ABSTRACT
RNA aptamers-artificially created RNAs with high affinity and selectivity for their target ligand generated from random sequence pools-are versatile tools in the fields of biotechnology and medicine. On a more fundamental level, they also further our general understanding of RNA-ligand interactions e. g. in regard to the relationship between structural complexity and ligand affinity and specificity, RNA structure and RNA folding. Detailed structural knowledge on a wide range of aptamer-ligand complexes is required to further our understanding of RNA-ligand interactions. Here, we present the atomic resolution structure of an RNA-aptamer binding to the fluorescent xanthene dye tetramethylrhodamine. The high resolution structure, solved by NMR-spectroscopy in solution, reveals binding features both common and different from the binding mode of other aptamers with affinity for ligands carrying planar aromatic ring systems such as the malachite green aptamer which binds to the tetramethylrhodamine related dye malachite green or the flavin mononucleotide aptamer.
Subject(s)
Aptamers, Nucleotide/chemistry , Nucleic Acid Conformation , RNA/chemistry , Rhodamines/chemistry , Ligands , Magnetic Resonance Spectroscopy , RNA FoldingABSTRACT
The saliva of blood-sucking leeches contains a plethora of anticoagulant substances. One of these compounds derived from Haementeria ghilianii, the 66mer three-disulfide-bonded peptide tridegin, specifically inhibits the blood coagulation factor FXIIIa. Tridegin represents a potential tool for antithrombotic and thrombolytic therapy. We recently synthesized two-disulfide-bonded tridegin variants, which retained their inhibitory potential. For further lead optimization, however, structure information is required. We thus analyzed the structure of a two-disulfide-bonded tridegin isomer by solution 2D NMR spectroscopy in a combinatory approach with subsequent MD simulations. The isomer was studied using two fragments, i.e., the disulfide-bonded N-terminal (Lys1-Cys37) and the flexible C-terminal part (Arg38-Glu66), which allowed for a simplified, label-free NMR-structure elucidation of the 66mer peptide. The structural information was subsequently used in molecular modeling and docking studies to provide insights into the structure-activity relationships. The present study will prospectively support the development of anticoagulant-therapy-relevant compounds targeting FXIIIa.
Subject(s)
Factor XIIIa/antagonists & inhibitors , Magnetic Resonance Spectroscopy/methods , Salivary Proteins and Peptides/pharmacology , Amino Acid Sequence , Animals , Disulfides/chemistry , Factor XIIIa/metabolism , Fibrinolytic Agents/pharmacology , Humans , Isomerism , Leeches/metabolism , Magnetic Resonance Imaging/methods , Models, Molecular , Molecular Dynamics Simulation , Salivary Proteins and Peptides/chemistry , Salivary Proteins and Peptides/metabolism , Structure-Activity RelationshipABSTRACT
Peptides and proteins carrying high numbers of cysteines can adopt various 3D structures depending on their disulfide connectivities. The unambiguous verification of such conformational isomers with more than two disulfide bonds is extremely challenging, and experimental strategies for their unequivocal structural analysis are largely lacking. We synthesized all 15 possible isomers of the 22mer conopeptide µ-PIIIA and applied 2D NMR spectroscopy and MS/MS for the elucidation of its structure. This study provides intriguing insights in how the disulfide connectivity alters the global fold of a toxin. We also show that analysis procedures involving comprehensive combinations of conventional methods are required for the unambiguous assignment of disulfides in cysteine-rich peptides and proteins and that standard compounds are crucially needed for the structural analysis of such complex molecules.
ABSTRACT
The cyclic undecapeptide cyclosporin A (CsA) is a widely used immunosuppressive agent. Its immunosuppressive properties arise from strong binding to cyclophilins (Cyp) followed by inhibition of the protein calcineurin (CaN) by the binary CsA/Cyp complex and subsequent negative regulation of T-cell activation. In the present study we show a novel way to modify the CsA ring by selective N-hydroxyalkylation of the residues Val5 and d-Ala8. Moreover, the influence of these structural CsA modifications on the ability of the CsA analogs to bind Cyp, to inhibit CaN and to penetrate membranes of living cells was investigated. Our results show that the Val5 N-substitution significantly improved compound cell-permeability and markedly diminished CaN inhibition by the binary CsA analog/CypA complex but to a lesser extent Cyp inhibition. In contrast, the N-alkylation of d-Ala8 gave a product with significantly reduced affinity for Cyp but its immunosuppressive effects remained similar to CsA. Possible explanations of the observed experimental data are provided by computational studies.
ABSTRACT
BACKGROUND: The occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements. METHODS: To understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV-vis, resonance Raman, cw-EPR and 2D NMR spectroscopy. RESULTS: We observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69±4.37 µM. CONCLUSIONS: It was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme. GENERAL SIGNIFICANCE: Understanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.
Subject(s)
Chloramphenicol O-Acetyltransferase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Heme/pharmacology , Amino Acid Motifs , Chloramphenicol O-Acetyltransferase/chemistry , Electron Spin Resonance Spectroscopy , Histidine , Magnetic Resonance Spectroscopy , Spectrum Analysis, Raman , TyrosineABSTRACT
NMR spectroscopy was used to study systematically the impact of imidazolium-based ionic liquid (IL) solutions on a TAT-derived model peptide containing Xaa-Pro peptide bonds. The selected IL anions cover a wide range of the Hofmeister series of ions. Based on highly resolved one- and two-dimensional NMR spectra individual 1H and 13C peptide chemical shift differences were analysed and a classification of IL anions according to the Hofmeister series was derived. The observed chemical shift changes indicate significant interactions between the peptide and the ILs. In addition, we examined the impact of different ILs towards the cis/trans equilibrium state of the Xaa-Pro peptide bonds. In this context, the IL cations appear to be of exceptional importance for inducing an alteration of the native cis/trans equilibrium state of Xaa-Pro bonds in favour of the trans-isomers.
ABSTRACT
The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical 'wings' of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Minichromosome Maintenance Proteins/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Archaeal Proteins/genetics , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Helicases/metabolism , Hydrolysis , Magnetic Resonance Spectroscopy , Methanobacteriaceae/genetics , Methanobacteriaceae/metabolism , Minichromosome Maintenance Proteins/genetics , Minichromosome Maintenance Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Mutation , Phylogeny , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Sulfolobus solfataricus/genetics , Sulfolobus solfataricus/metabolismABSTRACT
RNA tertiary structure motifs are stabilized by a wide variety of hydrogen-bonding interactions. Protonated A and C nucleotides are normally not considered to be suitable building blocks for such motifs since their pKa values are far from physiological pH. Here, we report the NMR solution structure of an inâ vitro selected GTP-binding RNA aptamer bound to GTP with an intricate tertiary structure. It contains a novel kind of base quartet stabilized by a protonated Aâ residue. Owing to its unique structural environment in the base quartet, the pKa value for the protonation of this Aâ residue in the complex is shifted by more than 5 pH units compared to the pKa for Aâ nucleotides in single-stranded RNA. This is the largest pKa shift for an Aâ residue in structured nucleic acids reported so far, and similar in size to the largest pKa shifts observed for amino acid side chains in proteins. Both RNA pre-folding and ligand binding contribute to the pKa shift.
Subject(s)
Adenine Nucleotides/chemistry , Aptamers, Nucleotide/chemistry , Guanosine Triphosphate/chemistry , Protons , Binding Sites , Hydrogen-Ion Concentration , Models, Molecular , Nucleic Acid ConformationABSTRACT
The RF pulse scheme RN[N-CA HEHAHA]NH, which provides a convenient approach to the acquisition of different multidimensional chemical shift correlation NMR spectra leading to backbone resonance assignments, including those of the proline residues of intrinsically disordered proteins (IDPs), is experimentally demonstrated. Depending on the type of correlation data required, the method involves the generation of in-phase ((15) N)(x) magnetisation via different magnetisation transfer pathways such as HâNâCOâN, HAâCAâCOâN, HâNâCAâN and HâCAâN, the subsequent application of (15) N-(13) C(α) heteronuclear Hartmann-Hahn mixing over a period of ≈100â ms, chemical-shift labelling of relevant nuclei before and after the heteronuclear mixing step and amide proton detection in the acquisition dimension. It makes use of the favourable relaxation properties of IDPs and the presence of (1) JCαN and (2) JCαN couplings to achieve efficient correlation of the backbone resonances of each amino acid residue "i" with the backbone amide resonances of residues "i-1" and "i+1". It can be implemented in a straightforward way through simple modifications of the RF pulse schemes commonly employed in protein NMR studies. The efficacy of the approach is demonstrated using a uniformly ((15) N,(13) C) labelled sample of α-synuclein. The different possibilities for obtaining the amino-acid-type information, simultaneously with the connectivity data between the backbone resonances of sequentially neighbouring residues, have also been outlined.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular , alpha-Synuclein/chemistryABSTRACT
Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.
Subject(s)
DNA/metabolism , RecQ Helicases/chemistry , RecQ Helicases/metabolism , Binding Sites , DNA/chemistry , DNA-Binding Proteins/chemistry , G-Quadruplexes , Humans , Intrinsically Disordered Proteins/chemistry , Protein Binding , Protein Structure, TertiaryABSTRACT
Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of â¼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.
Subject(s)
Heme , Kv1.4 Potassium Channel , Animals , Crystallography, X-Ray , Heme/chemistry , Heme/genetics , Heme/metabolism , Ion Transport/physiology , Kv1.4 Potassium Channel/chemistry , Kv1.4 Potassium Channel/genetics , Kv1.4 Potassium Channel/metabolism , Protein Binding , Protein Structure, Tertiary , Rats , Xenopus laevisABSTRACT
To ensure appropriate metabolic regulation, riboswitches must discriminate efficiently between their target ligands and chemically similar molecules that are also present in the cell. A remarkable example of efficient ligand discrimination is a synthetic neomycin-sensing riboswitch. Paromomycin, which differs from neomycin only by the substitution of a single amino group with a hydroxy group, also binds but does not flip the riboswitch. Interestingly, the solution structures of the two riboswitch-ligand complexes are virtually identical. In this work, we demonstrate that the local loss of key intermolecular interactions at the substitution site is translated through a defined network of intramolecular interactions into global changes in RNA conformational dynamics. The remarkable specificity of this riboswitch is thus based on structural dynamics rather than static structural differences. In this respect, the neomycin riboswitch is a model for many of its natural counterparts.
Subject(s)
Hydroxyl Radical/chemistry , Neomycin/analysis , Riboswitch , Ligands , Models, MolecularABSTRACT
A simple triple resonance NMR experiment that leads to the correlation of the backbone amide resonances of each amino acid residue 'i' with that of residues 'i-1' and 'i+1' in ((13)C, (15)N) labelled intrinsically disordered proteins (IDPs) is presented. The experimental scheme, {HN-NCA heteronuclear TOCSY-NH}, exploits the favourable relaxation properties of IDPs and the presence of (1) J CαN and (2) J CαN couplings to transfer the (15)N x magnetisation from amino acid residue 'i' to adjacent residues via the application of a band-selective (15)N-(13)C(α) heteronuclear cross-polarisation sequence of ~100 ms duration. Employing non-uniform sampling in the indirect dimensions, the efficacy of the approach has been demonstrated by the acquisition of 3D HNN chemical shift correlation spectra of α-synuclein. The experimental performance of the RF pulse sequence has been compared with that of the conventional INEPT-based HN(CA)NH pulse scheme. As the availability of data from both the HCCNH and HNN experiments will make it possible to use the information extracted from one experiment to simplify the analysis of the data of the other and lead to a robust approach for unambiguous backbone and side-chain resonance assignments, a time-saving strategy for the simultaneous collection of HCCNH and HNN data is also described.
Subject(s)
Intrinsically Disordered Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular/methodsABSTRACT
The importance of heme as a transient regulatory molecule has become a major focus in biochemical research. However, detailed information about the molecular basis of transient heme-protein interactions is still missing. We report an in-depth structural analysis of Fe(III) heme-peptide complexes by a combination of UV/Vis, resonance Raman, and 2D-NMR spectroscopic methods. The experiments reveal insights both into the coordination to the central iron ion and into the spatial arrangement of the amino acid sequences interacting with protoporphyrin IX. Cysteine-based peptides display different heme-binding behavior as a result of the existence of ordered, partially ordered, and disordered conformations in the heme-unbound state. Thus, the heme-binding mode is clearly the consequence of the nature and flexibility of the residues surrounding the iron ion coordinating cysteine. Our analysis reveals scenarios for transient binding of heme to heme-regulatory motifs in proteins and demonstrates that a thorough structural analysis is required to unravel how heme alters the structure and function of a particular protein.
Subject(s)
Cysteine/metabolism , Ferric Compounds/metabolism , Hemeproteins/chemistry , Hemeproteins/metabolism , Protoporphyrins/metabolism , Amino Acid Motifs , Binding Sites , Cysteine/chemistry , Ferric Compounds/chemistry , Protein Structure, Tertiary , Protoporphyrins/chemistryABSTRACT
An efficient approach to NMR assignments in intrinsically disordered proteins is presented, making use of the good dispersion of cross peaks observed in [(15) N,(13) C']- and [(13) C',(1) H(N) ]-correlation spectra. The method involves the simultaneous collection of {3D (H)NCO(CAN)H and 3D (HACA)CON(CA)HA} spectra for backbone assignments via sequential H(N) and H(α) correlations and {3D (H)NCO(CACS)HS and 3D (HS)CS(CA)CO(N)H} spectra for side-chain (1) H and (13) C assignments, employing sequential (1) H data acquisitions with direct detection of both the amide and aliphatic protons. The efficacy of the approach for obtaining resonance assignments with complete backbone and side-chain chemical shifts is demonstrated experimentally for the 61-residue [(13) C,(15) N]-labelled peptide of a voltage-gated potassium channel protein of the Kv1.4 channel subunit. The general applicability of the approach for the characterisation of moderately sized globular proteins is also demonstrated.
Subject(s)
Intrinsically Disordered Proteins/chemistry , Magnetic Resonance Spectroscopy/methods , Magnetic Resonance Spectroscopy/standards , Reference StandardsABSTRACT
The RecQL4 helicase is involved in the maintenance of genome integrity and DNA replication. Mutations in the human RecQL4 gene cause the Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. Mouse models and experiments in human and Xenopus have proven the N-terminal part of RecQL4 to be vital for cell growth. We have identified the first 54 amino acids of RecQL4 (RecQL4_N54) as the minimum interaction region with human TopBP1. The solution structure of RecQL4_N54 was determined by heteronuclear liquid-state nuclear magnetic resonance (NMR) spectroscopy (PDB 2KMU; backbone root-mean-square deviation 0.73 Å). Despite low-sequence homology, the well-defined structure carries an overall helical fold similar to homeodomain DNA-binding proteins but lacks their archetypical, minor groove-binding N-terminal extension. Sequence comparison indicates that this N-terminal homeodomain-like fold is a common hallmark of metazoan RecQL4 and yeast Sld2 DNA replication initiation factors. RecQL4_N54 binds DNA without noticeable sequence specificity yet with apparent preference for branched over double-stranded (ds) or single-stranded (ss) DNA. NMR chemical shift perturbation observed upon titration with Y-shaped, ssDNA and dsDNA shows a major contribution of helix α3 to DNA binding, and additional arginine side chain interactions for the ss and Y-shaped DNA.
Subject(s)
DNA/metabolism , Homeodomain Proteins/chemistry , RecQ Helicases/chemistry , Amino Acid Sequence , Carrier Proteins/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/metabolism , Protein Interaction Domains and Motifs , RecQ Helicases/metabolism , Sequence AlignmentABSTRACT
While the elucidation of regulatory mechanisms of folded proteins is facilitated due to their amenability to high-resolution structural characterization, investigation of these mechanisms in disordered proteins is more challenging due to their structural heterogeneity, which can be captured by a variety of biophysical approaches. Here, we used the transcriptional master corepressor CtBP, which binds the putative metastasis suppressor RAI2 through repetitive SLiMs, as a model system. Using cryo-electron microscopy embedded in an integrative structural biology approach, we show that RAI2 unexpectedly induces CtBP polymerization through filaments of stacked tetrameric CtBP layers. These filaments lead to RAI2-mediated CtBP nuclear foci and relieve its corepressor function in RAI2-expressing cancer cells. The impact of RAI2-mediated CtBP loss-of-function is illustrated by the analysis of a diverse cohort of prostate cancer patients, which reveals a substantial decrease in RAI2 in advanced treatment-resistant cancer subtypes. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions.
Subject(s)
Alcohol Oxidoreductases , Polymerization , Prostatic Neoplasms , Humans , Male , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Alcohol Oxidoreductases/metabolism , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/chemistry , Cryoelectron Microscopy , Cell Line, Tumor , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/chemistry , Protein Binding , HEK293 Cells , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/chemistry , Amino Acid Motifs , Co-Repressor Proteins/metabolism , Co-Repressor Proteins/geneticsABSTRACT
Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Methanobacteriaceae/enzymology , Methionine Sulfoxide Reductases/chemistry , Methionine Sulfoxide Reductases/metabolism , Amino Acid Sequence , Catalytic Domain , Methanobacteriaceae/chemistry , Methanobacteriaceae/metabolism , Methionine/metabolism , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Oxidation-Reduction , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Intrinsically disordered proteins (IDPs) or protein regions represent functionally important biomolecules without unique structure. Their inherent flexibility prevents high-resolution structure determination by X-ray or cryo-EM methods. In contrast, NMR spectroscopy provides an extensive and still growing set of experimental approaches to obtain detailed information on structure and dynamics of IDPs. Here, it is experimentally demonstrated that 15N-13Cα band-selective heteronuclear cross-polarisation that has been successfully employed recently to achieve the efficient transfer of 15Nx magnetisation from amino acid residue 'i' to 'i + 1' and 'i - 1' residues in uniformly (15N,13C)-labelled intrinsically disordered proteins can also be applied to transfer, without significant relaxation losses, 13Cαx magnetisation from an amino acid residue to its neighbouring residues. The possibility to obtain in one-shot correlation spectra arising from the simultaneous transfer of 15Nx and 13Cαx magnetisations from an amino acid residue to neighbouring residues is also demonstrated.
Subject(s)
Intrinsically Disordered Proteins , Amino Acids , Intrinsically Disordered Proteins/chemistry , Magnetic Resonance Imaging , Magnetic Resonance Spectroscopy/methods , Nuclear Magnetic Resonance, Biomolecular/methods , Protein ConformationABSTRACT
The dysbindin domain-containing protein 1 (DBNDD1) is a conserved protein among higher eukaryotes whose structure and function are poorly investigated so far. Here, we present the backbone and side chain nuclear magnetic resonance assignments for the human DBNDD1 protein. Our chemical-shift based secondary structure analysis reveals the human DBNDD1 as an intrinsically disordered protein.