Biology subjects

Rienstra, C. M.

Publications and source records attributed to Rienstra, C. M..

11 recordsLinked to original sources

bioRxiv2025

Backbone Assignment of a 28.5 kDa Class A Extended Spectrum β-Lactamase by High-Field, Carbon-Detected Solid-State NMR

13C and 15N backbone chemical shift assignments are reported for the 28.5 kDa protein Toho-1 {beta}-lactamase, a Class A extended spectrum {beta}-lactamase. A very high level of assignment completeness (97% of the backbone) is enabled by the combined sensitivity and resolution gains of ultrahigh-field NMR spectroscopy (1.1 GHz), improved probe technology, and optimized pulse sequences. The assigned chemical shifts agree well with our previous solution-state NMR assignments, indicating that the secondary structure is conserved in the solid state. These assignments provide a foundation for future investigations of sidechain chemical shifts and catalytic mechanism.

biochemistry↗

bioRxiv2025

Selective Detection of a Key Region in Chemotaxis Signaling Protein Complexes by Solid-State NMR

Understanding how large protein complexes function requires tools that can resolve both structure and dynamics in their native assembly states. Here, we apply solid-state NMR (SSNMR) to selectively detect rigid regions of a receptor protein fragment in the context of the >500 kDa chemoreceptor signaling complex found in chemotactic bacteria. These complexes assemble into hexagonal arrays that network multiple active units together. The cytoplasmic fragment of the E. coli aspartate chemoreceptor exhibits dynamics on multiple timescales across different regions of the protein, and these dynamics differ between signaling states. We apply 13C-15N dipolar coupling-based SSNMR experiments to selectively probe the rigid portion (motions slower than millisecond timescale) of this protein, in the context of the full array structure. We optimized assembly methods to form native-like, homogeneous complexes capable of maintaining activity and sample integrity during extended NMR experiments with low electric-field NMR probe designs. A subset of the protein, approximately 100 residues at the membrane-distal tip of the chemoreceptor where it interacts with its associated kinase, was detected and identified as the most rigid region. Chemical shift changes for many residues in this rigid region were observed between NMR spectra of the kinase-on and kinase-off signaling states. This suggests conformational changes occur at the chemoreceptor tip during signaling, which have not been observed in previous studies of this system. These findings demonstrate a dynamics-based NMR spectral editing approach to selectively examine a key region of a large signaling protein within its macromolecular assembly. Statement of SignificanceLarge protein complexes are challenging to study, but understanding their mechanism is vital to gaining insight into many important biological processes. In this work we demonstrate the use of solid-state NMR to study such a system, bacterial chemotaxis receptor signaling complexes. We identified an in vitro assembly method that creates reproducible and durable complexes that retain activity for weeks of data collection. Selective NMR detection of a key region of one protein reveals significant differences between signaling states, indicating there are changes in conformation and dynamics that were not previously seen by cryo-electron tomography and crystallography of this system. This highlights the potential of solid-state NMR as a key tool in mechanistic studies of multi-protein complexes.

biochemistry↗

bioRxiv2025

Top-Down Scoring of Spectral Fitness by Image Analysis for Protein Structure Validation

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for protein structure determination, but traditional approaches require extensive manual assignment of hundreds to thousands of resonances. Here we present NMRFAM-BPHON, a novel "top-down" approach that treats experimental NMR spectra as continuous grayscale images and quantitatively scores the agreement with simulated spectra generated from candidate protein structures. This method does not require complete resonance assignments, though it can incorporate experimental chemical shifts when available to improve performance. The simulated spectra are generated from postulated resonance assignments, which can be derived either from empirical database predictions, direct interpretation, or a hybrid combination. BPHON employs a physics-based approximate polarization transfer model to predict cross-peak intensities from the internuclear distances in the decoy structure, and models the peak lineshapes using empirical, bulk T2 relaxation rates and literature values for scalar couplings. The resulting simulated spectra are scored relative to the experimental data by normalized cross correlation, yielding a fitness score between 0 and 1. We demonstrate BPHONs ability to discriminate structural models, particularly in the case of 13C-detected magic angle spinning solid-state NMR spectra. The software is packaged with a user-friendly graphical user interface for ChimeraX, enabling advanced NMR analysis accessible without requiring extensive manual analysis.

biophysics↗

bioRxiv2025

NMR Spectral Alignment Utilizing a CryoEM Motion Correction Algorithm

With recent advances in magic-angle spinning (MAS) solid-state NMR (SSNMR) resolution, precise spectral alignment has become a critical bottleneck in data processing workflows. While solution NMR employs deuterium lock systems, most SSNMR probes still lack this capability; though a lock corrects for magnet drift and instabilities, it is not alone sufficient to account for field gradients, sample temperature differences, and pulse sequence effects that can contribute to referencing errors among several data sets. These offsets become particularly problematic in the lengthy multidimensional experiments that provide the foundation for resonance assignment and structure determination procedures. Currently, researchers rely on manual alignment through visual peak inspection--a qualitative approach that often overemphasizes prominent, outlying peaks while overlooking subtle, global patterns. This subjective process becomes increasingly impractical for use cases with lower sensitivity, such as large proteins with thousands of peaks. To address these challenges, here we present Automated NMR Spectral Alignment (ANSA), a program that adapts cryo-electron microscopy motion correction principles to NMR spectroscopy. ANSA treats NMR spectra as images and applies cross-correlation functions to determine optimal alignment, improving cross-correlation scores from 0.33 to 1.00 in controlled tests and achieving 0.96 correlation in real-world applications with previously misaligned spectra. The algorithm successfully aligns spectra across varying experimental conditions, corrects shifts in long-duration experiments, and works with 2D and 3D datasets, with approaches that can be readily extended to additional dimensions. By eliminating human bias and providing objective, consistent spectral alignment, ANSA enhances scientific rigor, improves reproducibility between experiments, and enables automation of critical data processing steps. The software is freely available as an open-source tool, ready for integration into existing NMR workflows.

biophysics↗

bioRxiv2025

Ultra-High Resolution Solid-State NMR for High Molecular Weight Proteins on GHz-Class Spectrometers

NMR spectroscopy is a powerful technique with broad impact across the physical and life sciences, and ultra-high field, GHz-class NMR spectrometers offer exceptional overall performance including superior resolution and sensitivity. While the resolution is fundamentally limited by molecular tumbling for solution NMR, solid-state NMR (SSNMR) is constrained only by instrumentation, making it well-suited for studying large and complex systems. To fully leverage UHF magnets for magic-angle-spinning SSNMR, it is essential to eliminate linebroadening arising from magnetic field drift and couplings among the nuclear spins. We address these challenges using an external 2H lock to compensate the field drift and Long-Observation-Window Band-Selective Homonuclear Decoupling (LOW-BASHD) to suppress 13C homonuclear couplings. We thereby achieve better than 0.2 ppm resolution in proteins up to 144 kDa, enabling unique site resolution for over 500 amide backbone pairs in 2D experiments. This exceeds the resolution available from solution NMR for large biological molecules, greatly expanding the potential of GHz-class NMR for research in life sciences. TeaserUltra-high field NMR enables scientists to observe the finest details of large biomolecules, and this study overcomes key challenges to achieve a new benchmark for resolution in solid-state NMR of high molecular weight proteins.

biophysics↗

bioRxiv2025

Alpha-Synuclein Fibril Structures Cluster into Distinct Classes

The accumulation of Alpha-synuclein (Asyn) fibrils is the defining pathologic feature in Parkinson Disease (PD), Lewy Body Dementia (LBD), and Multiple System Atrophy (MSA). As such, the process of Asyn fibril formation has been an important research area and fibrils themselves have become attractive targets for disease diagnosis and therapeutic intervention. Due to the presence of mixed populations of fibrillar proteins associated with neurodegenerative diseases in brain tissue, high-resolution structures of Asyn fibrils are essential for the design of high-specificity imaging and therapeutic agents. Approximately one hundred high-resolution solid-state NMR (SSNMR) spectroscopy and cryo-electron microscopy (cryo-EM) structures of Asyn fibrils have been deposited to the Protein Databank (PDB); intriguingly there is significant polymorphism among them. Understanding the molecular makeup and characteristic features of each structural polymorph can determine conserved structural motifs which can be used as templates to design ligands with high specificity for clinical use. Utilizing standard alignment tools and density-based clustering approaches, we objectively classify fibril structures by tertiary structure type. We find that 81% of the structures cluster into two polymorph classes. Within each class, additional subtle variations are observed which position sidechains in specific, conserved orientations, well poised as druggable targets. Furthermore, we find that the conserved structural motifs associated with each class are found in all but one published Asyn fibril structure. We consider these classifications and conserved motifs in the context of disease-relevant fibril structures and offer a perspective on the utility of in vitro fibrils as substrates for drug development and models for disease pathogenesis.

biophysics↗

bioRxiv2025

In situ light-driven pH modulation for NMR studies

Proton exchange is a fundamental chemical event, and NMR provides the most direct readout of protonation events with site-specific resolution. Conventional approaches require manual titration of sample pH to collect a series of NMR spectra at different pH values. This requires extensive sample handling and often results in significant sample loss, leading to reduced signal or the need to prepare additional samples. Here, we introduce a novel approach to control pH in NMR samples using water soluble photoacids, which alter the pH of the solution from near neutral to acidic pH upon in situ photo-illumination. We show that the solution pH can be precisely controlled by choice of illumination wavelength and intensity and sufficient protons are released from the photoacid to achieve meaningful pH change in samples where the molecule of interest has significant buffering capacity, such as a >100 M protein sample. The pH is monitored in situ using internal standards with pH-sensitive chemical shifts. This method enables precise, calibrated, non-invasive change of sample pH within an NMR magnet, dramatically reducing the necessary sample handling. These findings highlight the potential of light-induced pH control in NMR experiments and increase the robustness and reliability of pH-dependent studies. With pH playing a key role in modulating chemical behavior in both biological and synthetic systems, the ability to study protonation states and modulate sample pH in a simple and precise manner that is compatible with high-resolution NMR studies of molecular structure and function has wide applications. Entry for the Table of ContentsIn this work, we introduce a novel approach to control pH in NMR samples using light-activated photoacids. By combining light stimuli with pH-sensitive molecules, we demonstrate the ability to precisely modulate pH without physically manipulating the sample. This method enables non-invasive pH titration in NMR studies, where pH plays a key role in protein function. Our findings highlight the potential of light-induced pH control to overcome existing limitations in NMR, providing a powerful tool for advancing protein research under controlled pH conditions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/633412v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1bf9839org.highwire.dtl.DTLVardef@1a2d77eorg.highwire.dtl.DTLVardef@1f1a68dorg.highwire.dtl.DTLVardef@1ed73fa_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Backbone and Sidechain 1H, 15N and 13C 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 5.8, below the pKa of critical residues E14 and H110. This includes 1H, 15N, and 13C resonance assignments of the backbone, methyl groups (isoleucine, leucine, valine, threonine and alanine) from solution NMR experiments in bicelles, and backbone and side-chain assignments from solid-state NMR 13C-detected experiments in liposomes.

biophysics↗

bioRxiv2024

High-Resolution Cryo-EM Structure Determination of α-Synuclein - A Prototypical Amyloid Fibril

The physiological role of -synuclein (-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, -syn will self-assemble into {beta}-sheet rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient derived brain tissues have concluded that these inclusions are associated with Parkinsons disease, the second most common neurodegenerative disorder, and other synuclein related diseases called synucleinopathies. In addition, repetitions of and specific mutations to the SNCA gene, the gene that encodes -syn, results in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of -syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 [A]. Here, we provide a protocol for -syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step by step protocol for high resolution cryo-EM structure determination of -syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high resolution cryo-EM map. This workflow resulted in a 2.04 [A] structure of -syn fibrils with excellent resolution of residues 36 to 97 and an additional island of density for residues 15 to 22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of -synuclein and other amyloid fibrils. Key FeaturesO_LIIn vitro fibril amplification method yielding twisting fibrils that span several micrometers in length and are suitable for cryo-EM structure determination. C_LIO_LIHigh-throughput cryo-EM data collection of neurodegenerative fibrils, such as alpha-synuclein. C_LIO_LIUse of RELION implementations of helical reconstruction algorithms to generate high-resolution 3D structures of a-synuclein fibrils. C_LIO_LIBrief demonstration of the use of ChimeraX, Coot, and Phenix for molecular model building and refinement.s C_LI Graphical overview of -synuclein fibrilization and cryo-EM structure determination-synuclein protein expression and purification is followed by a fibrilization protocol yielding twisting filaments that span several micrometers in length and are validated by negative stain transmission electron microscopy (NS-TEM). The sample is then vitrified, followed by cryo-EM data collection. Real-space helical reconstruction is performed in RELION to generate an electron potential map that is used for model building. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/613698v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1b008forg.highwire.dtl.DTLVardef@c1c1e1org.highwire.dtl.DTLVardef@9fc539org.highwire.dtl.DTLVardef@14d3918_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.

neuroscience↗

bioRxiv2023

13C and 15N Resonance Assignments of Alpha Synuclein Fibrils Amplified from Lewy Body Dementia Tissue

Fibrils of the protein -synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

biophysics↗