RESUMEN
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.
Asunto(s)
Hibridación de Ácido Nucleico , ARN , Termodinámica , ARN/química , Cinética , Conformación de Ácido Nucleico , Emparejamiento Base , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Separación de FasesRESUMEN
Pathogenesis-related class 10 (PR-10) proteins play a crucial role in plant defense by acting as ribonucleases. The specific mechanism of action and substrate specificity of these proteins have remained largely unexplored so far. In this study, we elucidate the enzymatic activity of Pruâ p 1, a PR-10 protein from peach. We demonstrate that this protein catalyzes the endonucleolytic backbone cleavage of RNA substrates into short oligonucleotides. Initial cleavage products, identified through kinetic analysis, can bind again, priming them for further degradation. NMR binding site mapping reveals that the large internal cavity of Pruâ p 1, which is characteristic for PR-10 proteins, serves as an anchoring site for single-stranded ribonucleotide chains. We propose a structure-based mechanistic model that accounts for the observed cleavage patterns and the inhibitory effect of zeatin, a nucleoside analog, on the ribonuclease activity of Pruâ p 1.
Asunto(s)
Proteínas de Plantas , Sitios de Unión , Endonucleasas/metabolismo , Endonucleasas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , ARN/metabolismo , ARN/química , Especificidad por Sustrato , Prunus persicaRESUMEN
Here we report the efficient synthetic access to 13C/15N-labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75â nt E. coli tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis. 13C/15N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5'-monophosphate (ΨMP) by using YeiN, a Ψ-5'-monophosphate C-glycosidase. Removal of the 5'-phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ -building block carrying both a 13C and a 15N label was incorporated into a product RNA and the complex formation with a 63â nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75â nt E. coli tRNAGly CCmnm5U, identifying the TΨC-loop 5-methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI-labelled Ψ building blocks will allow the solution and solid-state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ-modified nucleic acids.
Asunto(s)
Escherichia coli , Marcaje Isotópico , Isótopos de Nitrógeno , Compuestos Organofosforados , Seudouridina , Seudouridina/química , Compuestos Organofosforados/química , Isótopos de Nitrógeno/química , Marcaje Isotópico/métodos , ARN/química , Isótopos de Carbono/química , Espectroscopía de Resonancia Magnética/métodosRESUMEN
Additive manufacturing (3D printing) has greatly revolutionized the way researchers approach certain technical challenges. Despite its outstanding print quality and resolution, stereolithography (SLA) printing is cost-effective and relatively accessible. However, applications involving mass spectrometry (MS) are few due to residual oligomers and additives leaching from SLA-printed devices that interfere with MS analyses. We identified the crosslinking agent urethane dimethacrylate as the main contaminant derived from SLA prints. A stringent washing and post-curing protocol mitigated sample contamination and rendered SLA prints suitable for MS hyphenation. Thereafter, SLA printing was used to produce 360 µm I.D. microcolumn chips with excellent structural properties. By packing the column with polystyrene microspheres and covalently immobilizing pepsin, an exceptionally effective microscale immobilized enzyme reactor (µIMER) was created. Implemented in an online liquid chromatography-MS/MS setup, the protease microcolumn enabled reproducible protein digestion and peptide mapping with 100% sequence coverage obtained for three different recombinant proteins. Additionally, when assessing the µIMER digestion efficiency for complex proteome samples, it delivered a 144-fold faster and significantly more efficient protein digestion compared to 24 h for bulk digestion. The 3D-printed µIMER withstands remarkably high pressures above 130 bar and retains its activity for several weeks. This versatile platform will enable researchers to produce tailored polymer-based enzyme reactors for various applications in analytical chemistry and beyond.
Asunto(s)
Enzimas Inmovilizadas , Espectrometría de Masas en Tándem , Cromatografía Liquida , Enzimas Inmovilizadas/química , Mapeo Peptídico/métodos , Impresión TridimensionalRESUMEN
PR-10 proteins constitute a major cause of food allergic reactions. Birch-pollen-related food allergies are triggered by the immunologic cross-reactivity of IgE antibodies with structurally homologous PR-10 proteins that are present in birch pollen and various food sources. While the three-dimensional structures of PR-10 food allergens have been characterized in detail, only a few experimental studies have addressed the structural flexibility of these proteins. In this study, we analyze the millisecond-timescale structural flexibility of thirteen PR-10 proteins from prevalent plant food sources by NMR relaxation-dispersion spectroscopy, in a comparative manner. We show that all the allergens in this study have inherently flexible protein backbones in solution, yet the extent of the structural flexibility appears to be strikingly protein-specific (but not food-source-specific). Above-average flexibility is present in the two short helices, α1 and α2, which form a V-shaped support for the long C-terminal helix α3, and shape the internal ligand-binding cavity, which is characteristic for PR-10 proteins. An in-depth analysis of the NMR relaxation-dispersion data for the PR-10 allergen from peanut reveals the presence of at least two subglobal conformational transitions on the millisecond timescale, which may be related to the release of bound low-molecular-weight ligands from the internal cavity.
Asunto(s)
Hipersensibilidad a los Alimentos , Polen , Alérgenos , Secuencia de Aminoácidos , Antígenos de Plantas , Betula/metabolismo , Reacciones Cruzadas , Proteínas de Plantas/metabolismo , Polen/metabolismoRESUMEN
Protein oligomerization processes are widespread and of crucial importance to understand degenerative diseases and healthy regulatory pathways. One particular case is the homo-oligomerization of folded domains involving domain swapping, often found as a part of the protein homeostasis in the crowded cytosol, composed of a complex mixture of cosolutes. Here, we have investigated the effect of a plethora of cosolutes of very diverse nature on the kinetics of a protein dimerization by domain swapping. In the absence of cosolutes, our system exhibits slow interconversion rates, with the reaction reaching the equilibrium within the average protein homeostasis timescale (24-48 h). In the presence of crowders, though, the oligomerization reaction in the same time frame will, depending on the protein's initial oligomeric state, either reach a pure equilibrium state or get kinetically trapped into an apparent equilibrium. Specifically, when the reaction is initiated from a large excess of dimer, it becomes unsensitive to the effect of cosolutes and reaches the same equilibrium populations as in the absence of cosolute. Conversely, when the reaction starts from a large excess of monomer, the reaction during the homeostatic timescale occurs under kinetic control, and it is exquisitely sensitive to the presence and nature of the cosolute. In this scenario (the most habitual case in intracellular oligomerization processes), the effect of cosolutes on the intermediate conformation and diffusion-mediated encounters will dictate how the cellular milieu affects the domain-swapping reaction.
Asunto(s)
Cinética , Difusión , Dimerización , Sustancias Macromoleculares , Multimerización de ProteínaRESUMEN
Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2'-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3'-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing.
Asunto(s)
Intrones/genética , Conformación de Ácido Nucleico , Empalme del ARN/fisiología , ARN/química , Azúcares/química , Sitios de Unión , Carbohidratos/química , Magnesio/química , Espectroscopía de Resonancia Magnética , ARN/metabolismo , Azúcares/metabolismoRESUMEN
Using chemical synthesis and solution NMR spectroscopy, RNA structural ensembles including a major ground state and minor populated excited states can be studied at atomic resolution. In this work, atom-specific 13C labeled RNA building blocks - a 5-13C-uridine and a 2,8-13C2-adenosine building block - are used to introduce isolated 13C-1H-spin topologies into a target RNA to probe such structural ensembles via NMR spectroscopy. First, the 5-13C-uridine 2'-O-TBDMS-phosphoramidite building block was introduced into a 21 nucleotide (nt) tP5c stem construct of the tP5abc subdomain of the Tetrahymena group I ribozyme. Then, the 2,8-13C2-adenosine 2'-O-TBDMS-phosphoramidite building block was incorporated into a 9â¯kDa and a 15â¯kD construct derived from the epsilon (ε) RNA element of the duck Hepatitis B virus. The 2,8-13C2-adenosine resonances of the 9â¯kDa 28 nt sequence could be mapped to the full-length 53â¯nt construct. The isolated NMR active nuclei pairs were used to probe for low populated excited states (<10%) via 13C-Carr-Purcell-Meiboom-Gill (CPMG)-relaxation dispersion NMR spectroscopy. The 13C-CPMG relaxation dispersion experiment recapitulated a secondary structure switching event in the P5c hairpin of the group I intron construct previously revealed by 15N relaxation dispersion experiments. In the ε-HBV RNA an unfolding event occurring on the millisecond time scale was found in the upper stem in-line with earlier observations. This unpaired conformational state is presumed to be important for the binding of the epsilon reverse transcriptase (RT) enzyme. Thus, a full description of an RNA's folding landscape helps to obtain a deeper understanding of its function, as these high energy conformational states often represent functionally important intermediates involved in (un)folding or ribozyme catalysis.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , ARN/análisis , ARN/genética , Radioisótopos de Carbono/análisis , Radioisótopos de Carbono/química , Conformación Proteica , ARN/síntesis químicaRESUMEN
We report the synthesis of atom-specifically 13C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-13C-modified pyrimidine and 8-13C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-13C-2'deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min-1. Finally, we used 13C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s-1 and with a backward rate constant of 10.6 s-1.
Asunto(s)
ADN Cruciforme/química , ADN/química , Duplicado del Terminal Largo de VIH , Proteínas de la Nucleocápside/química , Compuestos Organofosforados/química , ARN/química , Emparejamiento Base , Isótopos de Carbono , G-Cuádruplex , VIH-1/química , Marcaje Isotópico , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Imitación Molecular , Conformación de Ácido Nucleico , Compuestos Organofosforados/síntesis química , Técnicas de Síntesis en Fase SólidaRESUMEN
Watson-Crick like G-U mismatches with tautomeric Genol or Uenol bases can evade fidelity checkpoints and thereby contribute to translational errors. The 5-oxyacetic acid uridine (cmo5 U) modification is a base modification at the wobble position on tRNAs and is presumed to expand the decoding capability of tRNA at this position by forming Watson-Crick like cmo5 Uenol -G mismatches. A detailed investigation on the influence of the cmo5 U modification on structural and dynamic features of RNA was carried out by using solution NMR spectroscopy and UV melting curve analysis. The introduction of a stable isotope labeled variant of the cmo5 U modifier allowed the application of relaxation dispersion NMR to probe the potentially formed Watson-Crick like cmo5 Uenol -G base pair. Surprisingly, we find that at neutral pH, the modification promotes transient formation of anionic Watson-Crick like cmo5 U- -G, and not enolic base pairs. Our results suggest that recoding is mediated by an anionic Watson-Crick like species, as well as bring an interesting aspect of naturally occurring RNA modifications into focus-the fine tuning of nucleobase properties leading to modulation of the RNA structural landscape by adoption of alternative base pairing patterns.
RESUMEN
Nuclear magnetic resonance (NMR) spectroscopy provides a unique toolbox of experimental probes for studying dynamic processes on a wide range of timescales, ranging from picoseconds to milliseconds and beyond. Along with NMR hardware developments, recent methodological advancements have enabled the characterization of allosteric proteins at unprecedented detail, revealing intriguing aspects of allosteric mechanisms and increasing the proportion of the conformational ensemble that can be observed by experiment. Here, we present an overview of NMR spectroscopic methods for characterizing equilibrium fluctuations in free and bound states of allosteric proteins that have been most influential in the field. By combining NMR experimental approaches with molecular simulations, atomistic-level descriptions of the mechanisms by which allosteric phenomena take place are now within reach.
Asunto(s)
Enzimas/química , Enzimas/ultraestructura , Espectroscopía de Resonancia Magnética/métodos , Modelos Químicos , Simulación de Dinámica Molecular , Regulación Alostérica , Sitio Alostérico , Secuencia de Aminoácidos , Activación Enzimática , Datos de Secuencia Molecular , Unión Proteica , Análisis de Secuencia de ProteínaRESUMEN
BACKGROUND: The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties. OBJECTIVE: We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1. METHODS: By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model. RESULTS: Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment. CONCLUSIONS: Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.
Asunto(s)
Alérgenos/química , Antígenos de Plantas/química , Alérgenos/genética , Alérgenos/inmunología , Animales , Antígenos de Plantas/genética , Antígenos de Plantas/inmunología , Endosomas , Femenino , Concentración de Iones de Hidrógeno , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Ratones Endogámicos BALB C , Mutación , Polen/inmunología , Pliegue de Proteína , Estabilidad ProteicaRESUMEN
The protein Bet v 1 represents the main cause for allergic reactions to birch pollen in Europe and North America. Structurally homologous isoforms of Bet v 1 can have different properties regarding allergic sensitization and Th2 polarization, most likely due to differential susceptibility to proteolytic cleavage. Using NMR relaxation experiments and molecular dynamics simulations, we demonstrate that the initial proteolytic cleavage sites in two naturally occurring Bet v 1 isoforms, Bet v 1.0101 (Bet v 1a) and Bet v 1.0102 (Bet v 1d), are conformationally flexible. Inaccessible cleavage sites in helices and strands are highly flexible on the microsecond-millisecond time scale, whereas those located in loops display faster nanosecond-microsecond flexibility. The data consistently show that Bet v 1.0102 is more flexible and conformationally heterogeneous than Bet v 1.0101. Moreover, NMR hydrogen-deuterium exchange measurements reveal that the backbone amides in Bet v 1.0102 are significantly more solvent exposed, in agreement with this isoform's higher susceptibility to proteolytic cleavage. The differential conformational flexibility of Bet v 1 isoforms, along with the transient exposure of inaccessible sites to the protein surface, may be linked to proteolytic susceptibility, representing a potential structure-based rationale for the observed differences in Th2 polarization and allergic sensitization.
Asunto(s)
Antígenos de Plantas/química , Modelos Moleculares , Proteínas de Plantas/química , Conformación Proteica , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Isoformas de ProteínasRESUMEN
Allosteric regulation plays an important role in a myriad of biomacromolecular processes. Specifically, in a protein, the process of allostery refers to the transmission of a local perturbation, such as ligand binding, to a distant site. Decades after the discovery of this phenomenon, models built on static images of proteins are being reconsidered with the knowledge that protein dynamics plays an important role in its function. Molecular dynamics simulations are a valuable tool for studying complex biomolecular systems, providing an atomistic description of their structure and dynamics. Unfortunately, their predictive power has been limited by the complexity of the biomolecule free-energy surface and by the length of the allosteric timescale (in the order of milliseconds). In this work, we are able to probe the origins of the allosteric changes that transcription factor mixed lineage leukemia (MLL) causes to the interactions of KIX domain of CREB-binding protein (CBP) with phosphorylated kinase inducible domain (pKID), by combing all-atom molecular dynamics with enhanced sampling methods recently developed in our group. We discuss our results in relation to previous NMR studies. We also develop a general simulations protocol to study allosteric phenomena and many other biological processes that occur in the micro/milliseconds timescale.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Regulación Alostérica , Proteína de Unión a CREB/química , Modelos Moleculares , Simulación de Dinámica Molecular , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , FosforilaciónRESUMEN
In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear (13) C and (1) H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site-specific (2) H and (13) C labeling, spin topologies are introduced into DNA and RNA that make (1) H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A-site RNA was previously shown to undergo a two site exchange process in the micro- to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV-1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for (1) H RD experiments of nucleic acids.
Asunto(s)
ADN/química , Marcaje Isotópico/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , ARN/química , Secuencia de Bases , Conformación de Ácido Nucleico , ProtonesRESUMEN
Adiabatically swept pulses were originally designed for the purpose of broadband spin inversion. Later, unexpected advantages of their utilization were also found in other applications, such as refocusing to excite spin echoes, studies of chemical exchange or fragment-based drug design. Here, we present new experiments to characterize fast (ps-ns) protein dynamics, which benefit from little-known properties of adiabatic pulses. We developed a strategy for measuring cross-correlated cross-relaxation (CCCR) rates during adiabatic pulses. This experiment provides a linear combination of longitudinal and transverse CCCR rates, which is offset-independent across a typical amide (15)N spectrum. The pulse sequence can be recast to provide accurate transverse CCCR rates weighted by the populations of exchanging states. Sensitivity can be improved in systems in slow exchange. Finally, the experiments can be easily modified to yield residue-specific correlation times. The average correlation time of motions can be determined with a single experiment while at least two different experiments had to be recorded until now.
Asunto(s)
Proteína de Unión a CREB/química , Ubiquitina/química , Humanos , Resonancia Magnética Nuclear Biomolecular/métodos , Dominios y Motivos de Interacción de ProteínasRESUMEN
UNLABELLED: Nuclear magnetic resonance (NMR) is a powerful tool for observing the motion of biomolecules at the atomic level. One technique, the analysis of relaxation dispersion phenomenon, is highly suited for studying the kinetics and thermodynamics of biological processes. Built on top of the relax computational environment for NMR dynamics is a new dispersion analysis designed to be comprehensive, accurate and easy-to-use. The software supports more models, both numeric and analytic, than current solutions. An automated protocol, available for scripting and driving the graphical user interface (GUI), is designed to simplify the analysis of dispersion data for NMR spectroscopists. Decreases in optimization time are granted by parallelization for running on computer clusters and by skipping an initial grid search by using parameters from one solution as the starting point for another -using analytic model results for the numeric models, taking advantage of model nesting, and using averaged non-clustered results for the clustered analysis. AVAILABILITY AND IMPLEMENTATION: The software relax is written in Python with C modules and is released under the GPLv3+ license. Source code and precompiled binaries for all major operating systems are available from http://www.nmr-relax.com. CONTACT: edward@nmr-relax.com.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Programas Informáticos , Estadística como Asunto/métodos , Gráficos por Computador , Cinética , Termodinámica , Interfaz Usuario-ComputadorRESUMEN
An NMR-based approach to characterizing the binding kinetics of ligand molecules to biomolecules, like RNA or proteins, by ligand-detected Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments is described. A (15)N-modified preQ1 ligand is used to acquire relaxation dispersion experiments in the presence of low amounts of the Fsu classâ I preQ1 aptamer RNA, and increasing ligand concentrations to probe the RNA small molecule interaction. Our experimental data strongly support the conformational selection mechanism postulated. The approach gives direct access to two parameters of a ligand-receptor interaction: the off rate and the population of the small molecule-receptor complex. A detailed description of the kinetics underlying the ligand binding process is of crucial importance to fully understanding a riboswitch's function and to evaluate potential new antibiotics candidates targeting the noncoding RNA species. Ligand-detected NMR relaxation dispersion experiments represent a valuable diagnostic tool for the characterization of binding mechanisms.
Asunto(s)
Aptámeros de Nucleótidos/química , Resonancia Magnética Nuclear Biomolecular/métodos , LigandosRESUMEN
Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.