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TonB-dependent transporters (TBDTs) mediate energized transport of essential nutrients into gram-negative bacteria. TBDTs are increasingly being exploited for the delivery of antibiotics to drug-resistant bacteria. While much is known about ground state complexes of TBDTs, few details have emerged about the transport process itself. In this study, we exploit bacteriocin parasitization of a TBDT to probe the mechanics of transport. Previous work has shown that the N-terminal domain of Pseudomonas aeruginosa-specific bacteriocin pyocin S2 (PyoS2NTD) is imported through the pyoverdine receptor FpvAI. PyoS2NTD transport follows the opening of a proton-motive force-dependent pore through FpvAI and the delivery of its own TonB box that engages TonB. We use molecular models and simulations to formulate a complete translocation pathway for PyoS2NTD that we validate using protein engineering and cytotoxicity measurements. We show that following partial removal of the FpvAI plug domain which occludes the channel, the pyocin's N-terminus enters the channel by electrostatic steering and ratchets to the periplasm. Application of force, mimicking that exerted by TonB, leads to unraveling of PyoS2NTD as it squeezes through the channel. Remarkably, while some parts of PyoS2NTD must unfold, complete unfolding is not required for transport, a result we confirmed by disulfide bond engineering. Moreover, the section of the FpvAI plug that remains embedded in the channel appears to serve as a buttress against which PyoS2NTD is pushed to destabilize the domain. Our study reveals the limits of structural deformation that accompanies import through a TBDT and the role the TBDT itself plays in accommodating transport.
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Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.
Assuntos
Toxina da Cólera , Mutagênicos , Humanos , Gangliosídeo G(M1) , Ligantes , MutagêneseRESUMO
Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.
Assuntos
Biotinilação , Esteróis , Proteínas rab de Ligação ao GTP , Humanos , Colesterol/biossíntese , Colesterol/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Esteróis/biossíntese , Esteróis/metabolismo , Células Cultivadas , Técnicas de Silenciamento de Genes , Transporte Proteico/genéticaRESUMO
The C-type lectin receptor DC-SIGN has been highlighted as the coreceptor for the spike protein of the SARS-CoV-2 virus. A multivalent glycomimetic ligand, Polyman26, has been found to inhibit DC-SIGN-dependent trans-infection of SARS-CoV-2. The molecular details underlying avidity generation in such systems remain poorly characterized. In an effort to dissect the contribution of the known multivalent effects - chelation, clustering, and statistical rebinding - we studied a series of dendrimer constructs related to Polyman26 with a rod core rationally designed to engage simultaneously two binding sites of the tetrameric DC-SIGN. Binding properties of these compounds have been studied with a range of biophysical techniques, including recently developed surface plasmon resonance oriented-surface methodology. Using molecular modeling we addressed, for the first time, the impact of the carbohydrate recognition domains' flexibility of the DC-SIGN tetramer on the compounds' avidity. We were able to gain deeper insight into the role of different binding modes, which in combination produce a construct with a nanomolar affinity despite a limited valency. This multifaceted experimental-theoretical approach provides detailed understanding of multivalent ligand/multimeric protein interactions which can lead to future predictions. This work opens the way to the development of new virus attachment blockers adapted to different C-type lectin receptors of viruses.
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Intrinsically disordered proteins (IDPs) are important for a broad range of biological functions and are involved in many diseases. An understanding of intrinsic disorder is key to develop compounds that target IDPs. Experimental characterization of IDPs is hindered by the very fact that they are highly dynamic. Computational methods that predict disorder from the amino acid sequence have been proposed. Here, we present ADOPT (Attention DisOrder PredicTor), a new predictor of protein disorder. ADOPT is composed of a self-supervised encoder and a supervised disorder predictor. The former is based on a deep bidirectional transformer, which extracts dense residue-level representations from Facebook's Evolutionary Scale Modeling library. The latter uses a database of nuclear magnetic resonance chemical shifts, constructed to ensure balanced amounts of disordered and ordered residues, as a training and a test dataset for protein disorder. ADOPT predicts whether a protein or a specific region is disordered with better performance than the best existing predictors and faster than most other proposed methods (a few seconds per sequence). We identify the features that are relevant for the prediction performance and show that good performance can already be gained with <100 features. ADOPT is available as a stand-alone package at https://github.com/PeptoneLtd/ADOPT and as a web server at https://adopt.peptone.io/.
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The Staphylococcus aureus surface protein G (SasG) is associated with host colonisation and biofilm formation. As colonisation occurs at the liquid-substrate interface bacteria are subject to a myriad of external forces and, presumably as a consequence, SasG displays extreme mechanical strength. This mechanical phenotype arises from the B-domain; a repetitive region composed of alternating E and G5 subdomains. These subdomains have an unusual structure comprising collagen-like regions capped by triple-stranded ß-sheets. To identify the determinants of SasG mechanical strength, we characterised the mechanical phenotype and thermodynamic stability of 18 single substitution variants of a pseudo-wildtype protein. Visualising the mechanically-induced transition state at a residue-level by Ï-value analysis reveals that the main force-bearing regions are the N- and C-terminal 'Mechanical Clamps' and their side-chain interactions. This is tailored by contacts at the pseudo-hydrophobic core interface. We also describe a novel mechanical motif - the collagen-like region and show that glycine to alanine substitutions, analogous to those found in Osteogenesis Imperfecta (brittle bone disease), result in a significantly reduced mechanical strength.
Assuntos
Proteínas de Bactérias , Colágeno , Proteínas de Membrana , Humanos , Colágeno/genética , Colágeno/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/metabolismo , Fenótipo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Proteica , Substituição de Aminoácidos , Dobramento de Proteína , Domínios Proteicos , Conformação Proteica em Folha betaRESUMO
Experimental measurement of time-dependent spontaneous exchange of amide protons with deuterium of the solvent provides information on the structure and dynamical structural variation in proteins. Two experimental techniques are used to probe the exchange: NMR, which relies on different magnetic properties of hydrogen and deuterium, and MS, which exploits the change in mass due to deuteration. NMR provides residue-specific information, that is, the rate of exchange or, analogously, the protection factor (i.e., the unitless ratio between the rate of exchange for a completely unstructured state and the observed rate). MS provides information that is specific to peptides obtained by proteolytic digestion. The spatial resolution of HDX-MS measurements depends on the proteolytic pattern of the protein, the fragmentation method used, and the overlap between peptides. Different computational approaches have been proposed to extract residue-specific information from peptide-level HDX-MS measurements. Here, we demonstrate the advantages of a method recently proposed that exploits self-consistency and classifies the possible sets of protection factors into a finite number of alternative solutions compatible with experimental data. The degeneracy of the solutions can be reduced (or completely removed) by exploiting the additional information encoded in the shape of the isotopic envelopes. We show how sparse and noisy MS data can provide high-resolution protection factors that correlate with NMR measurements probing the same protein under the same conditions.
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Medição da Troca de Deutério , Hidrogênio , Deutério/química , Medição da Troca de Deutério/métodos , Hidrogênio/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas/métodos , Peptídeos/química , Proteínas/químicaRESUMO
Cytochrome P450cam (CYP101A1) catalyzes the regio- and stereo-specific 5-exo-hydroxylation of camphor via a multistep catalytic cycle that involves two-electron transfer steps, with an absolute requirement that the second electron be donated by the ferrodoxin, putidaredoxin (Pdx). Whether P450cam, once camphor has bound to the active site and the substrate entry channel has closed, opens up upon Pdx binding, during the second electron transfer step, or it remains closed is still a matter of debate. A potential allosteric site for camphor binding has been identified and postulated to play a role in the binding of Pdx. Here, we have revisited paramagnetic NMR spectroscopy data and determined a heterogeneous ensemble of structures that explains the data, provides a complete representation of the P450cam/Pdx complex in solution, and reconciles alternative hypotheses. The allosteric camphor binding site is always present, and the conformational changes induced by camphor binding to this site facilitates Pdx binding. We also determined that the state to which Pdx binds comprises an ensemble of structures that have features of both the open and closed state. These results demonstrate that there is a finely balanced interaction between allosteric camphor binding and the binding of Pdx at high camphor concentrations.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cânfora 5-Mono-Oxigenase/química , Cânfora 5-Mono-Oxigenase/metabolismo , Cânfora/química , Ferredoxinas/metabolismo , Pseudomonas putida/enzimologia , Regulação Alostérica , Cânfora/metabolismo , Domínio Catalítico , Cristalografia por Raios X/métodos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Pseudomonas putida/químicaRESUMO
Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.
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Proteínas de Bactérias , Proteínas de Membrana , Streptococcus gordonii , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Streptococcus gordonii/química , Streptococcus gordonii/genética , Streptococcus gordonii/metabolismoRESUMO
BACKGROUND: Single-molecule experimental techniques such as optical tweezers or atomic force microscopy are a direct probe of the mechanical unfolding/folding of individual proteins. They are also a means to investigate free energy landscapes. Protein force spectroscopy alone provides limited information; theoretical models relate measurements to thermodynamic and kinetic properties of the protein, but do not reveal atomic level information. By building a molecular model of the protein and probing its properties through numerical simulation, one can gauge the response to an external force for individual interatomic interactions and determine structures along the unfolding pathway. In combination, single-molecule force probes and molecular simulations contribute to uncover the rich behavior of proteins when subjected to mechanical force. SCOPE OF REVIEW: We focus on how simplified protein models have been instrumental in showing how general properties of the free energy landscape of a protein relate to its response to mechanical perturbations. We discuss the role of simple protein models to explore the complexity of free energy landscapes and highlight important conceptual issues that more chemically accurate models with all-atom representations of proteins and solvent cannot easily address. MAJOR CONCLUSIONS: Native-centric, coarse-grained models, despite simplifications in chemical detail compared to all-atom models, can reproduce and interpret experimental results. They also highlight instances where the theoretical framework used to interpret single-molecule data is too simple. However, these simple models are not able to reproduce experimental findings where non-native contacts are involved. GENERAL SIGNIFICANCE: Mechanical forces are ubiquitous in the cell and it is increasingly clear that the way a protein responds to mechanical perturbation is important.
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Modelos Moleculares , Proteínas/química , Cinética , TermodinâmicaRESUMO
Streptococcus groups A and B cause serious infections, including early onset sepsis and meningitis in newborns. Rib domain-containing surface proteins are found associated with invasive strains and elicit protective immunity in animal models. Yet, despite their apparent importance in infection, the structure of the Rib domain was previously unknown. Structures of single Rib domains of differing length reveal a rare case of domain atrophy through deletion of 2 core antiparallel strands, resulting in the loss of an entire sheet of the ß-sandwich from an immunoglobulin-like fold. Previously, observed variation in the number of Rib domains within these bacterial cell wall-attached proteins has been suggested as a mechanism of immune evasion. Here, the structure of tandem domains, combined with molecular dynamics simulations and small angle X-ray scattering, suggests that variability in Rib domain number would result in differential projection of an N-terminal host-colonization domain from the bacterial surface. The identification of 2 further structures where the typical B-D-E immunoglobulin ß-sheet is replaced with an α-helix further confirms the extensive structural malleability of the Rib domain.
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Cyclic nucleotide-binding (CNB) domains allosterically regulate the activity of proteins with diverse functions, but the mechanisms that enable the cyclic nucleotide-binding signal to regulate distant domains are not well understood. Here we use optical tweezers and molecular dynamics to dissect changes in folding energy landscape associated with cAMP-binding signals transduced between the two CNB domains of protein kinase A (PKA). We find that the response of the energy landscape upon cAMP binding is domain specific, resulting in unique but mutually coordinated tasks: one CNB domain initiates cAMP binding and cooperativity, whereas the other triggers inter-domain interactions that promote the active conformation. Inter-domain interactions occur in a stepwise manner, beginning in intermediate-liganded states between apo and cAMP-bound domains. Moreover, we identify a cAMP-responsive switch, the N3A motif, whose conformation and stability depend on cAMP occupancy. This switch serves as a signaling hub, amplifying cAMP-binding signals during PKA activation.
Assuntos
Regulação Alostérica , Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transdução de Sinais , Algoritmos , Sítio Alostérico , Animais , Sítios de Ligação , Bovinos , AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática , Simulação de Dinâmica Molecular , Pinças Ópticas , Ligação ProteicaRESUMO
Recombinant therapeutic proteins are playing an ever-increasing role in the clinic. High-affinity binding candidates can be produced in a high-throughput manner through the process of selection and evolution from large libraries, but the structures of the complexes with target protein can only be determined for a small number of them in a costly, low-throughput manner, typically by x-ray crystallography. Reliable modeling of complexes would greatly help to understand their mode of action and improve them by further engineering, for example, by designing bi-paratopic binders. Designed ankyrin repeat proteins (DARPins) are one such class of antibody mimetics that have proven useful in the clinic, in diagnostics and research. Here we have developed a standardized procedure to model DARPin-target complexes that can be used to predict the structures of unknown complexes. It requires only the sequence of a DARPin and a structure of the unbound target. The procedure includes homology modeling of the DARPin, modeling of the flexible parts of a target, rigid body docking to ensembles of the target and docking with a partially flexible backbone. For a set of diverse DARPin-target complexes tested it generated a single model of the complex that well approximates the native state of the complex. We provide a protocol that can be used in a semi-automated way and with tools that are freely available. The presented concepts should help to accelerate the development of novel bio-therapeutics for scaffolds with similar properties.
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Repetição de Anquirina , Animais , Cristalografia por Raios X , Bases de Dados de Proteínas , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Proteínas/química , Proteínas/metabolismoRESUMO
Pathogenic micro-organisms utilize protein receptors (lectins) in adhesion to host tissues, a process that in some cases relies on the interaction between lectins and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analysed by X-ray crystallography the structures of the LecB lectin from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 4% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we propose a general strategy which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors.
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Lectinas/metabolismo , Oligossacarídeos/metabolismo , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Cristalografia por Raios X , Glucosamina/química , Glucosamina/metabolismo , Lectinas/química , Lectinas/genética , Ligantes , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Oligossacarídeos/química , Ligação Proteica , Pseudomonas aeruginosa/químicaRESUMO
Hydrogen/deuterium exchange monitored by mass spectrometry is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time-dependent change in the mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens, but determining the latter from the former is generally not possible. Here, we show that, if time-resolved measurements are available for a number of overlapping peptides that cover the whole sequence, rate constants for each amide hydrogen exchange (or equivalently, their protection factors) may be extracted and the uniqueness of the solutions obtained depending on the degree of peptide overlap. However, in most cases, the solution is not unique, and multiple alternatives must be considered. We provide a statistical method that clusters the solutions to further reduce their number. Such analysis always provides meaningful constraints on protection factors and can be used in situations in which obtaining more refined experimental data is impractical. It also provides a systematic way to improve data collection strategies to obtain unambiguous information at single-residue level (e.g., for assessing protein structure predictions at atomistic level).
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Deutério/química , Espectrometria de Massas/métodos , Peptídeos/química , Amidas/química , Complemento C3/química , Ligação de Hidrogênio , Espectrometria de Massas/normasRESUMO
The self-assembly of proteins into higher order structures is ubiquitous in living systems. It is also an essential process for the bottom-up creation of novel molecular architectures and devices for synthetic biology. However, the complexity of protein-protein interaction surfaces makes it challenging to mimic natural assembly processes in artificial systems. Indeed, many successful computationally designed protein assemblies are prescreened for "designability", limiting the choice of components. Here, we report a simple and pragmatic strategy to assemble chosen multisubunit proteins into more complex structures. A coiled-coil domain appended to one face of the pentameric cholera toxin B-subunit (CTB) enabled the ordered assembly of tubular supra-molecular complexes. Analysis of a tubular structure determined by X-ray crystallography has revealed a hierarchical assembly process that displays features reminiscent of the polymorphic assembly of polyomavirus proteins. The approach provides a simple and straightforward method to direct the assembly of protein building blocks which present either termini on a single face of an oligomer. This scaffolding approach can be used to generate bespoke supramolecular assemblies of functional proteins. Additionally, structural resolution of the scaffolded assemblies highlight "native-state" forced protein-protein interfaces, which may prove useful as starting conformations for future computational design.
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Toxina da Cólera/química , Proteínas/química , Algoritmos , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Domínios ProteicosRESUMO
Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used here to provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts.
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Sítio Alostérico , Técnicas Biossensoriais , Desenho de Fármacos , Proteínas/química , Regulação Alostérica , Animais , Regulação da Expressão Gênica , Humanos , Redes e Vias Metabólicas , Simulação de Dinâmica Molecular , Proteínas/genética , Proteínas/metabolismo , Transdução de Sinais , Termodinâmica , Transcrição GênicaRESUMO
Ion pairs are key stabilizing interactions between oppositely charged amino acid side chains in proteins. They are often depicted as single conformer salt bridges (hydrogen-bonded ion pairs) in crystal structures, but it is unclear how dynamic they are in solution. Ion pairs are thought to be particularly important in stabilizing single α-helix (SAH) domains in solution. These highly stable domains are rich in charged residues (such as Arg, Lys, and Glu) with potential ion pairs across adjacent turns of the helix. They provide a good model system to investigate how ion pairs can contribute to protein stability. Using NMR spectroscopy, small-angle X-ray light scattering (SAXS), and molecular dynamics simulations, we provide here experimental evidence that ion pairs exist in a SAH in murine myosin 7a (residues 858-935), but that they are not fixed or long lasting. In silico modeling revealed that the ion pairs within this α-helix exhibit dynamic behavior, rapidly forming and breaking and alternating between different partner residues. The low-energy helical state was compatible with a great variety of ion pair combinations. Flexible ion pair formation utilizing a subset of those available at any one time avoided the entropic penalty of fixing side chain conformations, which likely contributed to helix stability overall. These results indicate the dynamic nature of ion pairs in SAHs. More broadly, thermodynamic stability in other proteins is likely to benefit from the dynamic behavior of multi-option solvent-exposed ion pairs.
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Miosinas/química , Miosinas/metabolismo , Animais , Cristalografia por Raios X , Camundongos , Simulação de Dinâmica Molecular , Miosina VIIa , Conformação Proteica em alfa-Hélice , Estabilidade ProteicaRESUMO
Helices are the most common structural pattern observed in structured proteins. Polypeptide sequences that form helices in isolation have been identified and extensively studied. These are generally rich in alanine, the amino acid with strongest helical propensity. Insertion of charged or polar amino acids has been shown to be necessary to make alanine-rich peptides soluble and sometimes even increase the helicity of the peptides. More recently sequences that contain mostly charged residues (E-R/K rich) have been found in naturally occurring proteins that are highly helical, soluble, and extended regardless their length. Artificial sequences composed mostly or exclusively of charged amino acids have been designed that are also highly helical, depending on the specific pattern of oppositely charged residues. Here we explore the thermodynamic properties of a number of 16-residue long peptides with varying helical propensity by performing equilibrium simulations over a broad range of temperatures. We observe quantitative differences in the peptides' helical propensities that can be related to qualitative differences in the free energy landscape, depending on the ampholytic patterns in the sequence. The results provide hints on how the specific physical properties of naturally occurring long sequences with similar patterns of charged residues may relate to their biological function.