RESUMO
Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution NMR spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1 interactions, focused on both unfolded and folded SOD1 states in mixed phase and demixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the SOD1 folding equilibrium toward the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates.
Assuntos
Solventes , Superóxido Dismutase-1 , Superóxido Dismutase-1/química , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/genética , Humanos , Solventes/química , Desdobramento de Proteína , Ligação Proteica , Dobramento de Proteína , Modelos Moleculares , Grânulos de Estresse/metabolismo , Grânulos de Estresse/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , Conformação Proteica , Espectroscopia de Ressonância MagnéticaRESUMO
NMR spectroscopy is an important tool for the measurement of the electrostatic properties of biomolecules. To this point, paramagnetic relaxation enhancements (PREs) of 1H nuclei arising from nitroxide cosolutes in biomolecular solutions have been used to measure effective near-surface electrostatic potentials (ÏENS) of proteins and nucleic acids. Here, we present a gadolinium (Gd)-based NMR method, exploiting Gd chelates with different net charges, for measuring ÏENS values and demonstrate its utility through applications to a number of biomolecular systems. The use of Gd-based cosolutes offers several advantages over nitroxides for ÏENS measurements. First, unlike nitroxide compounds, Gd chelates enable electrostatic potential measurements on oxidation-sensitive proteins that require reducing agents. Second, the large electron spin quantum number of Gd (7/2) results in notably larger PREs for Gd chelates when used at the same concentrations as nitroxide radicals. Thus, it is possible to measure ÏENS values exclusively from + and - charged compounds even for highly charged biomolecules, avoiding the use of neutral cosolutes that, as we further establish here, limits the accuracy of the measured electrostatic potentials. In addition, the smaller concentrations of cosolutes required minimize potential binding to sites on macromolecules. Fourth, the closer proximity of the paramagnetic center and charged groups within Gd chelates, in comparison to the corresponding nitroxide compounds, enables more accurate predictions of ÏENS potentials for cross-validation of the experimental results. The Gd-based method described here, thus, broadens the applicability of studies of biomolecular electrostatics using solution NMR spectroscopy.
Assuntos
Gadolínio , Eletricidade Estática , Gadolínio/química , Proteínas/química , Propriedades de Superfície , Ressonância Magnética Nuclear Biomolecular , Quelantes/química , Espectroscopia de Ressonância Magnética/métodosRESUMO
Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2'-O-methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA-RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA-RNA base pairing, resulting from a â¼270-fold decrease and a concomitant â¼15-fold increase in the on- and off-rates for duplex formation, respectively. The â¼30-fold slower diffusion of RNA single strands within the condensed phase partially accounts for the reduced on-rate, but the further â¼9-fold reduction likely reflects shedding of CAPRIN1 chains that are interacting with the RNA prior to hybridization. Our study emphasizes the important role of protein solvation in modulating nucleic acid recognition processes inside condensates.
Assuntos
Hibridização de Ácido Nucleico , RNA , Termodinâmica , RNA/química , Cinética , Conformação de Ácido Nucleico , Pareamento de Bases , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Separação de FasesRESUMO
The determination of a protein's structure is often a first step towards the development of a mechanistic understanding of its function. Considerable advances in computational protein structure prediction have been made in recent years, with AlphaFold2 (AF2) emerging as the primary tool used by researchers for this purpose. While AF2 generally predicts accurate structures of folded proteins, we present here a case where AF2 incorrectly predicts the structure of a small, folded and compact protein with high confidence. This protein, pro-interleukin-18 (pro-IL-18), is the precursor of the cytokine IL-18. Interestingly, the structure of pro-IL-18 predicted by AF2 matches that of the mature cytokine, and not the corresponding experimentally determined structure of the pro-form of the protein. Thus, while computational structure prediction holds immense promise for addressing problems in protein biophysics, there is still a need for experimental structure determination, even in the context of small well-folded, globular proteins.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Modelos Moleculares , Proteínas/química , Algoritmos , Interleucina-18/química , SoftwareRESUMO
Several interleukin-1 (IL-1) family members, including IL-1ß and IL-18, require processing by inflammasome-associated caspases to unleash their activities. Here, we unveil, by cryoelectron microscopy (cryo-EM), two major conformations of the complex between caspase-1 and pro-IL-18. One conformation is similar to the complex of caspase-4 and pro-IL-18, with interactions at both the active site and an exosite (closed conformation), and the other only contains interactions at the active site (open conformation). Thus, pro-IL-18 recruitment and processing by caspase-1 is less dependent on the exosite than the active site, unlike caspase-4. Structure determination by nuclear magnetic resonance uncovers a compact fold of apo pro-IL-18, which is similar to caspase-1-bound pro-IL-18 but distinct from cleaved IL-18. Binding sites for IL-18 receptor and IL-18 binding protein are only formed upon conformational changes after pro-IL-18 cleavage. These studies show how pro-IL-18 is selected as a caspase-1 substrate, and why cleavage is necessary for its inflammatory activity.
Assuntos
Caspase 1 , Microscopia Crioeletrônica , Interleucina-18 , Transdução de Sinais , Interleucina-18/metabolismo , Caspase 1/metabolismo , Humanos , Inflamassomos/metabolismo , Animais , Conformação Proteica , Ligação Proteica , Sítios de Ligação , Camundongos , Receptores de Interleucina-18/metabolismo , Modelos Moleculares , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
Solution NMR spectroscopy has tremendous potential for providing atomic resolution insights into the interactions between proteins and nucleic acids partitioned into condensed phases of phase-separated systems. However, the highly viscous nature of the condensed phase challenges applications, and in particular, the extraction of quantitative, site-specific information. Here, we present a delayed decoupling-based HMQC pulse sequence for methyl-TROSY studies of 'client' proteins and nucleic acids partitioned into 'scaffold' proteinaceous phase-separated solvents. High sensitivity and excellent quality spectra are recorded of a nascent form of superoxide dismutase and of a small RNA fragment partitioned into CAPRIN1 condensates.
Assuntos
Ressonância Magnética Nuclear Biomolecular , RNA , RNA/química , Ressonância Magnética Nuclear Biomolecular/métodos , Dobramento de Proteína , Proteínas/química , Superóxido Dismutase/química , Condensados Biomoleculares/química , AlgoritmosRESUMO
CPMG relaxation dispersion studies of biomolecular dynamics on the µs-ms timescale can provide detailed kinetic, thermodynamic, and structural insights into function. Frequently, the 15N spin serves as the probe of choice, as uniform incorporation of the 15N isotope is facile and cost-effective, and the interpretation of the resulting data is often relatively straightforward. In conventional CPMG relaxation dispersion experiments the application of CPMG pulses with constant radiofrequency (RF) phase can lead to artifactual dispersion profiles that result from off-resonance effects, RF field inhomogeneity, and pulse miscalibration. The development of CPMG experiments with the [0013]-phase cycle has significantly reduced the impact of pulse imperfections over a greater bandwidth of frequency offsets in comparison to constant phase experiments. Application of 15N-TROSY-based CPMG schemes to studies of the dynamics of large molecules is necessary for high sensitivity, yet the correct incorporation of the [0013]-phase cycle is non-trivial. Here we present TROSY- and anti-TROSY-based 15N CPMG experiments with the [0013]-phase cycling scheme and demonstrate, through comprehensive numerical simulations and experimental validation, enhanced resistance to pulse imperfections relative to traditional schemes utilizing constant phase CPMG pulses. Notably, exchange parameters derived from the new experiments are in good agreement with those obtained using other, more established, 15N-based CPMG approaches.
RESUMO
The DegP protease-chaperone operates within the periplasm of Gram-negative bacteria, where it assists in the regulation of protein homeostasis, promotes virulence, and is essential to survival under stress. To carry out these tasks, DegP forms a network of preorganized apo oligomers that facilitate the capture of substrates within distributions of cage-like complexes which expand to encapsulate clients of various sizes. Although the architectures of DegP cage complexes are well understood, little is known about the structures, dynamics, and interactions of client proteins within DegP cages and the relationship between client structural dynamics and function. Here, we probe host-guest interactions within a 600 kDa DegP cage complex throughout the DegP activation cycle using a model α-helical client protein through a combination of hydrodynamics measurements, methyl-transverse relaxation optimized spectroscopy-based solution nuclear magnetic resonance studies, and proteolytic activity assays. We find that in the presence of the client, DegP cages assemble cooperatively with few intermediates. Our data further show that the N-terminal half of the bound client, which projects into the interior of the cages, is predominantly unfolded and flexible, and exchanges between multiple conformational states over a wide range of time scales. Finally, we show that a concerted structural transition of the protease domains of DegP occurs upon client engagement, leading to activation. Together, our findings support a model of DegP as a highly cooperative and dynamic molecular machine that stabilizes unfolded states of clients, primarily via interactions with their C-termini, giving rise to efficient cleavage.
Assuntos
Proteínas de Choque Térmico , Hidrodinâmica , Proteínas Periplásmicas , Serina Endopeptidases , Humanos , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Espectroscopia de Ressonância MagnéticaRESUMO
Segments of proteins with high ß-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in ß-strand and ß-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein-peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid ß1-42 (Aß42). The Aß binders block the assembly of Aß fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aß42 species.
Assuntos
Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Ligação Proteica , Peptídeos/química , Peptídeos/farmacologia , Amiloide/química , Amiloide/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Desenho de Fármacos , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Proteínas tau/metabolismo , Proteínas tau/química , Pré-Albumina/química , Pré-Albumina/metabolismo , Sequência de AminoácidosRESUMO
Ras-related nuclear protein (Ran) is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) and a regulator of multiple cellular processes. In healthy cells, the GTP-bound form of Ran is concentrated at chromatin, creating a Ranâ¢GTP gradient that provides the driving force for nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation. The Ranâ¢GTP gradient is maintained by the regulator of chromatin condensation 1 (RCC1), a guanine nucleotide exchange factor that accelerates GDP/GTP exchange in Ran. RCC1 interacts with nucleosomes, which are the fundamental repeating units of eukaryotic chromatin. Here, we present a cryo-EM analysis of a trimeric complex composed of the nucleosome core particle (NCP), RCC1, and Ran. While the contacts between RCC1 and Ran in the complex are preserved compared with a previously determined structure of RCC1-Ran, our study reveals that RCC1 and Ran interact dynamically with the NCP and undergo rocking motions on the nucleosome surface. Furthermore, the switch 1 region of Ran, which plays an important role in mediating conformational changes associated with the substitution of GDP and GTP nucleotides in Ras family members, appears to undergo disorder-order transitions and forms transient contacts with the C-terminal helix of histone H2B. Nucleotide exchange assays performed in the presence and absence of NCPs are not consistent with an active role for nucleosomes in nucleotide exchange, at least in vitro. Instead, the nucleosome stabilizes RCC1 and serves as a hub that concentrates RCC1 and Ran to promote efficient Ranâ¢GDP to Ranâ¢GTP conversion.
Assuntos
Cromatina , Nucleossomos , Proteína ran de Ligação ao GTP , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Guanosina Trifosfato/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Nucleotídeos/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Humanos , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismoRESUMO
Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3. PARylation disrupted interactions between nucleosomes, preventing self-association. Addition of LIG3 and XRCC1 to PARylated nucleosomes generated condensates that selectively partition DNA repair-associated proteins in a PAR- and phosphorylation-dependent manner in vitro. Our results establish that PARylation influences nucleosomes across different length scales, extending from the atom-level motions of histone tails to the mesoscale formation of condensates with selective compositions.
Assuntos
Nucleossomos , Poli ADP Ribosilação , Nucleossomos/genética , Poli ADP Ribosilação/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Microscopia Crioeletrônica , Condensados Biomoleculares , Reparo do DNA , Histonas/genética , Histonas/metabolismo , DNA/genética , DNA/metabolismo , Dano ao DNA , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a 'visible' major conformer exchanges with two 'invisible' minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate-fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide 15N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although 15N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, 15N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of 15N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale ( k e x , F I 1 ~ 550 s-1, k e x , F I 2 ~ 1200 s-1, k e x , I 1 I 2 ~ 5000 s-1), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15 °C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.
Assuntos
Amidas , Humanos , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Espectroscopia de Ressonância Magnética , Amidas/química , TemperaturaRESUMO
Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.
Assuntos
Apoptossomas , Caspases , Caspase 9/metabolismo , Apoptossomas/química , Caspases/metabolismo , Apoptose , Espectroscopia de Ressonância Magnética , Caspase 3/metabolismoRESUMO
The periplasmic protein DegP, which is implicated in virulence factor transport leading to pathogenicity, is a bi-functional protease and chaperone that helps to maintain protein homeostasis in Gram-negative bacteria and is essential to bacterial survival under stress conditions. To perform these functions, DegP captures clients inside cage-like structures, which we have recently shown to form through the reorganization of high-order preformed apo oligomers, consisting of trimeric building blocks, that are structurally distinct from client-bound cages. Our previous studies suggested that these apo oligomers may allow DegP to encapsulate clients of various sizes under protein folding stresses by forming ensembles that can include extremely large cage particles, but how this occurs remains an open question. To explore the relation between cage and substrate sizes, we engineered a series of DegP clients of increasing hydrodynamic radii and analyzed their influence on DegP cage formation. We used dynamic light scattering and cryogenic electron microscopy to characterize the hydrodynamic properties and structures of the DegP cages that are adopted in response to each client. We present a series of density maps and structural models that include those for novel particles of approximately 30 and 60 monomers. Key interactions between DegP trimers and the bound clients that stabilize the cage assemblies and prime the clients for catalysis are revealed. We also provide evidence that DegP can form cages which approach subcellular organelles in terms of size.
Assuntos
Proteínas de Choque Térmico , Proteínas Periplásmicas , Humanos , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Peptídeo Hidrolases/metabolismo , Escherichia coli/metabolismo , Serina Endopeptidases/química , Proteínas Periplásmicas/química , Proteínas Periplásmicas/metabolismo , Chaperonas Moleculares/metabolismoRESUMO
Accumulation of α-synuclein into toxic oligomers or fibrils is implicated in dopaminergic neurodegeneration in Parkinson's disease. Here we performed a high-throughput, proteome-wide peptide screen to identify protein-protein interaction inhibitors that reduce α-synuclein oligomer levels and their associated cytotoxicity. We find that the most potent peptide inhibitor disrupts the direct interaction between the C-terminal region of α-synuclein and CHarged Multivesicular body Protein 2B (CHMP2B), a component of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III). We show that α-synuclein impedes endolysosomal activity via this interaction, thereby inhibiting its own degradation. Conversely, the peptide inhibitor restores endolysosomal function and thereby decreases α-synuclein levels in multiple models, including female and male human cells harboring disease-causing α-synuclein mutations. Furthermore, the peptide inhibitor protects dopaminergic neurons from α-synuclein-mediated degeneration in hermaphroditic C. elegans and preclinical Parkinson's disease models using female rats. Thus, the α-synuclein-CHMP2B interaction is a potential therapeutic target for neurodegenerative disorders.
Assuntos
Doença de Parkinson , Masculino , Feminino , Animais , Ratos , Humanos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Caenorhabditis elegans/metabolismo , Neurônios Dopaminérgicos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismoRESUMO
Epigenetic modifications of chromatin play a critical role in regulating the fidelity of the genetic code and in controlling the translation of genetic information into the protein components of the cell. One key posttranslational modification is acetylation of histone lysine residues. Molecular dynamics simulations, and to a smaller extent experiment, have established that lysine acetylation increases the dynamics of histone tails. However, a systematic, atomic resolution experimental investigation of how this epigenetic mark, focusing on one histone at a time, influences the structural dynamics of the nucleosome beyond the tails, and how this translates into accessibility of protein factors such as ligases and nucleases, has yet to be performed. Herein, using NMR spectroscopy of nucleosome core particles (NCPs), we evaluate the effects of acetylation of each histone on tail and core dynamics. We show that for histones H2B, H3, and H4, the histone core particle dynamics are little changed, even though the tails have increased amplitude motions. In contrast, significant increases to H2A dynamics are observed upon acetylation of this histone, with the docking domain and L1 loop particularly affected, correlating with increased susceptibility of NCPs to nuclease digestion and more robust ligation of nicked DNA. Dynamic light scattering experiments establish that acetylation decreases inter-NCP interactions in a histone-dependent manner and facilitates the development of a thermodynamic model for NCP stacking. Our data show that different acetylation patterns result in nuanced changes to NCP dynamics, modulating interactions with other protein factors, and ultimately controlling biological output.
Assuntos
Histonas , Nucleossomos , Histonas/metabolismo , Acetilação , Lisina/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
With the recent success in calculating protein structures from amino acid sequences using artificial intelligence-based algorithms, an important next step is to decipher how dynamics is encoded by the primary protein sequence so as to better predict function. Such dynamics information is critical for protein design, where strategies could then focus not only on sequences that fold into particular structures that perform a given task, but would also include low-lying excited protein states that could influence the function of the designed protein. Herein, we illustrate the importance of dynamics in modulating the function of C34, a designed α/ß protein that captures ß-strands of target ligands and is a member of a family of proteins designed to sequester ß-strands and ß hairpins of aggregation-prone molecules that lead to a variety of pathologies. Using a strategy to "see" regions of apo C34 that are invisible to NMR spectroscopy as a result of pervasive conformational exchange, as well as a mutagenesis approach whereby C34 molecules are stabilized into a single conformer, we determine the structures of the predominant conformations that are sampled by C34 and show that these attenuate the affinity for cognate peptide. Subsequently, the observed motion is exploited to develop an allosterically regulated peptide binder whose binding affinity can be controlled through the addition of a second molecule. Our study emphasizes the unique role that NMR can play in directing the design process and in the construction of new molecules with more complex functionality.
Assuntos
Inteligência Artificial , Proteínas , Conformação Proteica , Sequência de Aminoácidos , Peptídeos , LigantesRESUMO
The measurement of spin relaxation rates provides a unique avenue for quantifying dynamic processes in biomolecules. In order to simplify analysis of the measurements so that a few key intuitive parameters can be extracted, it is often the case that experiments are designed to eliminate interference effects between different classes of spin relaxation. One example emerges in the measurement of amide proton (1HN) transverse relaxation rates in 15N labeled proteins, where 15N inversion pulses are applied during a relaxation element to eliminate cross-correlated spin relaxation between 1HN-15N dipole-1HN CSA interactions. We show that unless these pulses are essentially perfect, significant oscillations in magnetization decay profiles can be obtained, due to the excitation of multiple-quantum coherences, leading potentially to errors in measured R2 rates. With the recent development of experiments for quantifying electrostatic potentials via amide proton relaxation rates, the need for highly accurate measurement schemes becomes critical. Straightforward modifications to existing pulse sequences are suggested to achieve this goal.
Assuntos
Amidas , Prótons , Ressonância Magnética Nuclear Biomolecular , ProteínasRESUMO
Electrostatic interactions can play important roles in regulating various biological processes. Quantifying surface electrostatics of biomolecules is, therefore, of significant interest. Recent advances in solution NMR spectroscopy have enabled site-specific measurements of de novo near-surface electrostatic potentials (ÏENS) based on a comparison of solvent paramagnetic relaxation enhancements generated from differently charged paramagnetic co-solutes with similar structures. Although the NMR-derived near-surface electrostatic potentials have been shown to agree with theoretical calculations in the context of folded proteins and nucleic acids, such benchmark comparisons may not always be possible, particularly in cases where high-resolution structural models are lacking, such as in the study of intrinsically disordered proteins. Cross-validation of ÏENS potentials can be achieved by comparing values obtained using three pairs of paramagnetic co-solutes, each with a different net charge. Notably we have found cases where agreement of ÏENS potentials between the three pairs is poor and herein we investigate the source of this discrepancy in some detail. We show that for the systems considered here ÏENS potentials obtained from cationic and anionic co-solutes are accurate and that the use of paramagnetic co-solutes with different structures can be a viable option for validation, although the optimal choice of paramagnetic compounds depends on the system of interest.
Assuntos
Proteínas Intrinsicamente Desordenadas , Ácidos Nucleicos , Solventes , Eletricidade Estática , Espectroscopia de Ressonância Magnética/métodos , Proteínas Intrinsicamente Desordenadas/química , Soluções , Ressonância Magnética Nuclear Biomolecular/métodosRESUMO
The HMQC pulse sequence and variants thereof have been exploited in studies of high molecular weight protein complexes, taking advantage of the fact that fast and slow relaxing magnetization components are sequestered along two distinct magnetization transfer pathways. Despite the simplicity of the HMQC scheme an even shorter version can be designed, based on elimination of the terminal refocusing period, as a further means of increasing signal. Here we present such an experiment, and show that significant sensitivity gains, in some cases by factors of two or more, are realized in studies of proteins varying in molecular masses from 100 kDa to 1 MDa.