Elastin-like polypeptides (ELPs) are biocompatible polymers exhibiting lower critical solution temperature (LCST) behavior, making them valuable in various applications, including drug delivery and tissue engineering. This study addresses the atomistic-level understanding of ELP self-assembly, focusing on their internal structural dynamics. Conventional proton-detected nuclear magnetic resonance (NMR) spectroscopy faces limitations in studying ELP aggregates due to accelerated proton exchange processes, which cause significant resonance broadening. Herein, we show how to overcome this hurdle by using carbon-13-detected NMR. This method mitigates issues related to amide proton exchange, allowing for a residue-resolved view of the internal configuration of ELP aggregates. With this method, we record residue-resolved 15N relaxation rates, revealing three features. (i) Proline residues within the PGXGV pentapeptide repeats (X being any amino acid except proline) of ELP become motional restricted upon aggregation, indicating their role as interchain contacts. (ii) Pentapeptides with alanine guest residue X display particularly significantly reduced motional freedom upon aggregation. (iii) Even within large ELP aggregates, fast internal dynamics characterize the peptide chains in a way that is reminiscent of condensed liquid phases. The presented study is the first proof of concept that 13C-direct detection is a viable tool to delineate the internal structural dynamics of condensed ELP phases by NMR. It might, thus, help to foster new investigations of their aggregation mechanisms.

• MeSH
• elastin * chemie   MeSH
• izotopy uhlíku chemie   MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• peptidy * chemie   MeSH
• polypeptidy podobné elastinu MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Carbon-13 MeSH Prohlížeč
• elastin * MeSH
• izotopy uhlíku MeSH
• peptidy * MeSH
• polypeptidy podobné elastinu MeSH

In mycobacteria, σA is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σA. The search revealed a number of proteins; prominent among them was MoaB2. The σA-MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors (e.g., closely related σB) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σA was identified to play a role in the σA-MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σA-dependent (but not σB-dependent) transcription and may increase the stability of σA in the cell. We propose that MoaB2, by sequestering σA, has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σA, paving the way for future investigation of this phenomenon.IMPORTANCEMycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σA, the primary sigma factor, and characterize its effects on transcription and σA stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.

• Klíčová slova
• MoaB2, RNA polymerase, mycobacteria, transcription, σA,
• MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• DNA řízené RNA-polymerasy metabolismus   genetika   MeSH
• genetická transkripce MeSH
• krystalografie rentgenová MeSH
• Mycobacterium smegmatis * metabolismus   genetika   MeSH
• regulace genové exprese u bakterií * MeSH
• sigma faktor * metabolismus   genetika   MeSH
• transkripční faktory * metabolismus   genetika   MeSH
• vazba proteinů * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• DNA řízené RNA-polymerasy MeSH
• sigma faktor * MeSH
• transkripční faktory * MeSH

Recently published in vivo observations have highlighted the presence of cruciform structures within the genome, suggesting their potential significance in the rapid recognition of the target sequence for transcription factor binding. In this in vitro study, we investigate the organization and stability of the sense (coding) strand within the Serum Response Element of the c-Fos gene promoter (c-Fos SRE), specifically focusing on segments spanning 12 to 36 nucleotides, centered around the CArG-box. Through a thorough examination of UV absorption patterns with varying temperatures, we identified the emergence of a remarkably stable structure, which we conclusively characterized as a hairpin using complementary 1H NMR experiments. Our research decisively ruled out the formation of homoduplexes, as confirmed by supplementary fluorescence experiments. Utilizing molecular dynamics simulations with atomic distance constraints derived from NMR data, we explored the structural intricacies of the compact hairpin. Notably, the loop consisting of the six-membered A/T sequence demonstrated substantial stabilization through extensive stacking, non-canonical inter-base hydrogen bonding, and hydrophobic clustering of thymine methyl groups. These findings suggest the potential of the c-Fos SRE to adopt a cruciform structure (consisting of two opposing hairpins), potentially providing a topological recognition site for the SRF transcription factor under cellular conditions. Our results should inspire further biochemical and in vivo studies to explore the functional implications of these non-canonical DNA structures.

• Publikační typ
• časopisecké články MeSH

The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the β-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity.

• MeSH
• ABC transportéry metabolismus   chemie   genetika   MeSH
• adenosintrifosfát metabolismus   MeSH
• Escherichia coli * metabolismus   genetika   MeSH
• molekulární chaperony * metabolismus   MeSH
• molekulární modely MeSH
• peptidylprolylisomerasa * metabolismus   genetika   MeSH
• proteinové domény MeSH
• proteiny vnější bakteriální membrány metabolismus   genetika   chemie   MeSH
• proteiny z Escherichia coli * metabolismus   genetika   chemie   MeSH
• rezonanční přenos fluorescenční energie MeSH
• sbalování proteinů MeSH
• transportní proteiny * metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ABC transportéry MeSH
• adenosintrifosfát MeSH
• molekulární chaperony * MeSH
• peptidylprolylisomerasa * MeSH
• proteiny vnější bakteriální membrány MeSH
• proteiny z Escherichia coli * MeSH
• SurA protein, E coli MeSH Prohlížeč
• transportní proteiny * MeSH

The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA.

• MeSH
• DNA * chemie   genetika   MeSH
• G-kvadruplexy * MeSH
• inzulin * chemie   genetika   MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• nukleotidové motivy MeSH
• polymorfismus genetický MeSH
• promotorové oblasti (genetika) * MeSH
• reportérové geny MeSH
• sekvence nukleotidů MeSH
• tandemové repetitivní sekvence genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA * MeSH
• inzulin * MeSH

Micro-Exon Genes are a widespread class of genes known for their high variability, widespread in the genome of parasitic trematodes such as Schistosoma mansoni. In this study, we present a strategy that allowed us to solve the structures of three alternatively spliced isoforms from the Schistoma mansoni MEG 2.1 family for the first time. All isoforms are hydrophobic, intrinsically disordered, and recalcitrant to be expressed in high yield in heterologous hosts. We resorted to the chemical synthesis of shorter pieces, before reconstructing the entire sequence. Here, we show that isoform 1 partially folds in a-helix in the presence of trifluoroethanol while isoform 2 features two rigid elbows, that maintain the peptide as disordered, preventing any structuring. Finally, isoform 3 is dominated by the signal peptide, which folds into a-helix. We demonstrated that combining biophysical techniques, like circular dichroism and nuclear magnetic resonance at natural abundance, with in silico molecular dynamics simulation for isoform 1 only, was the key to solve the structure of MEG 2.1. Our results provide a crucial piece to the puzzle of this elusive and highly variable class of proteins.

• MeSH
• exony genetika   MeSH
• peptidy * metabolismus   MeSH
• protein - isoformy genetika   MeSH
• Schistosoma mansoni * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• peptidy * MeSH
• protein - isoformy 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 (IDPs) or intrinsically disordered regions (IDRs) is a class of biologically important proteins exhibiting specific biophysical characteristics. They lack a hydrophobic core, and their conformational behavior is strongly influenced by electrostatic interactions. IDPs and IDRs are highly dynamic, and a characterization of the motions of IDPs and IDRs is essential for their physically correct description. NMR together with molecular dynamics simulations are the methods best suited to such a task because they provide information about dynamics of proteins with atomistic resolution. Here, we present a study of motions of a disordered C-terminal domain of the delta subunit of RNA polymerase from Bacillus subtilis. Positively and negatively charged residues in the studied domain form transient electrostatic contacts critical for the biological function. Our study is focused on investigation of ps-ns dynamics of backbone of the delta subunit based on analysis of amide 15N NMR relaxation data and molecular dynamics simulations. In order to extend an informational content of NMR data to lower frequencies, which are more sensitive to slower motions, we combined standard (high-field) NMR relaxation experiments with high-resolution relaxometry. Altogether, we collected data reporting the relaxation at 12 different magnetic fields, resulting in an unprecedented data set. Our results document that the analysis of such data provides a consistent description of dynamics and confirms the validity of so far used protocols of the analysis of dynamics of IDPs also for a partially folded protein. In addition, the potential to access detailed description of motions at the timescale of tens of ns with the help of relaxometry data is discussed. Interestingly, in our case, it appears to be mostly relevant for a region involved in the formation of temporary contacts within the disordered region, which was previously proven to be biologically important.

• MeSH
• amidy MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• konformace proteinů MeSH
• magnetická rezonanční spektroskopie MeSH
• simulace molekulární dynamiky MeSH
• vnitřně neuspořádané proteiny * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amidy MeSH
• DNA řízené RNA-polymerasy MeSH
• vnitřně neuspořádané proteiny * MeSH

Microtubule-associated protein 2 (MAP2) is an important neuronal target of extracellular signal-regulated kinase 2 (ERK2) involved in Raf signaling pathways, but mechanistic details of MAP2 phosphorylation are unclear. Here, we used NMR spectroscopy to quantitatively describe the kinetics of phosphorylation of individual serines and threonines in the embryonic MAP2 variant MAP2c. We carried out real-time monitoring of phosphorylation to discover major phosphorylation sites that were not identified in previous studies relying on specific antibodies. Our comparison with the phosphorylation of MAP2c by a model cyclin-dependent kinase CDK2 and with phosphorylation of the MAP2c homolog Tau revealed differences in phosphorylation profiles that explain specificity of regulation of biological functions of MAP2c and Tau. To probe the molecular basis of the regulatory effect of ERK2, we investigated the interactions of phosphorylated and unphosphorylated MAP2c by NMR with single-residue resolution. As ERK2 phosphorylates mostly outside the regions binding microtubules, we studied the binding of proteins other than tubulin, namely regulatory subunit RIIα of cAMP-dependent PKA, adapter protein Grb2, Src homology domain 3 of tyrosine kinases Fyn and Abl, and ERK2 itself. We found ERK2 phosphorylation interfered mostly with binding to proline-rich regions of MAP2c. Furthermore, our NMR experiments in SH-SY5Y neuroblastoma cell lysates showed that the kinetics of dephosphorylation are compatible with in-cell NMR studies and that residues targeted by ERK2 and PKA are efficiently phosphorylated in the cell lysates. Taken together, our results provide a deeper characterization of MAP2c phosphorylation and its effects on interactions with other proteins.

• Klíčová slova
• NMR, PKA, Src homology 3 domain, cyclin-dependent kinase, extracellular signal–regulated kinase, growth factor receptor-bound protein 2 (GRB2), microtubule-associated protein,
• MeSH
• extracelulárním signálem regulované MAP kinasy * metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• nádorové buněčné linie MeSH
• proteinkinasy řízené prolinem * metabolismus   MeSH
• proteiny asociované s mikrotubuly * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDK2 protein, human MeSH Prohlížeč
• extracelulárním signálem regulované MAP kinasy * MeSH
• MAP2 protein, human MeSH Prohlížeč
• MAPK1 protein, human MeSH Prohlížeč
• proteinkinasy řízené prolinem * MeSH
• proteiny asociované s mikrotubuly * MeSH

Nuclear magnetic resonance (NMR) spectroscopy is a key method for determining the structural dynamics of proteins in their native solution state. However, the low sensitivity of NMR typically necessitates nonphysiologically high sample concentrations, which often limit the relevance of the recorded data. We show how to use hyperpolarized water by dissolution dynamic nuclear polarization (DDNP) to acquire protein spectra at concentrations of 1 μM within seconds and with a high signal-to-noise ratio. The importance of approaching physiological concentrations is demonstrated for the vital MYC-associated factor X, which we show to switch conformations when diluted. While in vitro conditions lead to a population of the well-documented dimer, concentrations lowered by more than two orders of magnitude entail dimer dissociation and formation of a globularly folded monomer. We identified this structure by integrating DDNP with computational techniques to overcome the often-encountered constraint of DDNP of limited structural information provided by the typically detected one-dimensional spectra.

• Publikační typ
• časopisecké články MeSH