RESUMEN
In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1's LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we create LCR mutations, which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations that impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Gránulos Citoplasmáticos/química , Calor , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Mutagénesis , Proteínas de Unión a Poli(A)/química , Proteínas de Unión a Poli(A)/genética , Prolina/análisis , Prolina/metabolismo , Dominios Proteicos , Ribonucleasas/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Estrés FisiológicoRESUMEN
Eukaryotic cells form condensates to sense and adapt to their environment [S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen, Nat. Rev. Mol. Cell Biol. 18, 285-298 (2017), H. Yoo, C. Triandafillou, D. A. Drummond, J. Biol. Chem. 294, 7151-7159 (2019)]. Poly(A)-binding protein (Pab1), a canonical stress granule marker, condenses upon heat shock or starvation, promoting adaptation [J. A. Riback et al., Cell 168, 1028-1040.e19 (2017)]. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. We apply hydrogen-deuterium exchange/mass spectrometry to investigate the mechanism of Pab1's condensation. Pab1's four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe populations allows us to identify which regions contribute to the condensate's interaction network. Our data yield a picture of Pab1's stress-triggered condensation, which we term sequential activation (Fig. 1A), wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. Subsequent association is dictated more by the underlying free energy surface than specific interactions, an effect we refer to as thermodynamic specificity. Our study represents an advance for elucidating the interactions that drive condensation. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple stresses, a potentially general mechanism for stress-responsive proteins.
Asunto(s)
Proteínas de Choque Térmico , Proteínas de Unión a Poli(A) , Proteínas de Unión a Poli(A)/genética , Temperatura , Proteínas de Choque Térmico/metabolismo , Termodinámica , Respuesta al Choque Térmico , Medición de Intercambio de Deuterio/métodosRESUMEN
Lifeact is a popular peptide-based label of actin filaments in live cells. We have designed an improved Lifeact variant, LILAC, that binds to actin in light using the LOV2 protein. Light control allows the user to modulate actin labeling, enabling image analysis that leverages modulation for an enhanced view of F-actin dynamics in cells. Furthermore, the tool reduces actin perturbations and cell sickness caused by Lifeact overexpression.
Asunto(s)
Actinas , Optogenética , Citoesqueleto de Actina , Péptidos/metabolismoRESUMEN
To import large metabolites across the outer membrane of gram-negative bacteria, TonB-dependent transporters (TBDTs) undergo significant conformational change. After substrate binding in BtuB, the Escherichia coli vitamin B12 TBDT, TonB binds and couples BtuB to the inner-membrane proton motive force that powers transport [N. Noinaj, M. Guillier, T. J. Barnard, S. K. Buchanan, Annu. Rev. Microbiol. 64, 4360 (2010)]. However, the role of TonB in rearranging the plug domain of BtuB to form a putative pore remains enigmatic. Some studies focus on force-mediated unfolding [S. J. Hickman, R. E. M. Cooper, L. Bellucci, E. Paci, D. J. Brockwell, Nat. Commun. 8, 14804 (2017)], while others propose force-independent pore formation by TonB binding [T. D. Nilaweera, D. A. Nyenhuis, D. S. Cafiso, eLife 10, e68548 (2021)], leading to breakage of a salt bridge termed the "Ionic Lock." Our hydrogendeuterium exchange/mass spectrometry (HDX-MS) measurements in E. coli outer membranes find that the region surrounding the Ionic Lock, far from the B12 site, is fully destabilized upon substrate binding. A comparison of the exchange between the B12-bound and the B12+TonBbound complexes indicates that B12 binding is sufficient to unfold the Ionic Lock region, with the subsequent binding of a TonB fragment having much weaker effects. TonB binding accelerates exchange in the third substrate-binding loop, but pore formation does not obviously occur in this or any region. This study provides a detailed structural and energetic description of the early stages of B12 passage that provides support both for and against current models of the transport process.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Proteínas de la Membrana , Proteínas de Transporte de Membrana , Vitamina B 12 , Regulación Alostérica , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Vitamina B 12/metabolismoRESUMEN
Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.
Asunto(s)
Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Conformación Proteica , Biotinilación , Membrana Celular , Microscopía por Crioelectrón , Proteínas de Unión al ADN , Endopeptidasas , Escherichia coli , Proteínas de Escherichia coli/química , Modelos Moleculares , Desnaturalización Proteica , Pliegue de Proteína , EstreptavidinaRESUMEN
The dynamics and folding of potassium channel pore domain monomers are connected to the kinetics of tetramer assembly. In all-atom molecular dynamics simulations of Kv1.2 and KcsA channels, monomers adopt multiple nonnative conformations while the three helices remain folded. Consistent with this picture, NMR studies also find the monomers to be dynamic and structurally heterogeneous. However, a KcsA construct with a disulfide bridge engineered between the two transmembrane helices has an NMR spectrum with well-dispersed peaks, suggesting that the monomer can be locked into a native-like conformation that is similar to that observed in the folded tetramer. During tetramerization, fluoresence resonance energy transfer (FRET) data indicate that monomers rapidly oligomerize upon insertion into liposomes, likely forming a protein-dense region. Folding within this region occurs along separate fast and slow routes, with τfold â¼40 and 1,500 s, respectively. In contrast, constructs bearing the disulfide bond mainly fold via the faster pathway, suggesting that maintaining the transmembrane helices in their native orientation reduces misfolding. Interestingly, folding is concentration independent despite the tetrameric nature of the channel, indicating that the rate-limiting step is unimolecular and occurs after monomer association in the protein-dense region. We propose that the rapid formation of protein-dense regions may help with the assembly of multimeric membrane proteins by bringing together the nascent components prior to assembly. Finally, despite its name, the addition of KcsA's C-terminal "tetramerization" domain does not hasten the kinetics of tetramerization.
Asunto(s)
Canal de Potasio Kv.1.2/química , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Cinética , Cadenas de Markov , Simulación de Dinámica MolecularRESUMEN
Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis, as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/química , Bordetella pertussis/metabolismo , Desplegamiento Proteico , Factores de Virulencia de Bordetella/química , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bordetella pertussis/química , Bordetella pertussis/genética , Conformación Proteica , Pliegue de Proteína , Dispersión del Ángulo Pequeño , Factores de Virulencia de Bordetella/genética , Factores de Virulencia de Bordetella/metabolismoRESUMEN
Single-molecule force spectroscopy methods, such as AFM and magnetic tweezers, have proved extremely beneficial in elucidating folding pathways for soluble and membrane proteins. To identify factors that determine the force rupture levels in force-induced membrane protein unfolding, we applied our near-atomic-level Upside molecular dynamics package to study the vertical and lateral pulling of bacteriorhodopsin (bR) and GlpG, respectively. With our algorithm, we were able to selectively alter the magnitudes of individual interaction terms and identify that, for vertical pulling, hydrogen bond strength had the strongest effect, whereas other non-bonded protein and membrane-protein interactions had only moderate influences, except for the extraction of the last helix where the membrane-protein interactions had a stronger influence. The up-down topology of the transmembrane helices caused helices to be pulled out as pairs. The rate-limiting rupture event often was the loss of H-bonds and the ejection of the first helix, which then propagated tension to the second helix, which rapidly exited the bilayer. The pulling of the charged linkers across the membrane had minimal influence, as did changing the bilayer thickness. For the lateral pulling of GlpG, the rate-limiting rupture corresponded to the separation of the helices within the membrane, with the H-bonds generally being broken only afterward. Beyond providing a detailed picture of the rupture events, our study emphasizes that the pulling mode greatly affects the factors that determine the forces needed to unfold a membrane protein.
Asunto(s)
Bacteriorodopsinas , Bacteriorodopsinas/química , Simulación de Dinámica Molecular , Desplegamiento Proteico , Microscopía de Fuerza Atómica , Desnaturalización Proteica , Pliegue de ProteínaRESUMEN
The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (Rg) and end-to-end distance (Ree) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the Rg and Ree are "uncoupled" (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Hidrazinas/química , Proteínas/química , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Difracción de Rayos XRESUMEN
Myosins play countless critical roles in the cell, each requiring it to be activated at a specific location and time. To control myosin VI with this specificity, we created an optogenetic tool for activating myosin VI by fusing the light-sensitive Avena sativa phototropin1 LOV2 domain to a peptide from Dab2 (LOVDab), a myosin VI cargo protein. Our approach harnesses the native targeting and activation mechanism of myosin VI, allowing direct inferences on myosin VI function. LOVDab robustly recruits human full-length myosin VI to various organelles in vivo and hinders peroxisome motion in a light-controllable manner. LOVDab also activates myosin VI in an in vitro gliding filament assay. Our data suggest that protein and lipid cargoes cooperate to activate myosin VI, allowing myosin VI to integrate Ca2+, lipid, and protein cargo signals in the cell to deploy in a site-specific manner.
Asunto(s)
Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Actinas/genética , Actinas/metabolismo , Línea Celular Tumoral , Células HeLa , Humanos , Optogenética/métodos , Sistemas de Mensajero Secundario/genéticaRESUMEN
The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or ß-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.
Asunto(s)
Proteínas Anticongelantes/química , Proteínas de Artrópodos/química , Glicina/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Proteínas Anticongelantes/genética , Proteínas Anticongelantes/metabolismo , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/metabolismo , Cristalografía por Rayos X , Expresión Génica , Glicina/metabolismo , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Péptidos/metabolismo , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Siphonaptera/química , TermodinámicaRESUMEN
Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or "theft" mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein-coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein-Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.
Asunto(s)
Secuencia Conservada , Mapas de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Sustitución de Aminoácidos , Animales , Evolución Molecular , Humanos , Lisina/genética , Lisina/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/química , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Fosforilación , Unión Proteica , Serina/genética , Serina/metabolismo , Troponina C/química , Troponina C/genética , Troponina C/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Single-molecule force spectroscopy has proven extremely beneficial in elucidating folding pathways for membrane proteins. Here, we simulate these measurements, conducting hundreds of unfolding trajectories using our fast Upside algorithm for slow enough speeds to reproduce key experimental features that may be missed using all-atom methods. The speed also enables us to determine the logarithmic dependence of pulling velocities on the rupture levels to better compare to experimental values. For simulations of atomic force microscope measurements in which force is applied vertically to the C-terminus of bacteriorhodopsin, we reproduce the major experimental features including even the back-and-forth unfolding of single helical turns. When pulling laterally on GlpG to mimic the experiment, we observe quite different behavior depending on the stiffness of the spring. With a soft spring, as used in the experimental studies with magnetic tweezers, the force remains nearly constant after the initial unfolding event, and a few pathways and a high degree of cooperativity are observed in both the experiment and simulation. With a stiff spring, however, the force drops to near zero after each major unfolding event, and numerous intermediates are observed along a wide variety of pathways. Hence, the mode of force application significantly alters the perception of the folding landscape, including the number of intermediates and the degree of folding cooperativity, important issues that should be considered when designing experiments and interpreting unfolding data.
Asunto(s)
Proteínas de Unión al ADN/química , Endopeptidasas/química , Proteínas de Escherichia coli/química , Proteínas de la Membrana/química , Simulación de Dinámica Molecular , Pliegue de Proteína , Membrana Dobles de Lípidos/químicaRESUMEN
An ongoing challenge in protein chemistry is to identify the underlying interaction energies that capture protein dynamics. The traditional trade-off in biomolecular simulation between accuracy and computational efficiency is predicated on the assumption that detailed force fields are typically well-parameterized, obtaining a significant fraction of possible accuracy. We re-examine this trade-off in the more realistic regime in which parameterization is a greater source of error than the level of detail in the force field. To address parameterization of coarse-grained force fields, we use the contrastive divergence technique from machine learning to train from simulations of 450 proteins. In our procedure, the computational efficiency of the model enables high accuracy through the precise tuning of the Boltzmann ensemble. This method is applied to our recently developed Upside model, where the free energy for side chains is rapidly calculated at every time-step, allowing for a smooth energy landscape without steric rattling of the side chains. After this contrastive divergence training, the model is able to de novo fold proteins up to 100 residues on a single core in days. This improved Upside model provides a starting point both for investigation of folding dynamics and as an inexpensive Bayesian prior for protein physics that can be integrated with additional experimental or bioinformatic data.
Asunto(s)
Biología Computacional/métodos , Proteínas/química , Teorema de Bayes , Simulación por Computador , Aprendizaje Automático , Simulación de Dinámica Molecular/estadística & datos numéricos , Conformación Proteica , Pliegue de Proteína , Programas Informáticos , TermodinámicaRESUMEN
To address the large gap between time scales that can be easily reached by molecular simulations and those required to understand protein dynamics, we present a rapid self-consistent approximation of the side chain free energy at every integration step. In analogy with the adiabatic Born-Oppenheimer approximation for electronic structure, the protein backbone dynamics are simulated as preceding according to the dictates of the free energy of an instantaneously-equilibrated side chain potential. The side chain free energy is computed on the fly, allowing the protein backbone dynamics to traverse a greatly smoothed energetic landscape. This computation results in extremely rapid equilibration and sampling of the Boltzmann distribution. Our method, termed Upside, employs a reduced model involving the three backbone atoms, along with the carbonyl oxygen and amide proton, and a single (oriented) side chain bead having multiple locations reflecting the conformational diversity of the side chain's rotameric states. We also introduce a novel, maximum-likelihood method to parameterize the side chain interactions using protein structures. We demonstrate state-of-the-art accuracy for predicting χ1 rotamer states while consuming only milliseconds of CPU time. Our method enables rapidly equilibrating coarse-grained simulations that can nonetheless contain significant molecular detail. We also show that the resulting free energies of the side chains are sufficiently accurate for de novo folding of some proteins.
Asunto(s)
Simulación de Dinámica Molecular/estadística & datos numéricos , Proteínas/química , Aminoácidos/química , Entropía , Modelos Moleculares , Probabilidad , Conformación Proteica , TermodinámicaRESUMEN
The relationship between folding cooperativity and downhill, or barrier-free, folding of proteins under highly stabilizing conditions remains an unresolved topic, especially for proteins such as λ-repressor that fold on the microsecond timescale. Under aqueous conditions where downhill folding is most likely to occur, we measure the stability of multiple H bonds, using hydrogen exchange (HX) in a λYA variant that is suggested to be an incipient downhill folder having an extrapolated folding rate constant of 2 × 10(5) s(-1) and a stability of 7.4 kcal·mol(-1) at 298 K. At least one H bond on each of the three largest helices (α1, α3, and α4) breaks during a common unfolding event that reflects global denaturation. The use of HX enables us to both examine folding under highly stabilizing, native-like conditions and probe the pretransition state region for stable species without the need to initiate the folding reaction. The equivalence of the stability determined at zero and high denaturant indicates that any residual denatured state structure minimally affects the stability even under native conditions. Using our ψ analysis method along with mutational Ï analysis, we find that the three aforementioned helices are all present in the folding transition state. Hence, the free energy surface has a sufficiently high barrier separating the denatured and native states that folding appears cooperative even under extremely stable and fast folding conditions.
Asunto(s)
Aminoácidos/química , Hidrógeno/química , Espectroscopía de Resonancia Magnética/métodos , Pliegue de Proteína , Proteínas Represoras/química , Proteínas Represoras/ultraestructura , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/ultraestructura , Simulación por Computador , Enlace de Hidrógeno , Cinética , Modelos Químicos , Modelos Moleculares , Conformación Proteica , Desnaturalización Proteica , TermodinámicaRESUMEN
We use the statistics of a large and curated training set of transmembrane helical proteins to develop a knowledge-based potential that accounts for the dependence on both the depth of burial of the protein in the membrane and the degree of side-chain exposure. Additionally, the statistical potential includes depth-dependent energies for unsatisfied backbone hydrogen bond donors and acceptors, which are found to be relatively small, â¼2 RT. Our potential accurately places known proteins within the bilayer. The potential is applied to the mechanosensing MscL channel in membranes of varying thickness and curvature, as well as to the prediction of protein structure. The potential is incorporated into our new Upside molecular dynamics algorithm. Notably, we account for the exchange of protein-lipid interactions for protein-protein interactions as helices contact each other, thereby avoiding overestimating the energetics of helix association within the membrane. Simulations of most multimeric complexes find that isolated monomers and the oligomers retain the same orientation in the membrane, suggesting that the assembly of prepositioned monomers presents a viable mechanism of oligomerization.
Asunto(s)
Membrana Celular/química , Proteínas de la Membrana/química , Simulación de Dinámica Molecular , Enlace de Hidrógeno , Cinética , Conformación Proteica en Hélice alfa , Pliegue de Proteína , TermodinámicaRESUMEN
Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (â¼6 M) to low (â¼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.