Biology subjects

Schanda, P.

Publications and source records attributed to Schanda, P..

13 recordsLinked to original sources

bioRxiv2026

Integrated solid/solution NMR assignment allows mapping dynamics and ligand binding in a 134 kDa enzyme

Understanding enzyme function requires characterizing not only static structure but also dynamics and ligand interactions. NMR spectroscopy provides this insight at atomic resolution, yet for large proteins the difficulty of resonance assignment has largely confined such studies to systems below[~] 50 kDa, or to observing only methyl groups. Here we present an integrated magic-angle spinning (MAS) and solution NMR study of the 134 kDa tetrameric malate dehydrogenase from Ignicoccus islandicus (IiMDH), an enzyme of particular interest as an evolutionary intermediate between allosteric lactate dehydrogenases and non-allosteric malate dehydrogenases. By combining high-dimensional (up to 4D) MAS NMR experiments on sedimented protein with solution NMR, we achieved 92% backbone heavy-atom assignment and 91% assignment of all Ile-{delta}1, Leu-{delta}1/-{delta}2, Val-{gamma}1/-{gamma}2, Met-{varepsilon} and Thr-{gamma} methyl groups. Building on these assignments, MAS NMR 15N rotating-frame relaxation (R1p) measurements revealed pronounced microsecond-timescale backbone dynamics in functionally critical regions, including the catalytic loop and the mobile surface loop. Complementary methyl-axis order parameters from solution NMR identified additional flexible sites in the hydrophobic core. Chemical shift perturbation experiments upon addition of the substrate analogue oxamate, monitored via backbone 1H-15N TROSY, revealed both active-site contacts and rearrangements of helices a2F and a3G, regions implicated in allosteric signal transmission. The integrated approach demonstrated here exploits the distinct strengths of MAS and solution NMR, and provides a comprehensive view of structure, dynamics, and substrate interactions in a large oligomeric enzyme that would not be accessible by either technique alone.

biophysics↗

bioRxiv2026

A dynamic displacement mechanism drives protein import into mitochondria

Most mitochondrial proteins are produced in the cytosol and imported through the translocase of the outer mitochondrial membrane (TOM) to reach their final destination. Although this protein entry gate has been structurally characterized, it remains unclear how precursor proteins are handed off from the cytosolic receptor domains to the translocation pore. Here we show that the cytosolic domain of Tom22--traditionally viewed as the central TOM receptor--acts not as a structured scaffold but as a largely disordered, flexible segment that plays an active role in precursor transfer. Atomic-level structural techniques and in vivo experiments identified a conserved short linear motif that forms a transient !-helical element within this disordered domain. By binding to the canonical precursor protein binding sites of the receptors Tom20 and Tom70, this critical -helical segment acts as a precursor protein displacement element (PPDE). This competitive interaction facilitates the release of preproteins directly above the import pore, and thereby drives translocation across the outer mitochondrial membrane. These findings reveal that flexibility, rather than rigid structure, underlies the central transfer step of mitochondrial outermembrane protein translocation. Our results point to a versatile mechanism for ligand displacement in chaperone, receptor, and transport systems that must balance selective binding with efficient release.

cell biology↗

bioRxiv2025

Aromatic Ring Flips Reveal Reshaping of Protein Dynamics in Crystals and Complexes

Protein conformational energy landscapes are shaped not only by intramolecular interactions but also by their environment. In protein crystals and protein-protein complexes, intermolecular contacts alter this energy landscape, but the exact nature of this alteration is difficult to decipher. Understanding how the crystal lattice affects protein dynamics is crucial for crystallography-based studies of motion, yet its influence on collective motions remains unclear. Aromatic ring flips in the hydrophobic core represent sensitive probes of such dynamics. Here, we compare the kinetics of aromatic ring flips in the protein GB1 in crystals, in complex with its binding partner IgG, and in solution, combining advanced isotope labeling with quantitative NMR methods. We show that rings in the core flip nearly a thousand times less frequently in crystals than in solution. Enhanced-sampling molecular dynamics simulations, based on a new crystal structure, reproduce these elevated barriers and reveal how the crystal restrains motions.

biophysics↗

bioRxiv2024

Deuteration of proteins boosted by cell lysates: high-resolution amide and Hα MAS NMR without re-protonation bottleneck

Amide-proton detected magic-angle spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D2O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with H2O-based culture medium doped with deuterated cell lysate allow to overcome this "reprotonation bottleneck", while retaining a high level of deuteration (ca. 80 %) and narrow line widths. We quantified coherence life times of several proteins prepared with this labelling pattern over a range of MAS frequencies (40-100 kHz). We demonstrate that under commonly used conditions (50-60 kHz MAS), amide 1H line widths with our labelling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33-50 kDa size range many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and show that such media can be used identically to commercial media. The residual protonation of H sites allows for well-resolved H-detected spectra and H resonance assignment, exemplified by the de novo assignment of 168 H sites in a 39 kDa protein. The approach based on this H2O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm/100 kHz MAS experiments.

biochemistry↗

bioRxiv2023

Disulfide-bond-induced structural frustrationand dynamic disorder in a peroxiredoxin fromMAS NMR

Disulfide bond formation is fundamentally important for protein structure, and constitutes a key mechanism by which cells regulate the intracellular oxidation state. Peroxiredoxins (PRDXs) eliminate reactive oxygen species such as hydrogen peroxide by using a catalytic cycle of Cys oxidation and reduction. High molecular-weight assemblies of PRDXs have recently been shown to additionally act as molecular chaperones. The consequences of disulfide bonds on the dynamics of these large assemblies are poorly understood. We show that formation of disulfide bonds along the catalytic cycle induces extensive s time scale dynamics, as monitored by magic-angle spinning NMR of the 216 kDa-large Tsa1 decameric assembly and solution-NMR of a designed dimeric mutant. We ascribe the conformational dynamics to structural frustration, resulting from conflicts between the disulfide-constrained reduction of mobility and the desire to fulfil other favorable contacts.

biophysics↗

bioRxiv2022

Aromatic ring flips in differently packed protein crystals: MAS NMR and MD studies of 3 ubiquitin lattices

Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a proteins hydrophobic core report on breathing motion involving a large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. The question how packing of proteins in crystals affects ring flips has, thus, remained largely unexplored. Here we apply magic-angle spinning NMR, advanced phenylalanine 1H-13C/2H isotope labeling and MD simulation to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. The flips of the two Phe residues in ubiquitin, both surface exposed, appear are remarkably conserved in the different crystal forms, even though the intermolecular packing is quite different: Phe4 flips on a ca. 10-20 ns time scale, and Phe45 is broadened in all crystals, presumably due to s motion. Our findings suggest that intramolecular influences are more important for ring flips than intermolecular (packing) effects.

biophysics↗

bioRxiv2021

Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

Large oligomeric enzymes control a myriad of cellular processes, from protein synthesis and degradation to metabolism. The 0.5 MDa large TET2 aminopeptidase, a prototypical protease important for cellular homeostasis, degrades peptides within a ca. 60 [A] wide tetrahedral chamber with four lateral openings. The mechanisms of substrate trafficking and processing remain debated. Here, we integrate magic-angle spinning (MAS) NMR, mutagenesis, co-evolution analysis and molecular dynamics simulations and reveal that a loop in the catalytic chamber is a key element for enzymatic function. The loop is able to stabilize ligands in the active site and may additionally have a direct role in activating the catalytic water molecule whereby a conserved histidine plays a key role. Our data provide a strong case for the functional importance of highly dynamic - and often overlooked - parts of an enzyme, and the potential of MAS NMR to investigate their dynamics at atomic resolution.

biophysics↗

bioRxiv2020

Structural basis of client specificity in mitochondrial membrane-protein chaperones

Chaperones are essential for assisting protein folding, and for transferring poorly soluble proteins to their functional locations within cells. Hydrophobic interactions drive promiscuous chaperone-client binding, but our understanding how additional interactions enable client specificity is sparse. Here we decipher what determines binding of two chaperones (TIM8{middle dot}13, TIM9{middle dot}10) to different integral membrane proteins, the alltransmembrane mitochondrial carrier Ggc1, and Tim23 which has an additional disordered hydrophilic domain. Combining NMR, SAXS and molecular dynamics simulations, we determine the structures of Tim23/TIM8{middle dot}13 and Tim23/TIM9{middle dot}10 complexes. TIM8{middle dot}13 uses transient salt bridges to interact with the hydrophilic part of its client, but its interactions to the trans-membrane part are weaker than in TIM9{middle dot}10. Consequently, TIM9{middle dot}10 is outcompeting TIM8{middle dot}13 in binding hydrophobic clients, while TIM8{middle dot}13 is tuned to few clients with both hydrophilic and hydrophobic parts. Our study exemplifies how chaperones fine-tune the balance of promiscuity vs. specificity.

biophysics↗

bioRxiv2020

Architecture and subunit dynamics of the mitochondrial TIM9·10·12 chaperone

The mitochondrial Tim chaperones are responsible for the transport of membrane proteins across the inter-membrane space to the inner and outer mitochondrial membranes. TIM9{middle dot}10, a hexameric 70 kDa protein complex formed by 3 copies of Tim9 and Tim10, guides its clients across the aqueous compartment. The TIM9{middle dot}10{middle dot}12 complex is the anchor point at the inner-membrane insertase complex TIM22. The subunit composition of the TIM9{middle dot}10{middle dot}12 complex remains debated. Joint NMR, small-angle X-ray scattering and MD simulation data allow us to derive a structural model of the TIM9{middle dot}10{middle dot}12 assembly, which has a 2:3:1 stoichiometry (Tim9:Tim10:Tim12). We find that both TIM9{middle dot}10 and TIM9{middle dot}10{middle dot}12 hexamers are in a dynamic equilibrium with their constituent subunits, exchanging on a minutes time scale. Residue-resolved NMR data establish that the subunits exhibit large conformational dynamics: when the conserved cysteines of the CX3C-Xn-CX3C motifs are formed, short marginally stable -helices are formed, and these are fully stabilized only upon formation of the mature hexameric chaperone. We propose that the continuous subunit exchange is a means of mitochondria to control their level of inter-membrane space chaperones, and thus rapidly adapt to the cellular state.

biophysics↗

bioRxiv2019

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. ClpP protease is a tetradecameric complex that has emerged as a drug target against multiple pathogenic bacteria. During drug development efforts, the activation of different ClpPs by inhibitors was independently reported, but so far, no rationale for inhibitor-induced activation has been proposed. Using an integrated approach that included X-ray crystallography, solid-and solution-state NMR, MD simulations and ITC we show that the proteasome-inhibitor bortezomib binds to the ClpP active site serine mimicking a peptide substrate and induces the concerted allosteric activation of the complex. The bortezomib activated conformation also displays a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association as well as protein processive degradation.

biochemistry↗

bioRxiv2018

Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

Atomic-resolution structure determination is the key requirement for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enabled determination of the high-resolution structure of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 [A] by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and specifically labelled methyl groups, and a 4.1 [A] resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution (up to 8 [A]) cryo-EM data are available, thus paving avenues for the structure determination of challenging biological assemblies.

biophysics↗

bioRxiv2018

Dynamics and interactions of ADP/ATP transporter AAC3 in DPC detergent are not functionally relevant

A recent study1 used solution-state NMR spectroscopy to examine the interactions and dynamics of the yeast mitochondrial inner-membrane ADP/ATP carrier, yAAC3. Crystal structures of different AACs, including yAAC3, in a conformation locked with a strong inhibitor (CATR) had been determined before. This putative \"c-state\"2,3 is believed to represent one extreme conformation of an alternating access mechanism, which involves a further and yet elusive second state termed \"m-state\". Characterizing the dynamics between these states is of paramount importance to understand the transport mechanism. The authors refolded yAAC3 from inclusion bodies in the detergent dodecylphosphocholine (DPC), and observed micro-to-millisecond (s-ms) motion in part of yAAC3 using CPMG NMR experiments. The authors propose that this asymmetrically distributed dynamics, involving residues located in thr ...

biophysics↗

bioRxiv2017

Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Here we probe how the crystal packing alters microsecond dynamics, using solid-state NMR measurements and multi-microsecond MD simulations of different crystal forms of ubiquitin. In particular, NEar-Rotary-resonance Relaxation Dispersion (NERRD) experiments probe angular backbone motion, while Bloch-McConnell Relaxation Dispersion data report on fluctuations of the local electronic environment. These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. Moreover, we report small-amplitude reorientational motion of protein molecules in the crystal lattice with a [~]3-5{degrees} amplitude on a tens-of-microseconds time scale in one of the crystals, but not in others. An intriguing possibility arises that overall motion is to some extent coupled to local dynamics. Our study highlights the importance of considering the packing when analyzing dynamics of crystalline proteins.

biophysics↗