Biology subjects

Liebau, J.

Publications and source records attributed to Liebau, J..

5 recordsLinked to original sources

bioRxiv2026

Electrostatic control of conformational dynamics drives proton translocation in respiratory Complex I

Complex I is a large membrane protein that transduces redox energy into a proton motive force across a biological membrane, powering cellular respiration. Yet, despite significant experimental and computational efforts, its long-range proton translocation mechanism remains poorly understood. Here, we combine nuclear magnetic resonance spectroscopy and site-directed mutagenesis experiments with multiscale classical and hybrid quantum/classical (QM/MM) simulations to probe how conformational dynamics is linked to proton transfer reactions in the terminal antiporter module (Nqo12/NuoL/ND5) of Complex I. We show that protonation of a conserved proton-loading site (Lys329/His241Nqo12) in the terminal antiporter-like module gives rise to conformational changes that result in opening of the proton pathway. We further show that this protonation reaction is controlled by buried ion-pairs that modulate the barrier for proton translocation by intrinsic electric field effects. On a general level, our combined findings reveal how bioenergetic machines combine conformational dynamics with hydration transitions to establish efficient redox-driven proton pumps.

biophysics↗

bioRxiv2026

Functional dynamics of the eukaryotic RNA exosome upon complex formation

Rather than being static, proteins adopt multiple conformations and undergo dynamics on a wide range of timescales. Such conformational changes can be routinely probed by solution state NMR for protein sizes below 40 kDa and can be extended to large protein complexes with selective methyl-labeling strategies. Here, we expand the limit of methyl-NMR dynamics experiments to asymmetric protein complexes bigger than 300 kDa by investigating the inert core of the eukaryotic RNA exosome, Exo9. We demonstrate that isolated subunits are highly dynamic on the ms-s dynamics, which facilitates assembly. While ring subunits lose dynamics upon complex-formation, cap subunits and functional loops retain a large degree of flexibility as demonstrated by relaxation dispersion and chemical exchange saturation transfer experiments in the fully asymmetric Exo9 complex. Employing EPR spectroscopy we additionally revealed changes in the conformational ensemble and nanosecond dynamics of a spin-labeled functional loop region in the presence of the catalytic subunit Rrp44 and in response to substrate RNA binding. We thus demonstrate that an integrated NMR-EPR approach facilitates the characterization of functional dynamics that occur on a wide range of timescales in very large protein complexes.

biophysics↗

bioRxiv2026

Buried Ion-Pairs Reshape Folding Pathways of Artificial Proteins

Ion-pairs are central charged motifs that control the biological function of several proteins. Yet, the physical principles linked to the formation of ion-pairs during protein folding remain poorly understood. Here, we show that buried ion-pairs of de novo-designed helical bundle proteins reshape the folding pathways by introducing kinetic barriers that are overcome by a hydrophobic collapse linked to packing of the protein core. By combining protein design with co-translational folding studies, nuclear magnetic resonance (NMR) spectroscopy, and molecular dynamics simulations, we show that introduction of a buried ion-pair leads to a shifted folding transition that balances desolvation and electrostatic interactions within the protein and the bulk solvent. Moreover, we find that the folding pathway is sensitive to modifications in non-polar interactions and motifs that electrostatically shield the ion-pair from its surroundings. Our combined findings provide a blueprint for understanding how functional motifs affect protein folding and how mutation of charged networks alter the stability of proteins.

biophysics↗

bioRxiv2026

Nanosecond methyl dynamics in the eukaryotic RNA exosome core

Dynamics in proteins occur on a wide range of timescales and are crucial for protein function. On the fast end of that timescale, pico- to nanosecond dynamics have been extensively employed as proxies for entropy and their amplitude can be described by order parameters. Experimentally, NMR can be used to determine order parameters of the protein backbone and, via deuterium relaxation, of methyl groups, yet such experiments cannot be applied to large protein assemblies. In contrast, relaxation-violated coherence transfer experiments, that allow for the determination of side chain order parameters in highly deuterated, methyl-labeled proteins, are more sensitive. Here, we demonstrate that such experiments can be applied to very large, asymmetric protein assemblies by determining axial methyl order parameters for the 300 kDa fully asymmetric core of the eukaryotic RNA exosome complex. Ile-{delta}1[13CH3] methyl groups adopt a wide range of order parameters but highly flexible side chains are infrequent. High quality data, which we obtain for flexible regions, is required to observe subtle effects of RNA binding on order parameters. Local cryo-EM Q-scores correlate moderately with order parameters suggesting that Q-scores contain information on nanosecond motions. AF2{chi}, a recently described prediction tool for side-chain variability, provides good estimates of methyl order parameters, which are, in favorable cases, strongly correlated with experimental values. We thus demonstrate that relaxation-violated coherence transfer experiments can be employed to determine order parameters in large, asymmetric protein complexes that are difficult to capture by other methods, yet are crucial for the understanding of protein function. SignificanceNanosecond side chain dynamics contribute to the entropy of proteins and are therefore proxies for protein stability and binding. Here, we demonstrate that NMR can be employed to experimentally quantify nanosecond dynamics in large, asymmetric proteins paving the way to assess contributions of fast dynamics to the quality of static protein structures. Furthermore, we employ the experimental data to validate computational methods that provide structural insights into nanosecond dynamics.

biophysics↗

bioRxiv2024

Beyond static structures: quantitative dynamics in the eukaryotic RNA exosome complex

Molecular machines play pivotal roles in all biological processes. Most structural methods, however, are unable to directly probe molecular motions. Here, we demonstrate that dedicated NMR experiments can provide quantitative insights into functionally important dynamic regions in very large asymmetric protein complexes. We establish this for the 410 kDa eukaryotic RNA exosome complex that contains ten distinct protein chains. Methyl-group and fluorine NMR experiments reveal site-specific interactions among subunits and with an RNA substrate. Furthermore, we extract quantitative insights into conformational changes within the complex in response to substrate and subunit binding for regions that are invisible in static cryo-EM and crystal structures. In particular, we identified a flexible plug region that can block an aberrant route of RNA towards the active site. Based on molecular dynamics simulations and NMR data we provide a model that shows how the flexible plug is structured in the open and closed conformations. Our work thus demonstrates that a combination of state-of-the-art structural biology methods can provide quantitative insights into large molecular machines that go significantly beyond the well-resolved and static images of biomolecular complexes, thereby adding the time domain into structural biology.

molecular biology↗