Gert E. Folkers

Protein structures can now be predicted and designed at scale, yet experimental access to dynamics and conformational heterogeneity remains limited in throughput. This gap prevents a systematic understanding of how protein sequences encode motion and functional flexibility. Here, we establish a scalable experimental pipeline combining protein design, automated production, and nuclear magnetic resonance (NMR) spectroscopy to enable high-throughput characterization of protein structure and dynamics at atomic resolution. A single operator can produce and analyze hundreds of isotopically labeled proteins per week, with per-sample cost largely defined by DNA synthesis. To benchmark this approach, we experimentally characterized 384 de novo designed proteins spanning diverse regions of structure space. High-quality two-dimensional NMR spectra were obtained for 239 samples (62% of designs overall). NMR characterization confirmed that the designed proteins adopt their intended folds, and revealed unexpected local dynamics that are not captured by current computational models. Our approach establishes a foundation for data-driven modelling of sequence–structure–dynamics relationships and unlocks a new regime of statistical structural biology, where insight into protein biophysics is gained from experimental ensemble studies of suitably designed protein clusters.

Visualizing a protein's molecular motions has been a long standing topic of research in the biophysics community. Largely this has been done by exploiting nuclear magnetic resonance spectroscopy (NMR), and arguably no protein's molecular motions have been better characterized by NMR than that of ubiquitin (Ub), a 76 amino acid polypeptide essential in ubiquitination‐a key regulatory system within cells. Herein, we discuss ubiquitin's conformational plasticity as visualized, at atomic resolution, by more than 35 years of NMR work. In our discussions we point out the differences between data acquired in vitro, ex vivo, as well as in vivo and stress the need to investigate Ub's conformational plasticity in more biologically representative backgrounds.

Sensitivity enhanced dynamic nuclear polarization solid‐state NMR is emerging as a powerful technique for probing the structural properties of conformationally homogenous and heterogenous biomolecular species irrespective of size at atomic resolution within their native environments. Herein we detail advancements that have made acquiring such data, specifically within the confines of intact bacterial and eukaryotic cell a reality and further discuss the type of structural information that can presently be garnered by the technique's exploitation. Subsequently, we discuss bottlenecks that have thus far curbed cellular DNP‐ssNMR's broader adoption namely due a lack of sensitivity and spectral resolution. We also explore possible solutions ranging from utilization of new pulse sequences, design of better performing polarizing agents, and application of additional biochemical/ cell biological methodologies.

High-resolution NMR spectroscopy enabled us to characterize allosteric transitions between various functional states of the dimeric Escherichia coli Lac repressor. In the absence of ligands, the dimer exists in a dynamic equilibrium between DNA-bound and inducer-bound conformations. Binding of either effector shifts this equilibrium toward either bound state. Analysis of the ternary complex between repressor, operator DNA, and inducer shows how adding the inducer results in allosteric changes that disrupt the interdomain contacts between the inducer binding and DNA binding domains and how this in turn leads to destabilization of the hinge helices and release of the Lac repressor from the operator. Based on our data, the allosteric mechanism of the induction process is in full agreement with the well-known Monod–Wyman–Changeux model.

Solution‐state NMR spectroscopy has become a powerful tool to study soluble proteins in cells, provided that they tumble sufficiently fast. In addition, cryo‐electron tomography (cryo‐ET) has recently displayed a tremendous potential to probe structures of large proteins and assemblies in their native cellular environments. However, challenges remain to obtain atomic‐level information in native cell settings for proteins that are small, disordered, or are strongly engaged in intermolecular interactions. In this Minireview, we discuss recent progress in using sensitivity enhanced solid‐state NMR spectroscopy methods in the context of cellular structural biology.

Elucidating at atomic level how proteins interact and are chemically modified in cells represents a leading frontier in structural biology. We have developed a tailored solid‐state NMR spectroscopic approach that allows studying protein structure inside human cells at atomic level under high‐sensitivity dynamic nuclear polarization (DNP) conditions. We demonstrate the method using ubiquitin (Ub), which is critically involved in cellular functioning. Our results pave the way for structural studies of larger proteins or protein complexes inside human cells, which have remained elusive to in‐cell solution‐state NMR spectroscopy due to molecular size limitations.

Elucidating at atomic level how proteins interact and are chemically modified in cells represents a leading frontier in structural biology. We have developed a tailored solid‐state NMR spectroscopic approach that allows studying protein structure inside human cells at atomic level under high‐sensitivity dynamic nuclear polarization (DNP) conditions. We demonstrate the method using ubiquitin (Ub), which is critically involved in cellular functioning. Our results pave the way for structural studies of larger proteins or protein complexes inside human cells, which have remained elusive to in‐cell solution‐state NMR spectroscopy due to molecular size limitations.

Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways. Here we review the function of ERCC1-XPF in various DNA repair pathways and discuss human disorders associated with ERCC1-XPF deficiency. We also overview our molecular and structural understanding of XPF-ERCC1.