Biomolecular polyelectrolyte complexes can be formed between oppositely charged intrinsically disordered regions (IDRs) of proteins or between IDRs and nucleic acids. Highly charged IDRs are abundant in the nucleus, yet few have been functionally characterized. Here, we show that a positively charged IDR within the human ATP-dependent DNA helicase Q4 (RECQ4) forms coacervates with G-quadruplexes (G4s). We describe a three-step model of charge-driven coacervation by integrating equilibrium and kinetic binding data in a global numerical model. The oppositely charged IDR and G4 molecules form a complex in the solution that follows a rapid nucleation-growth mechanism leading to a dynamic equilibrium between dilute and condensed phases. We also discover a physical interaction with Replication Protein A (RPA) and demonstrate that the IDR can switch between the two extremes of the structural continuum of complexes. The structural, kinetic, and thermodynamic profile of its interactions revealed a dynamic disordered complex with nucleic acids and a static ordered complex with RPA protein. The two mutually exclusive binding modes suggest a regulatory role for the IDR in RECQ4 function by enabling molecular handoffs. Our study extends the functional repertoire of IDRs and demonstrates a role of polyelectrolyte complexes involved in G4 binding.

• MeSH
• G-kvadruplexy * MeSH
• helikasy RecQ * metabolismus   MeSH
• lidé MeSH
• nukleové kyseliny MeSH
• polyelektrolyty MeSH
• vnitřně neuspořádané proteiny * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• helikasy RecQ * MeSH
• nukleové kyseliny MeSH
• polyelektrolyty MeSH
• RECQL4 protein, human MeSH Prohlížeč
• vnitřně neuspořádané proteiny * MeSH

Defining dynamic protein-protein interactions in the ubiquitin conjugation reaction is a challenging research area. Generating peptide aptamers that target components such as ubiquitin itself, E1, E2, or E3 could provide tools to dissect novel features of the enzymatic cascade. Next-generation deep sequencing platforms were used to identify peptide sequences isolated from phage-peptide libraries screened against Ubiquitin and its ortholog NEDD8. In over three rounds of selection under differing wash criteria, over 13,000 peptides were acquired targeting ubiquitin, while over 10,000 peptides were selected against NEDD8. The overlap in peptides against these two proteins was less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8 toward linear peptide motifs. Two of these ubiquitin-binding peptides were identified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysis highlighted distinct modes of binding of the two different peptide aptamers. These data highlight the utility of using next-generation sequencing of combinatorial phage-peptide libraries to isolate peptide aptamers toward a protein target that can be used as a chemical tool in a complex multi-enzyme reaction.

• Klíčová slova
• aptamers, molecular dynamics, next-generation sequencing, phage-peptide, protein–peptide binding, ubiquitin,
• Publikační typ
• časopisecké články MeSH

Intrinsically disordered proteins are ubiquitous throughout all known proteomes, playing essential roles in all aspects of cellular and extracellular biochemistry. To understand their function, it is necessary to determine their structural and dynamic behavior and to describe the physical chemistry of their interaction trajectories. Nuclear magnetic resonance is perfectly adapted to this task, providing ensemble averaged structural and dynamic parameters that report on each assigned resonance in the molecule, unveiling otherwise inaccessible insight into the reaction kinetics and thermodynamics that are essential for function. In this review, we describe recent applications of NMR-based approaches to understanding the conformational energy landscape, the nature and time scales of local and long-range dynamics and how they depend on the environment, even in the cell. Finally, we illustrate the ability of NMR to uncover the mechanistic basis of functional disordered molecular assemblies that are important for human health.

• MeSH
• konformace proteinů MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• termodynamika MeSH
• vnitřně neuspořádané proteiny * chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• vnitřně neuspořádané proteiny * MeSH

Description of protein dynamics is known to be essential in understanding their function. Studies based on a well established [Formula: see text] NMR relaxation methodology have been applied to a large number of systems. However, the low dispersion of [Formula: see text] chemical shifts very often observed within intrinsically disordered proteins complicates utilization of standard 2D HN correlated spectra because a limited number of amino acids can be characterized. Here we present a suite of triple resonance HNCO-type NMR experiments for measurements of five [Formula: see text] relaxation parameters ([Formula: see text], [Formula: see text], NOE, cross-correlated relaxation rates [Formula: see text] and [Formula: see text]) in doubly [Formula: see text],[Formula: see text]-labeled proteins. We show that the third spectral dimension combined with non-uniform sampling provides relaxation rates for almost all residues of a protein with extremely poor chemical shift dispersion, the C terminal domain of [Formula: see text]-subunit of RNA polymerase from Bacillus subtilis. Comparison with data obtained using a sample labeled by [Formula: see text] only showed that the presence of [Formula: see text] has a negligible effect on [Formula: see text], [Formula: see text], and on the cross-relaxation rate (calculated from NOE and [Formula: see text]), and that these relaxation rates can be used to calculate accurate spectral density values. Partially [Formula: see text]-labeled sample was used to test if the observed increase of [Formula: see text] [Formula: see text] in the presence of [Formula: see text] corresponds to the [Formula: see text] dipole-dipole interactions in the [Formula: see text],[Formula: see text]-labeled sample.

• Klíčová slova
• Intrinsically disordered proteins, Non-uniform sampling, Nuclear magnetic resonance, Relaxation,
• MeSH
• Bacillus subtilis enzymologie   MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• vnitřně neuspořádané proteiny chemie   MeSH
• vodík MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Carbon-13 MeSH Prohlížeč
• DNA řízené RNA-polymerasy MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• Nitrogen-15 MeSH Prohlížeč
• vnitřně neuspořádané proteiny MeSH
• vodík MeSH

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.

• Klíčová slova
• Adiabatic sweep, Cross-correlated cross relaxation, NMR, Protein dynamics,
• MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• protein vázající CREB chemie   MeSH
• ubikvitin chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CREBBP protein, human MeSH Prohlížeč
• protein vázající CREB MeSH
• ubikvitin MeSH

Merozoite surface protein 2 (MSP2) of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design.

• MeSH
• antigeny protozoální chemie   imunologie   MeSH
• konformace proteinů MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• Plasmodium falciparum imunologie   MeSH
• protozoální proteiny chemie   imunologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny protozoální MeSH
• merozoite surface protein 2, Plasmodium MeSH Prohlížeč
• protozoální proteiny MeSH

Spectral density mapping represents the method of choice for investigations of molecular motions of intrinsically disordered proteins (IDPs). However, the current methodology has been developed for well-folded proteins. In order to find conditions for a reliable analysis of relaxation of IDPs, accuracy of the current reduced spectral density mapping protocols applied to IDPs was examined and new spectral density mapping methods employing cross-correlated relaxation rates have been designed. Various sources of possible systematic errors were analyzed theoretically and the presented approaches were tested on a partially disordered protein, delta subunit of bacterial RNA polymerase. Results showed that the proposed protocols provide unbiased description of molecular motions of IDPs and allow to separate slow exchange from fast dynamics.

• MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• sbalování proteinů MeSH
• vnitřně neuspořádané proteiny analýza   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• vnitřně neuspořádané proteiny MeSH

Backbone dynamics of mouse major urinary protein I (MUP-I) was studied by (15)N NMR relaxation. Data were collected at multiple temperatures for a complex of MUP-I with its natural pheromonal ligand, 2- sec -4,5-dihydrothiazole, and for the free protein. The measured relaxation rates were analyzed using the reduced spectral density mapping. Graphical analysis of the spectral density values provided an unbiased qualitative picture of the internal motions. Varying temperature greatly increased the range of analyzed spectral density values and therefore improved reliability of the analysis. Quantitative parameters describing the dynamics on picosecond to nanosecond time scale were obtained using a novel method of simultaneous data fitting at multiple temperatures. Both methods showed that the backbone flexibility on the fast time scale is slightly increased upon pheromone binding, in accordance with the previously reported results. Zero-frequency spectral density values revealed conformational changes on the microsecond to millisecond time scale. Measurements at different temperatures allowed to monitor temperature dependence of the motional parameters.

• MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• myši MeSH
• pohyb těles MeSH
• proteiny chemie   MeSH
• teplota * MeSH
• terciární struktura proteinů MeSH
• thiazoly chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• 2-(sec-butyl)-4,5-dihydrothiazole MeSH Prohlížeč
• major urinary proteins MeSH Prohlížeč
• proteiny MeSH
• thiazoly MeSH