Backbone and Sidechain H, N and C Resonance Assignments of a Multidrug Efflux Membrane Protein using Solution and Solid-State NMR.

EmrE is a bacterial membrane-embedded multidrug transporter that functions as an asymmetric homodimer. EmrE is implicated in antibiotic resistance, but is now known to confer either resistance or susceptibility depending on the identity of the small molecule substrate. Here, we report both solution- and solid-state NMR assignments of S64V-EmrE at pH 5.

OPTO: Automated Optimization for Solid-State NMR Spectroscopy.

NMR spectroscopy presents boundless opportunities for understanding the structure, dynamics, and function for a broad range of scientific applications. Solid-state NMR (SSNMR), in particular, provides novel insights into biological and material systems that are not amenable to other approaches. However, a major bottleneck is the extent of user training and the difficulty of obtaining reproducible, high-quality experimental results, especially for the sophisticated multidimensional pulse sequences that are essential to provide site-resolved measurements in large biomolecules.

Enhancing Spectrometer Performance with Unsupervised Machine Learning.

Solid-state NMR spectroscopy (SSNMR) is a powerful technique to probe structural and dynamic properties of biomolecules at an atomic level. Modern SSNMR methods employ multidimensional pulse sequences requiring data collection over a period of days to weeks. Variations in signal intensity or frequency due to environmental fluctuation introduce artifacts into the spectra.

Tetrakis(trimethylsilyl)silane as a standard compound for fast spinning Solid-State NMR experiments.

The development of magic angle spinning (MAS) at rates ranging from 30 kHz to greater than 100 kHz has substantially advanced solid-state nuclear magnetic resonance (SSNMR) spectroscopy H-detection methods. The small rotors required for such MAS rates have a limited sample volume and low C-detection sensitivity, rendering the traditional set of standard compounds for SSNMR insufficient or highly inconvenient for shimming and magic-angle calibration. Additionally, the reproducibility of magic angle setting, chemical shift referencing, and probe position can be especially critical for SSNMR experiments at high fields.

Higher Order Structure Differences Among Insulin Crystalline Drugs Revealed by 2D heteronuclear NMR.

During therapeutic protein development, two-dimensional (2D) heteronuclear NMR spectra can be a powerful analytical method for measuring protein higher order structure (HOS) in solution since the spectra exhibit much higher resolution than homonuclear H spectra. However, 2D NMR capabilities for characterizing protein HOS in crystalline states remain to be assessed, given the low C natural abundance and intrinsically broader lines in solid-state NMR (SSNMR). Herein, high-resolution heteronuclear correlation (HETCOR) SSNMR was utilized to directly measure intact crystal drug products of insulin human, insulin analogs of insulin lispro and insulin aspart.

Low power supercycled TPPM decoupling.

Improving the spectral sensitivity and resolution of biological solids is one of the long-standing problems in nuclear magnetic resonance (NMR) spectroscopy. In this report, we introduce low-power supercycled variants of two-pulse phase-modulated (TPPM) sequence for heteronuclear decoupling. The utility of the sequence is shown by improvements in the transverse relaxation time of observed nuclei (with H decoupling) with its application to different samples (uniformly C, N, H-labeled GB1 back-exchanged with 25% HO and 75% DO, uniformly C, N, H-labeled human derived Asyn fibril back-exchanged with 100% HO and uniformly C, N -labeled human derived Asyn fibril) at fast MAS using low radiofrequency (RF) fields.

Cross polarization stability in multidimensional NMR spectroscopy of biological solids.

Magic-angle spinning (MAS) solid-state nuclear magnetic resonance (SSNMR) spectroscopy is a powerful and versatile technique for probing structure and dynamics in large, insoluble biological systems at atomic resolution. With many recent advances in instrumentation and polarization methods, technology development in SSNMR remains an active area of research and presents opportunities to further improve data collection, processing, and analysis of samples with low sensitivity and complex tertiary and quaternary structures. SSNMR spectra are often collected as multidimensional data, requiring stable experimental conditions to minimize signal fluctuations (t noise).

Magnetic Susceptibility Modeling of Magic-Angle Spinning Modules for Part Per Billion Scale Field Homogeneity.

Magic-angle spinning (MAS) solid-state NMR methods are crucial in many areas of biology and materials science. Conventional probe designs have often been specified with 0.1 part per million (ppm) or 100 part per billion (ppb) magnetic field resolution, which is a limitation for many modern scientific applications.

Biologically Representative Lipid-Coated Gold Nanoparticles and Phospholipid Vesicles for the Study of Alpha-Synuclein/Membrane Interactions.

Alpha-synuclein is an intrinsically disordered protein whose formation of beta-sheet-rich protein aggregates in the brain is implicated in the development of Parkinson's disease. Due to its believed role in synaptic vesicle trafficking and neurotransmission, many studies have employed simple, synthetic model systems to investigate alpha-synuclein/membrane interactions in an attempt to gain a better understanding of the protein's native and pathogenic functions. Interestingly, these studies seem to suggest that alpha-synuclein interacts differently with rigid vesicle mimics in comparison to malleable vesicle mimics.

Sterol Sponge Mechanism Is Conserved for Glycosylated Polyene Macrolides.

Amphotericin-like glycosylated polyene macrolides (GPMs) are a clinically and industrially important family of natural products, but the mechanisms by which they exert their extraordinary biological activities have remained unclear for more than half a century. Amphotericin B exerts fungicidal action primarily via self-assembly into an extramembranous sponge that rapidly extracts ergosterol from fungal membranes, but it has remained unclear whether this mechanism is applicable to other GPMs. Using a highly conserved polyene-hemiketal region of GPMs that we hypothesized to represent a conserved ergosterol-binding domain, we bioinformatically mapped the entirety of the GPM sequence-function space and expanded the number of GPM biosynthetic gene clusters (BGCs) by 10-fold.

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

The highly infectious disease COVID-19 caused by the SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail.

Interaction of Alpha-Synuclein and Its Mutants with Rigid Lipid Vesicle Mimics of Varying Surface Curvature.

Abnormal aggregation of alpha-synuclein (α-syn), an intrinsically disordered neuronal protein, is strongly implicated in the development of Parkinson's disease. Efforts to better understand α-syn's native function and its pathogenic role in neurodegeneration have revealed that the protein interacts with anionic lipid vesicles adoption of an amphipathic α-helical structure; however, the ability of α-syn to remodel lipid membranes has made it difficult to decipher the role of vesicle surface curvature in protein binding behavior. In this study, sodium dodecyl sulfate (SDS)-coated gold nanoparticles (AuNPs), which mimic bilayer vesicle architecture, were synthesized in order to conduct a systematic investigation into the binding interaction of α-syn and two of its mutants (A30P and E46K) with rigid lipid vesicle mimics of defined surface curvature.

Polymer-Peptide Conjugates Convert Amyloid into Protein Nanobundles through Fragmentation and Lateral Association.

The assembly of proteins into amyloid fibrils has become linked not only with the progression of myriad human diseases, but also important biological functions. Understanding and controlling the formation, structure, and stability of amyloid fibrils is therefore a major scientific goal. Here we utilize electron microscopy-based approaches combined with quantitative statistical analysis to show how recently developed kind of amyloid modulators-multivalent polymer-peptide conjugates (mPPCs)-can be applied to control the structure and stability of amyloid fibrils.

Multivalent Polymer-Peptide Conjugates-A General Platform for Inhibiting Amyloid Beta Peptide Aggregation.

Protein aggregation is implicated in multiple deposition diseases including Alzheimer's Disease, which features the formation of toxic aggregates of amyloid beta (Aβ) peptides. Many inhibitors have been developed to impede or reverse Aβ aggregation. Multivalent inhibitors, however, have been largely overlooked despite the promise of high inhibition efficiency endowed by the multivalent nature of Aβ aggregates.