Decoding Protein Dynamics in Cells Using Chemical Cross‐Linking and Hierarchical Analysis**

Protein dynamics play a crucial role in their diverse functions. The intracellular environment significantly influences protein dynamics, particularly for intrinsically disordered proteins (IDPs). To comprehensively capture structural information from various proteins wihtin cells and characterize protein dynamics, chemical cross-linking mass spectrometry was employed. In this study, we introduce a hierarchical decoding strategy that enables the investigation of protein dynamics in vivo. Computational analysis based on distance restraints derived from cross-links is used to infer protein dynamics in cells. To facilitate this analysis, we leverage the prior structure obtained from AlphaFold2. By employing this strategy, we can characterize the full-length structure of multi-domain proteins taking into account their distinct dynamic features. Furthermore, by combining restraint sampling with an unbiased sampling and evaluation approach, we can provide a comprehensive description of the intrinsic motion of IDPs. Consequently, the hierarchical strategy we propose holds significant potential in advancing our understanding of the molecular mechanisms that undelie protein functions in cells.

Incoherent quasielastic neutron scattering (iQENS) is a fascinating technique for investigating the internal dynamics of protein. However, low flux of neutron beam, low signal to noise ratio of QENS spectrometers and unavailability of well-established analyzing method have been obstacles for studying internal dynamics under physiological condition (in solution). The recent progress of neutron source and spectrometer provide the fine iQENS profile with high statistics and as well the progress of computational technique enable us to quantitatively reveal the internal dynamic from the obtained iQENS profile. The internal dynamics of two proteins, globular domain protein (GDP) and intrinsically disordered protein (IDP) in solution, were measured with the state-of-the art QENS spectrometer and then revealed with the newly developed analyzing method. It was clarified that the average relaxation rate of IDP was larger than that of GDP and the fraction of mobile H atoms of IDP was also much higher than that of GDP. Combined with the structural analysis and the calculation of solvent accessible surface area of amino acid residue, it was concluded that the internal dynamics were related to the highly solvent exposed amino acid residues depending upon protein’s structure.

Deciphering Protein Dynamics from NMR Data Using Explicit Structure Sampling and Selection

AlphaFold 2 (AF2) has placed Molecular Biology in a new era where we can visualize, analyze and interpret the structures and functions of all proteins solely from their primary sequences. We performed AF2 structure predictions for various protein systems, including globular proteins, a multi-domain protein, an intrinsically disordered protein (IDP), a randomized protein, two larger proteins (> 1000 AA), a heterodimer and a homodimer protein complex. Our results show that along with the three dimensional (3D) structures, AF2 also decodes protein sequences into residue flexibilities via both the predicted local distance difference test (pLDDT) scores of the models, and the predicted aligned error (PAE) maps. We show that PAE maps from AF2 are correlated with the distance variation (DV) matrices from molecular dynamics (MD) simulations, which reveals that the PAE maps can predict the dynamical nature of protein residues. Here, we introduce the AF2-scores, which are simply derived from pLDDT scores and are in the range of [0, 1]. We found that for most protein models, including large proteins and protein complexes, the AF2-scores are highly correlated with the root mean square fluctuations (RMSF) calculated from MD simulations. However, for an IDP and a randomized protein, the AF2-scores do not correlate with the RMSF from MD, especially for the IDP. Our results indicate that the protein structures predicted by AF2 also convey information of the residue flexibility, i.e., protein dynamics.

Molecular dynamics in proteins animate and play a vital role for biologically relevant processes of these biomacromolecules. Quasielastic incoherent neutron scattering (QENS) is a well-suited experimental method to study protein dynamics from the picosecond to several nanoseconds and in the Ångström length-scale. In QENS experiments of protein solutions hydrogens act as reporters for the motions of methyl groups or amino acids to which they are bound. Neutron Spin-Echo spectroscopy (NSE) offers the highest energy resolution in the field of neutron spectroscopy and allows the study of slow collective motions in proteins up to several hundred nanoseconds and in the nanometer length-scale. In the following manuscript I will review recent studies that stress the relevance of molecular dynamics for protein folding and for conformational transitions of intrinsically disordered proteins (IDPs). During the folding collapse the protein is exploring its accessible conformational space via molecular motions. A large flexibility of partially folded and unfolded proteins, therefore, is mandatory for rapid protein folding. IDPs are a special case as they are largely unstructured under physiological conditions. A large flexibility is a characteristic property of IDPs as it allows, for example, the interaction with various binding partners or the rapid response to different conditions.

Crowded, Cell-like Environment Induces Shape Changes In Aspherical Protein

The ability of proteins to regulate their own enzymatic activity can be facilitated by changes in structure or protein dynamics in response to external regulators. Because many proteins contain SH2 and SH3 domains, transmission of information between the domains is a potential method of allosteric regulation. To determine if ligand binding to one modular domain may alter structural dynamics in an adjacent domain, allowing potential transmission of information through the protein, we used hydrogen exchange and mass spectrometry to measure changes in protein dynamics in the SH3 and SH2 domains of hematopoietic cell kinase (Hck). Ligand binding to either domain had little or no effect on hydrogen exchange in the adjacent domain, suggesting that changes in protein structure or dynamics are not a means of SH2/SH3 crosstalk. Furthermore, ligands of varying affinity covalently attached to SH3/SH2 altered dynamics only in the domain to which they bind. Such results demonstrate that ligand binding may not structurally alter adjacent SH3/SH2 domains and implies that other aspects of protein architecture contribute to the multiple levels of regulation in proteins containing SH3 and SH2 domains.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Prediction of the Effects of the Val66Met Polymorphism on the Conformational Ensemble of an Intrinsically Disordered Protein, Brain-Derived Neurotrophic Factor

During the last decade, network approaches became a powerful tool to describe protein structure and dynamics. Here we review the links between disordered proteins and the associated networks, and describe the consequences of local, mesoscopic and global network disorder on changes in protein structure and dynamics. We introduce a new classification of protein networks into 'cumulus-type', i.e., those similar to puffy (white) clouds, and 'stratus-type', i.e., those similar to flat, dense (dark) low-lying clouds, and relate these network types to protein disorder dynamics and to differences in energy transmission processes. In the first class, there is limited overlap between the modules, which implies higher rigidity of the individual units; there the conformational changes can be described by an 'energy transfer' mechanism. In the second class, the topology presents a compact structure with significant overlap between the modules; there the conformational changes can be described by 'multi-trajectories'; that is, multiple highly populated pathways. We further propose that disordered protein regions evolved to help other protein segments reach 'rarely visited' but functionally-related states. We also show the role of disorder in 'spatial games' of amino acids; highlight the effects of intrinsically disordered proteins (IDPs) on cellular networks and list some possible studies linking protein disorder and protein structure networks.

Molecular chaperones are diligent helpers of the cell that ensure a well-balanced proteome by regulating protein production, protein folding, refolding and holding as well as protein degradation. In doing so, molecular chaperones assist proteins throughout their lifetime. Within the proteome, i.e. the entirety of all proteins, a special feature is reserved for the group of intrinsically disordered proteins (IDPs). They inherently lack a distinct three-dimensional structure so that, when unbound, their side chains are unprotected. Thereby, IDPs are able to participate in a wide range of interactions and react sensitively to external changes. At the same time this feature bears a particularly high risk for unfavorable contacts that may in the worst case engender protein aggregation. The importance of molecular chaperones to shelter IDPs from such undesired interactions manifests in yet incurable neurodegenerations such as Alzheimer's or Parkinson's disease. In each of these disorders, there are disease specific IDPs that attach to each other, clump together, and deposit as huge protein aggregates. With regard to IDP chaperoning, a special role is assigned to the heat shock protein (Hsp) family, as they possess the unique capability of protein holding to protect proteins from undesired interactions. Thus, the binding of Hsps to IDPs gained increasing research interest. So far, mainly the interaction of IDPs, including the Alzheimer’s disease related, IDP prime example protein Tau, with individual Hsps has been studied. However, for foldable substrates, it is known that, in addition to their discrete function, Hsps can team up to drive protein turnover. And so also Hsp70 and Hsp90, which together can form the Hsp70/Hsp90 chaperone machinery to particularly prevent protein misfolding. Hence, being on the one side equipped with the marvelous capacity to counteract aggregation via the Hsp70/Hsp90 chaperone machinery and observing on the other side the deposition of insoluble Tau aggregates, poses the central question on whether the cellular defense system of the Hsp70/Hsp90 chaperone machinery isn’t likewise involved in the chaperoning of Tau. The Hsp70/Hsp90 chaperone machinery comprises the minimal system of the five proteins Hsp70, Hsp40, Hsp90, Hop and p23. In order to understand the role of each of these proteins in the Hsp70/Hsp90 chaperone machinery mediated Tau chaperoning, its stepwise assembly with distinct intermediate states was reconstituted in vitro. The analysis showed that (i) one Hop molecule stabilizes the Hsp902 dimer in a V-shaped conformation, (ii) Hsp70, Hop and Hsp90 together form the Hsp70/Hsp90 chaperone machinery comprising the Hsp702:Hop1:Hsp902 complex, (iii) Tau associates with the preassembled Hsp70/Hsp90 chaperone machinery creating a 710 kDa large (Hsp701:Hop1:Hsp902:Tau1)2 dimeric complex and that (iv) the addition of p23 stabilizes the Hsp70/Hsp90 chaperone machinery:Tau interaction generating a stable 750 kDa large (Hsp701:Hop1:Hsp902:Tau1:p231)2 complex. (v) By means of the co-chaperone CHIP, which marks proteins for proteasomal degradation, it was further shown that the (Hsp701:Hop1:Hsp902:Tau1:p231)2 complex is no irreversible deadlock but part of a co-chaperone-mediated dynamic equilibrium between protein holding via Hop and p23, and protein degradation induced by CHIP. (vi) The Hsp70/Hsp90 chaperone machinery was revealed to control normal as well as abnormal Tau, as pathologically modified, hyperphosphorylated Tau (PTau) equally associated with the Hsp70/Hsp90 chaperone machinery forming Hsp70:Hop:Hsp90:PTau and Hsp70:Hop:Hsp90:PTau:p23 complexes. Altogether, the Hsp70/Hsp90 chaperone machinery was revealed as interaction partner and thus potential chaperone system for the intrinsically disordered protein Tau. An integrated approach of biochemical and biophysical methods including native page, tryptophane fluorescence, isothermal titration calorimetry, nuclear magnetic resonance, dynamic light scattering, sucrose density gradient centrifugation, chromatography and chemical cross-linking coupled to mass spectrometry was used to describe a detailed model of stepwise machinery assembly accompanied with Tau binding. The knowledge gained about binding sites, conformational changes, stoichiometries and affinities presents novel structural and biochemical insights into the IDP chaperoning by the Hsp70/Hsp90 chaperone machinery. The obtained data thus may serve as fundamental basis for high-resolution structure determination and future research directions deciphering why the Hsp70/Hsp90 chaperone machinery fails to protect against Tau aggregation during disease.

Capturing the Cooperativity of Backbone Hydrogen Bonding with Polarizable Force Fields

The DNA mismatch repair (MMR) factor Mlh1–Pms1 contains long intrinsically disordered regions (IDRs) whose exact functions remain elusive. We performed cross-linking mass spectrometry to identify interactions within Mlh1–Pms1 and used this information to insert FRB and FKBP dimerization domains into their IDRs. Baker's yeast strains bearing these constructs were grown with rapamycin to induce dimerization. A strain containing FRB and FKBP domains in the Mlh1 IDR displayed a complete defect in MMR when grown with rapamycin. but removing rapamycin restored MMR functions. Strains in which FRB was inserted into the IDR of one MLH subunit and FKBP into the other subunit were also MMR defective. The MLH complex containing FRB and FKBP domains in the Mlh1 IDR displayed a rapamycin-dependent defect in Mlh1–Pms1 endonuclease activity. In contrast, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization inappropriately activated Mlh1–Pms1 endonuclease activity. We conclude that dynamic and coordinated rearrangements of the MLH IDRs both positively and negatively regulate how the MLH complex acts in MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.

Theoretical perspectives on nonnative interactions and intrinsic disorder in protein folding and binding

Hydrogen exchange rates at peptide backbone amide linkages of proteins are highly sensitive to changes in protein structure and dynamics. Hydrogen exchange-mass spectrometry (HX-MS) can be used to measure hydrogen/deuterium exchange in protein fragments and allows the study of large proteins with high sensitivity and at very low concentrations as well as determination of the intermolecular distribution of deuterium. This unit provides a brief discussion of the interplay between protein dynamics and hydrogen exchange, a detailed prescription for designing and performing typical HX-MS experiments, and common procedures for processing and interpreting HX-MS data.