Aug 2026· Angewandte Chemie· pp.
e4052471
· 0 citations· 24 references
Medicine
TL;DR
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.
Abstract
Recently, the application of deep learning to structural data deposited in the Protein Data Bank has enabled the reliable and accurate prediction of 3D folded structures of proteins from their sequences. However, this approach is not applicable to highly dynamic proteins, where multiple structures interconvert. Furthermore, the mechanistic details of protein folding and unfolding remain challenging to study. Herein, we present a set of data highlighting these complexities. By chemical incorporation of stereoisomeric 4-fluoroproline residues at selected sites in the sequence of a folded, multi-conformational protein ("molten-globule"), we were able to modify its structural properties that propagated to highly distinct functional features, such as modulation of ligand binding affinities or misfolding and aggregation into amyloids. Application of NMR methods, notably 19F NMR spectroscopy, provided detailed molecular insights into the observed phenomena. 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.
Tryptophan residues play a critical role in protein-ligand recognition owing to their unique aromatic character, high polarizability, and ability to participate in diverse noncovalent interactions, including π-π stacking, cation-π interactions, hydrogen bonding, and hydrophobic contacts. In this study, a two-stage approach was employed to elucidate the functional significance of tryptophan residues within ligand-binding sites across diverse protein systems. First, a large-scale statistical analysis was performed using a curated dataset of protein-ligand complexes extracted from the BioLiP database. The dataset was analysed with respect to enzymatic classification and Gene Ontology enrichment, revealing a pronounced enrichment of tryptophan residues in the binding sites of hydrolases, oxidoreductases, and transferases, as well as strong associations with metal-ion binding, redox-related functions, and membrane or cytoplasmic localization. Within the G protein-coupled receptor (GPCR) superfamily, rhodopsin-like class A receptors were identified as the most prominently represented group featuring tryptophan-mediated ligand interactions. In the second stage, molecular dynamics simulations were conducted for selected serotonin 5-HT₂ receptor subtypes (5-HT₂A, 5-HT₂B, and 5-HT₂C) to provide atomistic insights into the role of the conserved W6.48 residue. The simulations reveal a stable spatial relationship between the ligand and W6.48, characterized by distances consistent with aromatic anchoring, supporting its role in maintaining ligand orientation and binding pocket organization. Collectively, these results highlight tryptophan as a key determinant of ligand recognition and stabilization across both enzymatic and receptor-mediated systems.
Karina Pakosz, Paweł Śliwa· Science Technology and Innov...· 0 citations
This work investigates how protein folding landscapes are altered inside condensates, using the protein α-helix as a model folded domain and develops a chemically specific, residue-resolution model for quantification of α-helical folding and applies it to characterize diverse helices within condensates of varying physicochemical properties.
Nathaniel Hess, Jerelle A. Joseph· Journal of the American Chem...· 0 citations
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.
Joshua L. Wort, Xinyu Liu, James Pullen et al.· Current Opinion in Structura...· 0 citations
The consistency principle represents a physicochemical condition requisite for ideal protein folding. It assumes that any pair of amino acid residues in partially folded structures has an attractive short-range interaction only if the two residues are in contact within the native structure. The residue-specific equilibrium constant, K, and the residue-specific rate constant, k (forward and backward), can be determined by NMR and hydrogen-deuterium exchange studies. Linear free energy relationships (LFER) in the rate-equilibrium free energy relationship (REFER) plots (i.e., log k vs. log K) are widely seen in protein-related phenomena, but our REFER plot differs from them in that the data points are derived from one polypeptide chain under a single condition. Here, we examined the theoretical basis of the residue-based LFER. First, we derived a basic equation, ρij = ½(ϕi + ϕj), from the consistency principle, where ρij is the slope of the line segment that connects residues i and j in the REFER plot, and ϕi and ϕj are the local fractions of the native state in the transient state ensemble (TSE). Next, we showed that the general solution is the alignment of the (log K, log k) data points on a parabolic curve in the REFER plot. Importantly, unlike LFER, the quadratic free energy relationship (QFER) is compatible with the heterogenous formation of local structures in the TSE. Residue-based LFER/QFER provides a unique insight into the TSE: A foldable polypeptide chain consists of several folding units, which are consistently coupled to undergo smooth structural changes. Significance The physicochemical basis of smooth protein folding has been theoretically explained by the consistency principle. We propose 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. The quadratic relationship offers a procedure for the experimental verification of the consistency principle. One application is a ϕ-value analysis, free from the adverse effects of mutations. These results will trigger the development of experimental techniques that enable the determination of accurate residue-specific equilibrium and kinetic parameters for analyzing the transition states of structural changes in proteins.
Peptide–lipid membrane interactions underlie many essential biological processes, yet the molecular determinants of peptide partitioning and dissociation from lipid bilayers remain incompletely understood. Here, we combine coarse-grained molecular dynamics (CG MD) simulations and atomic force microscopy (AFM)-based force spectroscopy to study the structural dynamics, energetics, and kinetics of penta-X5 peptides (WLLLX, with X = R or I) interacting with POPC bilayers. To elucidate how the position and identity of a single guest residue X modulates peptide–membrane interactions, we present these results in the context of the canonical Wimley–White penta-X (WLXLL) motif. Our findings from CG simulations are consistent with penta-X5 peptides adopting snorkeling conformations beneath the bilayer surface and with a dissociation scenario in which the final two or three residues detach almost simultaneously under the applied pulling force. Ensemble analyses of the reconstructed potential of mean force profiles lead to multiple energetic dissociation pathways. In combination with kinetic modeling of AFM rupture force distributions, the data reveal that both the mechanics (dissociation force) and kinetics (off rate) of peptide detachment are sensitive to the identity and sequence position of individual residues. These results highlight 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
Glycosaminoglycans (GAGs) play diverse and fundamental roles in physiology by regulating the function of large classes of proteins. Despite their importance, knowledge of how GAGs are organized in protein-bound complexes remains limited. This can be attributed to the linear structure, conformational flexibility, and high negative charge of GAGs, all of which disfavor structure determination by crystallography or NMR spectroscopy. A hybrid approach based on GAG-binding-induced changes in NMR protein chemical shifts, computational docking, and molecular dynamics simulations has proven to be valuable in providing structural models. However, these approaches can identify multiple plausible GAG geometries, making it difficult to determine whether the observed geometries reflect intrinsic plasticity or limitations of the NMR data and docking methods. In the case of chemokine CXCL8, two GAG-binding modes have been proposed, one within a monomer and the other across the dimer interface. Here, we determined low-resolution solution structures of heparin and chondroitin sulfate octasaccharides bound to the CXCL8 dimer using small-angle X-ray scattering (SAXS). SAXS analyses show that both heparin and chondroitin sulfate bind to a surface within a monomer and are incompatible with binding across the dimer. NMR paramagnetic relaxation enhancement measurements for heparin-bound CXCL8 dimer and monomer complexes show that heparin engages a similar surface within the monomer in both complexes, consistent with the SAXS models. Together, these studies establish how GAGs are organized in the CXCL8-bound complex and highlight the value of complementary low-resolution structural methods for characterizing GAG-protein complexes.
M. A. White, B. Mahler, P. R. B. Joseph et al.· Biochemical Journal· 0 citations
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