RESUMEN
When two proteins bind to each other, this process is often accompanied by a change in their structural states (from disordered to ordered or vice versa). As it turns out, there are 10 distinct possibilities for such binding-related order/disorder transitions. Out of this number, seven scenarios have been experimentally observed, while another three remain hitherto unreported. As an example, we discuss the so-called mutual synergistic folding, whereby two disordered proteins come together to form a fully structured complex. Our bioinformatics analysis of the Protein Databank found potential new examples of this remarkable binding mechanism.
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TipA, a MerR family transcription factor from Streptomyces lividans, promotes antibiotic resistance by sequestering broad-spectrum thiopeptide-based antibiotics, thus counteracting their inhibitory effect on ribosomes. TipAS, a minimal binding motif which is expressed as an isoform of TipA, harbors a partially disordered N-terminal subdomain that folds upon binding multiple antibiotics. The extent and nature of the underlying molecular heterogeneity in TipAS that shapes its promiscuous folding-function landscape is an open question and is critical for understanding antibiotic-sequestration mechanisms. Here, combining equilibrium and time-resolved experiments, statistical modeling, and simulations, we show that the TipAS native ensemble exhibits a pre-equilibrium between binding-incompetent and binding-competent substates, with the fully folded state appearing only as an excited state under physiological conditions. The binding-competent state characterized by a partially structured N-terminal subdomain loses structure progressively in the physiological range of temperatures, swells on temperature increase, and displays slow conformational exchange across multiple conformations. Binding to the bactericidal antibiotic thiostrepton follows a combination of induced-fit and conformational-selection-like mechanisms, via partial binding and concomitant stabilization of the binding-competent substate. These ensemble features are evolutionarily conserved across orthologs from select bacteria that infect humans, underscoring the functional role of partial disorder in the native ensemble of antibiotic-sequestering proteins belonging to the MerR family.
Asunto(s)
Antibacterianos , Proteínas Bacterianas , Pliegue de Proteína , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Streptomyces lividans/metabolismo , Streptomyces lividans/genética , Unión Proteica , Conformación Proteica , Modelos Moleculares , Factores de Transcripción/metabolismo , Factores de Transcripción/químicaRESUMEN
The spindle checkpoint complex is a key surveillance mechanism in cell division that prevents premature separation of sister chromatids. Mad2 is an integral component of this spindle checkpoint complex that recognizes cognate substrates such as Mad1 and Cdc20 in its closed (C-Mad2) conformation by fastening a "seatbelt" around short peptide regions that bind to the substrate recognition site. Mad2 is also a metamorphic protein that adopts not only the fold found in C-Mad2, but also a structurally distinct open conformation (O-Mad2) which is incapable of binding substrates. Here, we show using chemical exchange saturation transfer (CEST) and relaxation dispersion (CPMG) NMR experiments that Mad2 transiently populates three other higher free energy states with millisecond lifetimes, two in equilibrium with C-Mad2 (E1 and E2) and one with O-Mad2 (E3). E1 is a mimic of substrate-bound C-Mad2 in which the N-terminus of one C-Mad2 molecule inserts into the seatbelt region of a second molecule of C-Mad2, providing a potential pathway for autoinhibition of C-Mad2. E2 is the "unbuckled" conformation of C-Mad2 that facilitates the triage of molecules along competing fold-switching and substrate binding pathways. The E3 conformation that coexists with O-Mad2 shows fluctuations at a hydrophobic lock that is required for stabilizing the O-Mad2 fold and we hypothesize that E3 represents an early intermediate on-pathway towards conversion to C-Mad2. Collectively, the NMR data highlight the rugged free energy landscape of Mad2 with multiple low-lying intermediates that interlink substrate-binding and fold-switching, and also emphasize the role of molecular dynamics in its function.
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Molecular recognition events mediated by glycans play pivotal roles in controlling the fate of diverse biological processes such as cellular communication and the immune response. The affinity of glycans for their target receptors is governed primarily by the hydrogen bonds formed by hydroxyl groups decorating the glycan surface. Hydroxyl exchange rate constants are therefore vital parameters that report on glycan structure and dynamics. Here we present a strategy for characterizing hydroxyl hydrogen/deuterium (H/D) exchange in glycans that employs a synergistic combination of 13C chemical exchange saturation transfer (CEST) and Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG) NMR methods. We show that the combination of CEST and CPMG experiments facilitates the sensitive detection of the small (â¼0.1 ppm) two-bond deuterium isotope shift on a 13C nucleus when the attached hydroxyl group fluctuates between protonated and deuterated states. This shift is leveraged for measuring site-specific kinetic H/D exchange rate constants as well as thermodynamic free energies of isotope fractionation. The CEST and CPMG modules are integrated with a selective J-cross-polarization scheme that provides the flexibility for rapid characterization of H/D exchange at a specific hydroxyl site. Moreover, our approach enables the precise isothermal measurement of hydroxyl exchange rate constants without the need for cumbersome isotope labeling. The H/D exchange rate constants of three different glycans assessed using this method highlight its potential for detecting transient intra- and intermolecular hydrogen bonds. In addition, the trends in H/D exchange rate constants establish site-specific steric accessibility as a key determinant of solvent exchange dynamics in glycans.
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Proteínas Portadoras , Imagen por Resonancia Magnética , Deuterio , Espectroscopía de Resonancia Magnética/métodos , Radical Hidroxilo , Polisacáridos , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
Over 40% of eukaryotic proteomes and 15% of bacterial proteomes are predicted to be intrinsically disordered based on their amino acid sequence. Intrinsically disordered proteins (IDPs) exist as heterogeneous ensembles of interconverting conformations and pose a challenge to the structure-function paradigm by apparently functioning without possessing stable structural elements. IDPs play a prominent role in biological processes involving extensive intermolecular interaction networks and their inherently dynamic nature facilitates their promiscuous interaction with multiple structurally diverse partner molecules. NMR spectroscopy has made pivotal contributions to our understanding of IDPs because of its unique ability to characterize heterogeneity at atomic resolution. NMR methods such as Chemical Exchange Saturation Transfer (CEST) and relaxation dispersion have enabled the detection of 'invisible' excited states in biomolecules which are transiently and sparsely populated, yet central for function. Here, we develop a 1Hα CEST pulse sequence which overcomes the resonance overlap problem in the 1Hα-13Cα plane of IDPs by taking advantage of the superior resolution in the 1H-15N correlation spectrum. In this sequence, magnetization is transferred after 1H CEST using a triple resonance coherence transfer pathway from 1Hα (i) to 1HN(i + 1) during which the 15N(t1) and 1HN(t2) are frequency labelled. This approach is integrated with spin state-selective CEST for eliminating spurious dips in CEST profiles resulting from dipolar cross-relaxation. We apply this sequence to determine the excited state 1Hα chemical shifts of the intrinsically disordered DNA binding domain (CytRN) of the bacterial cytidine repressor (CytR), which transiently acquires a functional globally folded conformation. The structure of the excited state, calculated using 1Hα chemical shifts in conjunction with other excited state NMR restraints, is a three-helix bundle incorporating a helix-turn-helix motif that is vital for binding DNA.
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Proteínas Intrínsecamente Desordenadas , Proteoma , Secuencia de Aminoácidos , Citidina , EucariontesRESUMEN
Over the last decade amide 15N CEST experiments have emerged as a popular tool to study protein dynamics that involves exchange between a 'visible' major state and sparsely populated 'invisible' minor states. Although initially introduced to study exchange between states that are in slow exchange with each other (typical exchange rates of, 10 to 400 s-1), they are now used to study interconversion between states on the intermediate to fast exchange timescale while still using low to moderate (5 to 350 Hz) 'saturating' B1 fields. The 15N CEST experiment is very sensitive to exchange as the exchange delay TEX can be quite long (~0.5 s) allowing for a large number of exchange events to occur making it a very powerful tool to detect minor sates populated ([Formula: see text]) to as low as 1%. When systems are in fast exchange and the 15N CEST data has to be described using a model that contains exchange, the exchange parameters are often poorly defined because the [Formula: see text] versus [Formula: see text] and [Formula: see text] versus exchange rate ([Formula: see text]) plots can be quite flat with shallow or no minima and the analysis of such 15N CEST data can lead to wrong estimates of the exchange parameters due to the presence of 'spurious' minima. Here we show that the inclusion of experimentally derived constraints on the intrinsic transverse relaxation rates and the inclusion of visible state peak-positions during the analysis of amide 15N CEST data acquired with moderate B1 values (~50 to ~350 Hz) results in convincing minima in the [Formula: see text] versus [Formula: see text] and the [Formula: see text] versus [Formula: see text] plots even when exchange occurs on the 100 µs timescale. The utility of this strategy is demonstrated on the fast-folding Bacillus stearothermophilus peripheral subunit binding domain that folds with a rate constant ~104 s-1. Here the analysis of 15N CEST data alone results in [Formula: see text] versus [Formula: see text] and [Formula: see text] versus [Formula: see text] plots that contain shallow minima, but the inclusion of visible-state peak positions and restraints on the intrinsic transverse relaxation rates of both states during the analysis of the 15N CEST data results in pronounced minima in the [Formula: see text] versus [Formula: see text] and [Formula: see text] versus [Formula: see text] plots and precise exchange parameters even in the fast exchange regime ([Formula: see text]~5). Using this strategy we find that the folding rate constant of PSBD is invariant (~10,500 s-1) from 33.2 to 42.9 °C while the unfolding rates (~70 to ~500 s-1) and unfolded state populations (~0.7 to ~4.3%) increase with temperature. The results presented here show that protein dynamics occurring on the 10 to 104 s-1 timescale can be studied using amide 15N CEST experiments.
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Amidas , Amidas/química , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
A longstanding goal in the field of intrinsically disordered proteins (IDPs) is to characterize their structural heterogeneity and pinpoint the role of this heterogeneity in IDP function. Here, we use multinuclear chemical exchange saturation (CEST) nuclear magnetic resonance to determine the structure of a thermally accessible globally folded excited state in equilibrium with the intrinsically disordered native ensemble of a bacterial transcriptional regulator CytR. We further provide evidence from double resonance CEST experiments that the excited state, which structurally resembles the DNA-bound form of cytidine repressor (CytR), recognizes DNA by means of a "folding-before-binding" conformational selection pathway. The disorder-to-order regulatory switch in DNA recognition by natively disordered CytR therefore operates through a dynamical variant of the lock-and-key mechanism where the structurally complementary conformation is transiently accessed via thermal fluctuations.
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Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/química , Pliegue de Proteína , Unión Proteica , Espectroscopía de Resonancia Magnética , ADN/química , Conformación ProteicaRESUMEN
How proteins switch between various ligand-free and ligand-bound structures has been a key biophysical question ever since the postulation of the Monod-Wyman-Changeux and Koshland-Nemethy-Filmer models over six decades ago. The ability of NMR spectroscopy to provide structural and kinetic information on biomolecular conformational exchange places it in a unique position as an analytical tool to interrogate the mechanisms of biological processes such as protein folding and biomolecular complex formation. In addition, recent methodological developments in the areas of saturation transfer and relaxation dispersion have expanded the scope of NMR for probing the mechanics of transitions in systems where one or more states constituting the exchange process are sparsely populated and 'invisible' in NMR spectra. In this review, we highlight some of the strategies available from NMR spectroscopy for examining the nature of multi-site conformational exchange, using five case studies that have employed NMR, either in isolation, or in conjunction with other biophysical tools.
RESUMEN
We investigate the conformational properties of the intrinsically disordered DNA-binding domain of CytR in the presence of the polymeric crowder polyethylene glycol (PEG). Integrating circular dichroism, nuclear magnetic resonance, and single-molecule Förster resonance energy transfer measurements, we demonstrate that disordered CytR populates a well-folded minor conformation in its native ensemble, while the unfolded ensemble collapses and folds with an increase in crowder density independent of the crowder size. Employing a statistical-mechanical model, the effective reduction in the accessible conformational space of a residue in the unfolded state is estimated to be 10% at 300 mg/mL PEG8000, relative to dilute conditions. The experimentally consistent PEG-temperature phase diagram thus constructed reveals that entropic effects can stabilize disordered CytR by 10 kJ mol-1, driving the equilibrium toward folded conformations under physiological conditions. Our work highlights the malleable conformational landscape of CytR, the presence of a folded conformation in the disordered ensemble, and proposes a scaling relation for quantifying excluded volume effects on protein stability.
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Pliegue de Proteína , Proteínas , Dicroismo Circular , Entropía , Conformación Molecular , Conformación ProteicaRESUMEN
Lectins are sugar-binding proteins that have shown considerable promise as antiviral agents because of their ability to interact with envelope glycoproteins present on the surface of viruses such as HIV-1. However, their therapeutic potential has been compromised by their mitogenicity that stimulates uncontrolled division of T-lymphocytes. Horcolin, a member of the jacalin family of lectins, tightly binds the HIV-1 envelope glycoprotein gp120 and neutralizes HIV-1 particles but is nonmitogenic. In this report, we combine X-ray crystallography and NMR spectroscopy to obtain atomic-resolution insights into the structure of horcolin and the molecular basis for its carbohydrate recognition. Each protomer of the horcolin dimer adopts a canonical ß-prism I fold with three Greek key motifs and carries two carbohydrate-binding sites. The carbohydrate molecule binds in a negatively charged pocket and is stabilized by backbone and side chain hydrogen bonds to conserved residues in the ligand-binding loop. NMR titrations reveal a two-site binding mode and equilibrium dissociation constants for the two binding sites determined from two-dimensional (2D) lineshape modeling are 4-fold different. Single-binding-site variants of horcolin confirm the dichotomy in binding sites and suggest that there is allosteric communication between the two sites. An analysis of the horcolin structure shows a network of hydrogen bonds linking the two carbohydrate-binding sites directly and through a secondary binding site, and this coupling between the two sites is expected to assume importance in the interaction of horcolin with high-mannose glycans found on viral envelope glycoproteins.
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VIH-1 , Lectinas , Sitios de Unión , Carbohidratos , Cristalografía por Rayos X , VIH-1/metabolismo , Lectinas/metabolismo , Manosa/químicaRESUMEN
The application of NMR spectroscopy for studying molecular and reaction dynamics relies crucially on the measurement of the magnitude of radiofrequency (RF) fields that are used to nutate or lock the nuclear magnetization. Here, we report a method for measuring RF field amplitudes that leverages the intrinsic modulations observed in offset-dependent NMR nutation profiles of small molecules. Such nutation profiles are exquisitely sensitive to the magnitude of the RF field, and B1 values ranging from 1 to 2000 Hz, as well the inhomogeneity in B1 distributions, can be determined with high accuracy and precision using this approach. In order to measure B1 fields associated with NMR experiments carried out on protein or nucleic acids, where these modulations are obscured by the large transverse relaxation rate constants of the analyte, our approach can be used in conjunction with a suitable external small molecule standard, expanding the scope of the method for large biomolecules.
RESUMEN
The structural paradigm that the sequence of a protein encodes for a unique three-dimensional native fold does not acknowledge the intrinsic plasticity encapsulated in conformational free energy landscapes. Metamorphic proteins are a recently discovered class of biomolecules that illustrate this plasticity by folding into at least two distinct native state structures of comparable stability in the absence of ligands or cofactors to facilitate fold-switching. The expanding list of metamorphic proteins clearly shows that these proteins are not mere aberrations in protein evolution, but may have actually been a consequence of distinctive patterns in selection pressure such as those found in virus-host co-evolution. In this review, we describe the structure-function relationships observed in well-studied metamorphic protein systems, with specific focus on how functional residues are sequestered or exposed in the two folds of the protein. We also discuss the implications of metamorphosis for protein evolution and the efforts that are underway to predict metamorphic systems from sequence properties alone.
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Modelos Moleculares , Conformación Proteica , Proteínas/química , Secuencia de Aminoácidos , Evolución Biológica , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Análisis Espectral , Relación Estructura-ActividadRESUMEN
The diversity of the cellular proteome substantially exceeds the number of genes coded by the DNA of an organism because one or more residues in a majority of eukaryotic proteins are post-translationally modified (PTM) by the covalent conjugation of specific chemical groups. We now know that PTMs alter protein conformation and function in ways that are not entirely understood at the molecular level. NMR spectroscopy has been particularly successful as an analytical tool in elucidating the themes underlying the structural role of PTMs. In this Perspective, we focus on the NMR-based characterization of three abundant PTMs: phosphorylation, acetylation, and glycosylation. We detail NMR methods that have found success in detecting these modifications at a site-specific level. We also highlight NMR studies that have mapped the conformational changes ensuing from these PTMs as well as evaluated their relation to function. The NMR toolbox is expanding rapidly with experiments available to probe not only the average structure of biomolecules but also how this structure changes with time on time scales ranging from picoseconds to seconds. The atomic resolution insights into the biomolecular structure, dynamics, and mechanism accessible from NMR spectroscopy ensure that NMR will continue to be at the forefront of research in the structural biology of PTMs.
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Glicoproteínas/metabolismo , Fosfoproteínas/metabolismo , Acetilación , Bacterias/química , Glicoproteínas/química , Glicosilación , Humanos , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosforilación , Conformación Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Structural disorder in proteins arises from a complex interplay between weak hydrophobicity and unfavorable electrostatic interactions. The extent to which the hydrophobic effect contributes to the unique and compact native state of proteins is, however, confounded by large compensation between multiple entropic and energetic terms. Here we show that protein structural order and cooperativity arise as emergent properties upon hydrophobic substitutions in a disordered system with non-intuitive effects on folding and function. Aided by sequence-structure analysis, equilibrium, and kinetic spectroscopic studies, we engineer two hydrophobic mutations in the disordered DNA-binding domain of CytR that act synergistically, but not in isolation, to promote structure, compactness, and stability. The double mutant, with properties of a fully ordered domain, exhibits weak cooperativity with a complex and rugged conformational landscape. The mutant, however, binds cognate DNA with an affinity only marginally higher than that of the wild type, though nontrivial differences are observed in the binding to noncognate DNA. Our work provides direct experimental evidence of the dominant role of non-additive hydrophobic effects in shaping the molecular evolution of order in disordered proteins and vice versa, which could be generalized to even folded proteins with implications for protein design and functional manipulation.
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Proteínas de Escherichia coli/química , Proteínas Intrínsecamente Desordenadas/química , Proteínas Represoras/química , Sitios de Unión , Rastreo Diferencial de Calorimetría , Escherichia coli/química , Proteínas de Escherichia coli/genética , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Intrínsecamente Desordenadas/genética , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Mutación Puntual , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estabilidad Proteica , Proteínas Represoras/genética , Electricidad EstáticaRESUMEN
Biological molecules are often highly dynamic, and this flexibility can be critical for function. The large range of sampled timescales and the fact that many of the conformers that are continually explored are only transiently formed and sparsely populated challenge current biophysical approaches. Solution nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for characterizing biomolecular dynamics in detail, even in cases where excursions involve short-lived states. Here, we briefly review a number of NMR experiments for studies of biomolecular dynamics on the microsecond-to-second timescale and focus on applications to protein and nucleic acid systems that clearly illustrate the functional relevance of motion in both health and disease.
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Espectroscopía de Resonancia Magnética , Humanos , Imagen por Resonancia Magnética , Proteínas/químicaRESUMEN
Proteins are not locked in a single structure but often interconvert with other conformers that are critical for function. When such conformers are sparsely populated and transiently formed they become invisible to routine biophysical methods, however they can be studied in detail by NMR spin-relaxation experiments. Few experiments are available in the NMR toolkit, however, for characterizing the hydrodynamic properties of invisible states. Herein we describe a CPMG-based experiment for measuring translational diffusion constants of invisible states using a pulsed-field gradient approach that exploits methyl 1 H triple-quantum coherences. An example, involving diffusion of a sparsely populated and hence invisible unfolded protein ensemble is presented, without the need for the addition of denaturants that tend to destroy weak interactions that can be involved in stabilizing residual structure in the unfolded state.
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Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Difusión , Hidrodinámica , Conformación Proteica , Teoría CuánticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating fatal syndrome characterized by very rapid degeneration of motor neurons. A leading hypothesis is that ALS is caused by toxic protein misfolding and aggregation, as also occurs in many other neurodegenerative disorders, such as prion, Alzheimer's, Parkinson's, and Huntington's diseases. A prominent cause of familial ALS is mutations in the protein superoxide dismutase (SOD1), which promote the formation of misfolded SOD1 conformers that are prone to aberrant interactions both with each other and with other cellular components. We have shown previously that immature SOD1, lacking bound Cu and Zn metal ions and the intrasubunit disulfide bond (apoSOD12SH), has a rugged free-energy surface (FES) and exchanges with four other conformations (excited states) that have millisecond lifetimes and sparse populations on the order of a few percent. Here, we examine further states of SOD1 along its maturation pathway, as well as those off-pathway resulting from metal loss that have been observed in proteinaceous inclusions. Metallation and disulfide bond formation lead to structural transformations including local ordering of the electrostatic loop and native dimerization that are observed in rare conformers of apoSOD12SH; thus, SOD1 maturation may occur via a population-switch mechanism whereby posttranslational modifications select for preexisting structures on the FES. Metallation and oxidation of SOD1 stabilize the native, mature conformation and decrease the number of detected excited conformational states, suggesting that it is the immature forms of the protein that contribute to misfolded conformations in vivo rather than the highly stable enzymatically active dimer.
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Pliegue de Proteína , Superóxido Dismutasa-1/química , Cobre/química , Cobre/metabolismo , Dimerización , Entropía , Humanos , Oxidación-Reducción , Conformación Proteica , Superóxido Dismutasa-1/metabolismo , Zinc/química , Zinc/metabolismoRESUMEN
Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution. In such a process the bound structure can be selected from the ensemble of interconverting ligands a priori (conformational selection, CS) or may form once the ligand is bound (induced fit, IF). Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins. We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies. Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures, suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecular dynamics in this recognition event.
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Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Bacterias , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Conformación Proteica , Especificidad por SustratoRESUMEN
Membrane encapsulation is frequently used by the cell to sequester biomolecules and compartmentalize their function. Cells also concentrate molecules into phase-separated protein or protein/nucleic acid "membraneless organelles" that regulate a host of biochemical processes. Here, we use solution NMR spectroscopy to study phase-separated droplets formed from the intrinsically disordered N-terminal 236 residues of the germ-granule protein Ddx4. We show that the protein within the concentrated phase of phase-separated Ddx4, [Formula: see text], diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing [Formula: see text] to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of [Formula: see text] have significant mobility, explaining why high-resolution spectra are observed, but motion is reduced compared with an equivalently concentrated nonphase-separating control. Observation of a network of interchain interactions, as established by NOE spectroscopy, shows the importance of Phe and Arg interactions in driving the phase separation of Ddx4, while the salt dependence of both low- and high-concentration regions of phase diagrams establishes an important role for electrostatic interactions. The diffusion of a series of small probes and the compact but disordered 4E binding protein 2 (4E-BP2) protein in [Formula: see text] are explained by an excluded volume effect, similar to that found for globular protein solvents. No changes in structural propensities of 4E-BP2 dissolved in [Formula: see text] are observed, while changes to DNA and RNA molecules have been reported, highlighting the diverse roles that proteinaceous solvents play in dictating the properties of dissolved solutes.
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ARN Helicasas DEAD-box/química , Hidrodinámica , Proteínas Intrínsecamente Desordenadas/química , Orgánulos/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Gránulos Citoplasmáticos/química , Células Germinativas/metabolismo , Células HeLa , Humanos , Espectroscopía de Resonancia MagnéticaRESUMEN
Understanding the extent to which information is transmitted through the intramolecular interaction network of proteins upon a perturbation, that is, an allosteric effect, has long remained an unsolved problem. Through an analysis of high-resolution NMR data from the literature on 28 different proteins and 49 structural perturbations, we show that the extent of induced structural changes through mutations and molecular events including protein-protein, protein-peptide, protein-ligand binding, and post-translational modifications exhibit a near-universal exponential functional form. The extent of percolation into the protein structures can be up to 20-25 Å despite no apparent change in the 3D structures. These observations are also consistent with theoretical expectations, elementary graph theoretic analysis of protein structures, detailed molecular dynamics simulations, and experimental double-mutant cycles. Our analysis highlights that most molecular events would contribute to allosteric effects independent of protein structure, topology, or identity and provides a simple avenue to test and potentially model their effects.