibers is of great importance for our understanding of disease progression and design of agents for diagnostics or potential prevention of protein aggregation. In lack of 3D crystal ordering, solid-state NMR forms the most suited method to determine the structures of the fibrils with atomic resolution. To exploit this potential, large amounts of isotopic-labeled protein need to be obtained through recombinant protein expression. However, expression and purification of amyloidogenic proteins in large amounts remains challenging due to their aggregation potential, toxicity for cells and difficult purification. In this work, we report a method for the production of large amounts of uniformly labeled (13)C,(15)N-human amylin, being one of the most amyloidogenic peptides known. This method utilizes inclusion bodies-directed expression and cheap chemical cleavage with cyanogen bromide in order to minimize the cost of the procedure compared to the use of less efficient proteolytic enzymes. We demonstrate the formation of amylin fibrils in vitro characterized using biophysical methods and electron microscopy, show toxicity towards human cells, and demonstrate that produced material may form the basis for structure determination using solid-state NMR.

Designing dipolar recoupling and decoupling experiments for biological solid-state NMR using interleaved continuous wave and RF pulse irradiation.

Rapid developments in solid-state NMR methodology have boosted this technique into a highly versatile tool for structural biology. The invention of increasingly advanced rf pulse sequences that take advantage of better hardware and sample preparation have played an important part in these advances. In the development of these new pulse sequences, researchers have taken advantage of analytical tools, such as average Hamiltonian theory or lately numerical methods based on optimal control theory. In this Account, we focus on the interplay between these strategies in the systematic development of simple pulse sequences that combines continuous wave (CW) irradiation with short pulses to obtain improved rf pulse, recoupling, sampling, and decoupling performance. Our initial work on this problem focused on the challenges associated with the increasing use of fully or partly deuterated proteins to obtain high-resolution, liquid-state-like solid-state NMR spectra. Here we exploit the overwhelming presence of (2)H in such samples as a source of polarization and to gain structural information. The (2)H nuclei possess dominant quadrupolar couplings which complicate even the simplest operations, such as rf pulses and polarization transfer to surrounding nuclei. Using optimal control and easy analytical adaptations, we demonstrate that a series of rotor synchronized short pulses may form the basis for essentially ideal rf pulse performance. Using similar approaches, we design (2)H to (13)C polarization transfer experiments that increase the efficiency by one order of magnitude over standard cross polarization experiments. We demonstrate how we can translate advanced optimal control waveforms into simple interleaved CW and rf pulse methods that form a new cross polarization experiment. This experiment significantly improves (1)H-(15)N and (15)N-(13)C transfers, which are key elements in the vast majority of biological solid-state NMR experiments. In addition, we demonstrate how interleaved sampling of spectra exploiting polarization from (1)H and (2)H nuclei can substantially enhance the sensitivity of such experiments. Finally, we present systematic development of (1)H decoupling methods where CW irradiation of moderate amplitude is interleaved with strong rotor-synchronized refocusing pulses. We show that these sequences remove residual cross terms between dipolar coupling and chemical shielding anisotropy more effectively and improve the spectral resolution over that observed in current state-of-the-art methods.

Refocused continuous-wave decoupling: a new approach to heteronuclear dipolar decoupling in solid-state NMR spectroscopy.

A novel strategy for heteronuclear dipolar decoupling in magic-angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy is presented, which eliminates residual static high-order terms in the effective Hamiltonian originating from interactions between oscillating dipolar and anisotropic shielding tensors. The method, called refocused continuous-wave (rCW) decoupling, is systematically established by interleaving continuous wave decoupling with appropriately inserted rotor-synchronized high-power π refocusing pulses of alternating phases. The effect of the refocusing pulses in eliminating residual effects from dipolar coupling in heteronuclear spin systems is rationalized by effective Hamiltonian calculations to third order. In some variants the π pulse refocusing is supplemented by insertion of rotor-synchronized π/2 purging pulses to further reduce the residual dipolar coupling effects. Five different rCW decoupling sequences are presented and their performance is compared to state-of-the-art decoupling methods. The rCW decoupling sequences benefit from extreme broadbandedness, tolerance towards rf inhomogeneity, and improved potential for decoupling at relatively low average rf field strengths. In numerical simulations, the rCW schemes clearly reveal superior characteristics relative to the best decoupling schemes presented so far, which we to some extent also are capable of demonstrating experimentally. A major advantage of the rCW decoupling methods is that they are easy to set up and optimize experimentally.

In†situ solid-state NMR spectroscopy of protein in heterogeneous membranes: the baseplate antenna complex of Chlorobaculum tepidum.

A clever combination: an in situ solid-state NMR analysis of CsmA proteins in the heterogeneous environment of the photoreceptor of Chlorobaculum tepidum is reported. Using different combinations of 2D and 3D solid-state NMR spectra, 90 % of the CsmA resonances are assigned and provide on the basis of chemical shift data information about the structure and conformation of CsmA in the CsmA-bacteriochlorophyll a complex.