Catalysis of proline isomerization and molecular chaperone activity in a tug-of-war

Catalysis of cis/trans isomerization of prolines is important for the activity and misfolding of intrinsically disordered proteins. Catalysis is achieved by peptidylprolyl isomerases, a superfamily of molecular chaperones. Here, we provide atomic insight into a tug-of-war between cis/trans isomerization and molecular chaperone activity. Catalysis of proline isomerization by cyclophilin A lowers the energy barrier for \u3b1-synuclein misfolding, while isomerase-binding to a separate, disease-associated protein region opposes aggregation. We further show that cis/trans isomerization outpowers the holding activity of cyclophilin A. Removal of the proline isomerization barrier through posttranslational truncation of \u3b1-synuclein reverses the action of the proline isomerase and turns it into a potent molecular chaperone that inhibits protein misfolding. The data reveal a conserved mechanism of dual functionality in cis/trans isomerases and define its molecular determinants acting on intrinsically disordered proteins

Resolution and Polarization Distribution in Cryogenic DNP/MAS Experiments

This contribution addresses four potential misconceptions associated with high-resolution dynamic nuclear polarization/magic angle spinning (DNP/MAS) experiments. First, spectral resolution is not generally compromised at the cryogenic temperatures at which DNP experiments are performed. As we demonstrate at a modest field of 9 T (380 MHz [superscript 1]H), 1 ppm linewidths are observed in DNP/MAS spectra of a membrane protein in its native lipid bilayer, and <0.4 ppm linewidths are reported in a crystalline peptide at 85 K. Second, we address the concerns about paramagnetic broadening in DNP/MAS spectra of proteins by demonstrating that the exogenous radical polarizing agents utilized for DNP are distributed in the sample in such a manner as to avoid paramagnetic broadening and thus maintain full spectral resolution. Third, the enhanced polarization is not localized around the polarizing agent, but rather is effectively and uniformly dispersed throughout the sample, even in the case of membrane proteins. Fourth, the distribution of polarization from the electron spins mediated via spin diffusion between [superscript 1]H–[superscript 1]H strongly dipolar coupled spins is so rapid that shorter magnetization recovery periods between signal averaging transients can be utilized in DNP/MAS experiments than in typical experiments performed at ambient temperature.National Institutes of Health (U.S.) (Grant EB002804)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB002026)National Institutes of Health (U.S.) (Grant EB001965)National Institutes of Health (U.S.) (Grant EB004866)National Science Foundation (U.S.). Graduate Research Fellowship Progra

Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning

Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR

Altimetry for the future: Building on 25 years of progress

In 2018 we celebrated 25 years of development of radar altimetry, and the progress achieved by this methodology in the fields of global and coastal oceanography, hydrology, geodesy and cryospheric sciences. Many symbolic major events have celebrated these developments, e.g., in Venice, Italy, the 15th (2006) and 20th (2012) years of progress and more recently, in 2018, in Ponta Delgada, Portugal, 25 Years of Progress in Radar Altimetry. On this latter occasion it was decided to collect contributions of scientists, engineers and managers involved in the worldwide altimetry community to depict the state of altimetry and propose recommendations for the altimetry of the future. This paper summarizes contributions and recommendations that were collected and provides guidance for future mission design, research activities, and sustainable operational radar altimetry data exploitation. Recommendations provided are fundamental for optimizing further scientific and operational advances of oceanographic observations by altimetry, including requirements for spatial and temporal resolution of altimetric measurements, their accuracy and continuity. There are also new challenges and new openings mentioned in the paper that are particularly crucial for observations at higher latitudes, for coastal oceanography, for cryospheric studies and for hydrology. The paper starts with a general introduction followed by a section on Earth System Science including Ocean Dynamics, Sea Level, the Coastal Ocean, Hydrology, the Cryosphere and Polar Oceans and the ‘‘Green” Ocean, extending the frontier from biogeochemistry to marine ecology. Applications are described in a subsequent section, which covers Operational Oceanography, Weather, Hurricane Wave and Wind Forecasting, Climate projection. Instruments’ development and satellite missions’ evolutions are described in a fourth section. A fifth section covers the key observations that altimeters provide and their potential complements, from other Earth observation measurements to in situ data. Section 6 identifies the data and methods and provides some accuracy and resolution requirements for the wet tropospheric correction, the orbit and other geodetic requirements, the Mean Sea Surface, Geoid and Mean Dynamic Topography, Calibration and Validation, data accuracy, data access and handling (including the DUACS system). Section 7 brings a transversal view on scales, integration, artificial intelligence, and capacity building (education and training). Section 8 reviews the programmatic issues followed by a conclusion

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Altimetry for the future: building on 25 years of progress

In 2018 we celebrated 25 years of development of radar altimetry, and the progress achieved by this methodology in the fields of global and coastal oceanography, hydrology, geodesy and cryospheric sciences. Many symbolic major events have celebrated these developments, e.g., in Venice, Italy, the 15th (2006) and 20th (2012) years of progress and more recently, in 2018, in Ponta Delgada, Portugal, 25 Years of Progress in Radar Altimetry. On this latter occasion it was decided to collect contributions of scientists, engineers and managers involved in the worldwide altimetry community to depict the state of altimetry and propose recommendations for the altimetry of the future. This paper summarizes contributions and recommendations that were collected and provides guidance for future mission design, research activities, and sustainable operational radar altimetry data exploitation. Recommendations provided are fundamental for optimizing further scientific and operational advances of oceanographic observations by altimetry, including requirements for spatial and temporal resolution of altimetric measurements, their accuracy and continuity. There are also new challenges and new openings mentioned in the paper that are particularly crucial for observations at higher latitudes, for coastal oceanography, for cryospheric studies and for hydrology. The paper starts with a general introduction followed by a section on Earth System Science including Ocean Dynamics, Sea Level, the Coastal Ocean, Hydrology, the Cryosphere and Polar Oceans and the “Green” Ocean, extending the frontier from biogeochemistry to marine ecology. Applications are described in a subsequent section, which covers Operational Oceanography, Weather, Hurricane Wave and Wind Forecasting, Climate projection. Instruments’ development and satellite missions’ evolutions are described in a fourth section. A fifth section covers the key observations that altimeters provide and their potential complements, from other Earth observation measurements to in situ data. Section 6 identifies the data and methods and provides some accuracy and resolution requirements for the wet tropospheric correction, the orbit and other geodetic requirements, the Mean Sea Surface, Geoid and Mean Dynamic Topography, Calibration and Validation, data accuracy, data access and handling (including the DUACS system). Section 7 brings a transversal view on scales, integration, artificial intelligence, and capacity building (education and training). Section 8 reviews the programmatic issues followed by a conclusion

Structure and function of the Influenza membrane protein M2 by magic angle spinning NMR and dynamic nuclear polarization

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2014.Cataloged from PDF version of thesis. Vita.Includes bibliographical references.Determination of the 3D structure of membrane proteins is a frontier that is rapidly being explored due to the importance of membrane proteins in regulating cellular processes and because they are the target of many drugs. In addition, measuring and understanding how these proteins interact with ligands and other molecules is of critical importance to the design of the next generation of therapeutic agents. With this motivation, we report new methods for the structural characterization of proteins using magic angle spinning (MAS) nuclear magnetic resonance (NMR) applied to the membrane protein M2 from Influenza A, a small helical transmembrane proton transporter that is the target of adamantane based inhibitors but which is now drug resistant due primarily to the point mutation S31N. The development of techniques that boost the sensitivity of NMR are key to its extension to larger molecules such as membrane proteins, and two such methods, dynamic nuclear polarization (DNP) and proton detection, are applied herein. We report the measurement of the distance between the amine of the inhibitor rimantadine and the pore of M2 using DNP. We have applied recoupling techniques to assign the spectra and measure internuclear distances in the drug resistant S31N mutant of M2 in lipid bilayers. Attenuation of strong proton dipole couplings with 60 kHz spinning has allowed us to detect well-resolved proton spectra, and with the higher receptivity of protons, we measured interhelical distances with a methyl-methyl 4D spectrum. Synthesis of the information resulted in a high resolution structure of S3 IN M2.by Loren B. Andreas.Ph. D

Determination of Global Structure from Distance and Orientation Constraints in Biological Solids Using Solid-State NMR Spectroscopy

We report the results from a new solid-state NMR experiment, DANTE−REDOR, which can determine global secondary structure in uniformly (13C,15N)-enriched systems by simultaneously measuring distance and orientation constraints. Following a heteronuclear spin-pair selection using a DANTE pulse train, the magnitude and orientation of the internuclear dipole vector, within the chemical shift anisotropy (CSA) frame of the observed nucleus, are determined by tracking the dephasing of individual spinning sidebands under magic angle spinning. The efficacy of the experiment is demonstrated by measuring the imidazole side-chain orientation in U-[13C6,15N3]-l-histidine·HCl·H2O

Proton Association Constants of His 37 in the Influenza-A M218–60 Dimer-of-Dimers

The membrane protein M2 from influenza-A forms a single-pass transmembrane helix that assembles in lipid membrane as homotetramers whose primary function is to act as a proton transporter for viral acidification. A single residue, histidine 37 (His 37), is known to be responsible for selectivity and plays an integral role in the protein’s function. We report pH-dependent 15N MAS NMR spectra of His 37 within the influenza-A proton conduction domain of M2, M218–60, which has been previously shown to be a fully functional construct and was recently determined to adopt a dimer-of-dimers structure in lipids. By extracting the ratio of [His]/[HisH+] as a function of pH, we obtained two doubly degenerate proton disassociation constants, 7.63 ± 0.15 and 4.52 ± 0.15, despite a possible maximum of four. We also report the 1HNε chemical shifts at pH 6.5 recorded at 60 kHz MAS in a CP-based 1H–15N spectrum. We were unable to detect resonances indicative of direct proton sharing among His 37 side chains when the tetramer is in the +2 state. In the neutral state, His 37 is exclusively in the τ tautomer, indicating that the δ nitrogen is protonated solely as a function of pH. We also found that the plot of [HisH+]/[His] as a function of pH is qualitatively similar to previously reported proton conduction rates, indicating that proton conduction rate is proportional to the level of histidine protonation within the channel. Two-dimensional 13C–13C and 13C–15N correlations suggest that at low pH multiple conformations are populated as the spectra broaden and eventually disappear as the acidity is increased. A second highly resolved state at low pH was not observed