Aug 2026· International Journal of Molecular Sciences· 0 citations· 99 references
TL;DR
Computed collisional cross-sections from compacted “o-shape” and extended “l-shape” protein G three-dimensional (3D) structures match experimental data, indicating equilibrium, and suggest a dynamic “o2l” flip process.
Abstract
Studying protein structure dynamics is key to understanding protein function modulation. Alternative protein conformations are well discriminated from each other by nanoESI mass spectrometry and ion mobility measurements. Experimentally determined collisional cross-sections were compared to calculated collisional cross-sections of fifteen peptides, single-domain proteins, and protein complexes. The multi-domain proteins investigated here, four immunoglobulin G (IgG) antibodies and protein G, are present as compacted/folded “native” conformations in neutral buffered solutions, and they are identified by molecular ions with narrow charge-state distributions, relatively few charges, and small collisional cross-sections. Simultaneously present extended/folded but nevertheless “native” conformations produced additional ions with higher charge states, different charge-state distributions, and larger collisional cross-sections. Computed collisional cross-sections from compacted “o-shape” and extended “l-shape” protein G three-dimensional (3D) structures match experimental data, indicating equilibrium, and suggest a dynamic “o2l” flip process. Likewise, “m-shape” (compacted) and “Y-shape” (extended) IgGs are regarded as two supposedly reversibly adopted antibody conformations which may interchange by an “m2Y” flip. Adopting an m-shape would prevent an antibody-based initiation of humoral and cellular immune system responses, such as opsonophagocytosis, prior to antigen contact, which stands in line with the rearrangement hypothesis.
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
It is proposed that the consistency principle is formulated by the quadratic relationship in the double logarithm plot of the residue-specific equilibrium and rate constants of a polypeptide chain.
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
Understanding the mechanical stability and architecture of viral proteins can provide valuable information about their biological function, but it remains a significant biophysical challenge. This study employs single-molecule force spectroscopy (SMFS) to investigate the multi-domain architecture of the Orsay virus δ protein, which lacks repeat structures and exhibits weak unfolding peaks. We engineered a construct using titin (I27)4 domains as an internal molecular ruler, enabling us to bracket the δ protein peaks to determine domain length and identify unfolding forces with an atomic force microscope (AFM). To address limitations of one-dimensional (1D) force distributions in resolving overlapping structural states, we created a two-dimensional (2D) mechano-structural signature map. By plotting kinetic stability (unfolding force F) against physical structural footprint (domain length L), we distinguished distinct unfolding domains, successfully separating degenerate 1D data into two statistically distinct populations corresponding to the δ protein’s internal domain (I) and C-terminal domain (C). This label-free method provides the first mechanical evidence of the δ protein’s multi-domain architecture. It establishes a robust, multi-dimensional framework for decoding the mechanics of complex biomolecular assemblies in their native state.
Cynthia S. Deem, Sithara S. Wijeratne, Tsung-Cheng Lin et al.· bioRxiv· 0 citations
The data reveal that both the mechanics and kinetics of peptide detachment are sensitive to the identity and sequence position of individual residues, highlighting the power of integrating CG MD and single-molecule force spectroscopy to unravel residue-specific, sequence-dependent factors underlying peptide–lipid interactions.
Ryan S. Smith, Krishna P Sigdel, D. R. Weaver et al.· Langmuir· 0 citations
RNA interactions are a key contributor to the formation and disassembly of intracellular protein condensates. Although some proteins utilize specific RNA-binding domains, these processes can also be mediated by charge interactions with intrinsically disordered regions. Due to the dynamic nature of these systems, investigating the underlying specificity and stoichiometry remains challenging. Here, we demonstrate that single-molecule mass measurements with mass photometry can capture RNA-protein interactions in phase-separated protein systems. Using the approach to investigate RNA-mediated phase shifts of tau condensates, we find that increasing the RNA concentration, which promotes phase re-entry, results in RNA-mediated tau multimerization, where each tau monomer binds a linear RNA sequence of approximately 30 nucleotides. Solution NMR and native mass spectrometry confirm the formation of stable complexes between RNA and the basic proline-rich and repeat domains of tau, which have a net charge of -29. Our findings demonstrate that mass photometry can distinguish between charge neutralization, which drives coacervation, and complex formation, which mediates phase re-entry, making it a highly complementary tool for the study of RNA-mediated phase separation.
Axel Leppert, Jesper Shiapan, Irena Papageorgiou et al.· bioRxiv· 0 citations
Related blog posts
MIT News · Artificial Intelligence· news.mit.eduAug 27, 2026
A new machine-learning framework aims to improve the success rate of computational protein design while moving away from results that reproduce sequences found in nature.
