Jul 2026· Current Opinion in Structural Biology· Vol 99, pp.
103328
· 0 citations· 80 references
Medicine
Abstract
The advent of machine learning structural prediction tools has largely accelerated bottom-up structural biology and provides static Anfinsenian models with near-experimental accuracy, complementing X-ray crystallography and cryogenic electron microscopy. However, these techniques often overlook two fundamental aspects of biomolecular structure-function relationships: (i) conformational equilibria and (ii) transient noncovalent interactions. Indeed, these considerations are particularly important for integral membrane proteins (MPs), frequently serving mechanistic roles, such as conformational plasticity in ion channels facilitating heterogeneous state transitions, or dynamic binding partners modulating signal transduction in receptors and small-molecule transporters. In this vein, electron paramagnetic resonance (EPR) spectroscopy, an ensemble method, provides a powerful toolbox to guide high-resolution structural data by directly reporting on conformational subensembles and noncovalent binding events. Herein, we highlight recent EPR applications to integral MPs and emerging synergies with bottom-up atomistic approaches to enhance insights into structure-dynamics-function relationships.
Heat shock protein 90 kDa (Hsp90) is an ATP-dependent molecular chaperone whose function relies on conformational rearrangements and interactions with cochaperones and clients. Binding of nucleotides, cochaperones, and clients leads to functional structural rearrangements, often associated with heterogeneous structural ensembles that are challenging to characterize using high-resolution structure determination methods alone. Electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach for tracking the conformational landscape of proteins. However, despite its great potential, its application to Hsp90 homologs has so far remained limited, and the insights gained have not yet been comprehensively reviewed. In this mini-review, we summarize EPR studies on cytosolic, mitochondrial, and endoplasmic reticulum Hsp90 with the aim of highlighting how EPR has advanced our understanding of nucleotide- and partner-dependent conformations. Specifically, we summarize how continuous wave EPR (CW-EPR), double electron–electron resonance (DEER), and electron nuclear double resonance (ENDOR) spectroscopies revealed conformational equilibria, symmetry breaking, dissociation constants, and the structural role of intrinsically disordered regions within Hsp90, while also discussing methodological advances and recent extensions to cellular contexts. Overall, these studies establish EPR spectroscopy as a valuable biophysical tool for resolving conformational ensembles and structural heterogeneity underlying Hsp90’s function, with general relevance to the structural biology, spectroscopy, and chaperone fields, and pave the way for future in vitro and in-cell investigations of this exciting molecular chaperone.
Unknown authors· Journal of Physical Chemistr...· 0 citations
Proteins are intrinsically dynamic molecules that continuously explore conformational ensembles to execute biological functions. Conventional structural biology methods rely on in vitro reconstitution of purified components and therefore capture predominantly static snapshots, often overlooking the regulatory roles of the cellular microenvironment, such as molecular crowding, weak interaction networks, and post-translational modifications. This limitation has driven an urgent need to transition from in vitro reconstruction to in vivo characterization within living cells. Nuclear magnetic resonance (NMR) spectroscopy provides atomic-resolution insights into structure and motions spanning multiple timescales, yet its application is constrained by molecular weight limits, isotopic labeling requirements, and inherently low throughput. Cross-linking mass spectrometry (XL-MS) complements NMR by delivering sparse but long-range spatial restraints without an upper molecular weight limit. The integration of NMR and XL-MS establishes a powerful synergistic framework that bridges atomic-resolution local structures and large-scale interaction topologies, thereby enabling comprehensive characterization of protein dynamic conformations and interaction networks in native environments. Here, we review how this integrative strategy advances the understanding of intrinsically disordered proteins, multi-domain proteins, and dynamic protein-protein interaction networks in native cellular environments. We further discuss emerging technological frontiers, including hyperpolarized NMR, photo-cross-linking, organelle-resolved analysis, and artificial intelligence-guided integrative modeling, which together promise to transform our ability to resolve the true functional states of proteins inside cells.
Zhou Gong, Qun Zhao, Min Sun et al.· Magnetic Resonance Letters· 0 citations
Determining the higher order structure (HOS) of proteins and protein complexes is central to understanding their functions, dynamics, and interactions. Traditional structural biology approaches, such as X-ray crystallography and nuclear magnetic resonance, provide high-resolution snapshots but often require large amounts of homogeneous samples and may miss dynamic or heterogeneous states. Mass spectrometry (MS) has become an indispensable tool for sensitive and rapid analysis of intact proteins and assemblies under native or near-native conditions. This review discusses the major MS-based strategies for probing HOS. Native mass spectrometry (nMS) preserves non-covalent interactions and exhibits characteristic charge-state distributions that report on folding, while native top-down fragmentation and ion mobility spectrometry provide sequence-specific and conformation-specific information. Hydrogen–deuterium exchange MS measures backbone amide exchange rates to map regions of solvent accessibility, ligand binding, and allosteric regulation in solution. Covalent labeling MS irreversibly modifies solvent-accessible side chains, allowing epitope mapping and detection of subtle conformational changes, while fast photochemical oxidation of proteins offers microsecond snapshots of transient structures. Chemical cross-linking MS applies bifunctional reagents to capture proximity between residues or subunits, providing distance restraints for integrative modeling and proteome-wide interaction mapping. We outline recent advances in instrumentation, software, labeling chemistry and in-cell techniques across these modalities, and we illustrate their applications to characterizing membrane proteins, large assemblies, therapeutic antibodies, intrinsically disordered proteins, and protein–ligand complexes. Together, these tools offer complementary insights into HOS that are reshaping structural biology, biopharmaceutical development and mechanistic studies.
Dulakshi Herath, Kaitlyn N. Walls, Ashlyn N. Dollar et al.· In Analysis· 0 citations
This study illustrates how subtle residue-localized conformational bias can affect the overall protein conformational dynamics influencing protein-protein interactions that are important for cellular functions and related to diseases.
Abir Ben Bouzayene, M. Sai, Alexis Jouin et al.· Angewandte Chemie· 0 citations
It is demonstrated that BioEmu can generate plausible conformational ensembles for relatively large, six-and seven-pass membrane proteins, sampling rare states at a fraction of the computational cost of conventional MD simulations, suggesting that AI-based ensemble generation could provide an accessible approach for exploring membrane protein dynamics and complement conventional molecular modelling approaches.
B. Clifton, Adam G Grieve, Robin A. Corey· bioRxiv· 0 citations
Recent developments in integrative modeling of protein-RNA complexes are reviewed, highlighting advances in in-cell, 4D and condensate structural biology, and how these approaches shape the understanding of RNP assembly, regulation, and function in physiologically relevant environments are discussed.
S. Heber, Janosch Hennig· Current Opinion in Structura...· 1 citation
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