RESUMEN
Prolyl-hydroxylation is an oxygen-dependent posttranslational modification (PTM) that is known to regulate fibril formation of collagenous proteins and modulate cellular expression of hypoxia-inducible factor (HIF) α subunits. However, our understanding of this important but relatively rare PTM has remained incomplete due to the lack of biophysical methodologies that can directly measure multiple prolyl-hydroxylation events within intrinsically disordered proteins. Here, we describe a real-time 13C-direct detection NMR-based assay for studying the hydroxylation of two evolutionarily conserved prolines (P402 and P564) simultaneously in the intrinsically disordered oxygen-dependent degradation domain of hypoxic-inducible factor 1α by exploiting the "proton-less" nature of prolines. We show unambiguously that P564 is rapidly hydroxylated in a time-resolved manner while P402 hydroxylation lags significantly behind that of P564. The differential hydroxylation rate was negligibly influenced by the binding affinity to prolyl-hydroxylase enzyme, but rather by the surrounding amino acid composition, particularly the conserved tyrosine residue at the +1 position to P564. These findings support the unanticipated notion that the evolutionarily conserved P402 seemingly has a minimal impact in normal oxygen-sensing pathway.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia , Proteínas Intrínsecamente Desordenadas , Prolina , Hidroxilación , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Prolina/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Humanos , Procesamiento Proteico-Postraduccional , Espectroscopía de Resonancia Magnética/métodosRESUMEN
Intrinsically disordered proteins (IDPs) are significantly enriched in proline residues, which can populate specific local secondary structural elements called PPII helices, characterized by small packing densities. Proline is often thought to promote disorder, but it can participate in specific π·CH interactions with aromatic side chains resulting in reduced conformational flexibilities of the polypeptide. Differential local motional dynamics are relevant for the stabilization of preformed structural elements and can serve as nucleation sites for the establishment of long-range interactions. NMR experiments to probe the dynamics of proline ring systems would thus be highly desirable. Here we present a pulse scheme based on 13C detection to quantify dipole-dipole cross-correlated relaxation (CCR) rates at methylene CH2 groups in proline residues. Applying 13C-CON detection strategy provides exquisite spectral resolution allowing applications also to high molecular weight IDPs even in conditions approaching the physiological ones. The pulse scheme is illustrated with an application to the 220 amino acids long protein Osteopontin, an extracellular cytokine involved in inflammation and cancer progression, and a construct in which three proline-aromatic sequence patches have been mutated.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Humanos , Imagen por Resonancia Magnética , Frecuencia Cardíaca , Inflamación , Conformación MolecularRESUMEN
As a direct consequence of technological advancements, the interest in direct detection of low-gamma/low-sensitivity heteronuclei for NMR experiments has been revived. Until recently, experimental development of 13C/15N detected experiments has been focused on protein NMR. In the present report, we extend the use of 13C-detected experiments to structural studies of glycans in natural abundance. The narrow 1H and wider 13C signal dispersion make glycans ideal candidates for heteronuclear detection. We show that 13C-detected HSQC offers a ten-fold increase in 13C dimension resolution compared to the analogous 1H-detected HSQC, when the experiments are acquired for the same amount of time. The enhanced resolution comes at the expense of 2 to 3-fold loss in SNR; however, the observed signal loss is a fraction of the theoretical 8-fold difference expected between experiments. Further, we show that by combining a 1H constant time element (CT), SMILE data reconstruction and 13C-direct detection, complete resonance assignments of highly degenerate glycan signals are possible. Finally, we demonstrate the potential of our strategy to aid in the assignment of complex glycans, by using a novel 13C-detected version of the CT-HSQC-TOCSY experiment performed on sialyl Lewis X pentasaccharide model system.
Asunto(s)
Isótopos de Carbono , Resonancia Magnética Nuclear Biomolecular/métodos , Polisacáridos/química , Algoritmos , Celobiosa , Hidrógeno/química , Oligosacáridos/química , Antígeno Sialil Lewis X/químicaRESUMEN
Up to now, NMR spectroscopic investigations of RNA have utilized imino proton resonances as reporters for base pairing and RNA structure. The nucleobase amino groups are often neglected, since most of their resonances are broadened beyond detection due to rotational motion around the C-NH2 bond. Here, we present 13 C-detected NMR experiments for the characterization of all RNA amino groups irrespective of their motional behavior. We have developed a C(N)H-HDQC experiment that enables the observation of a complete set of sharp amino resonances through the detection of proton-NH2 double quantum coherences. Further, we present an "amino"-NOESY experiment to detect NOEs to amino protons, which are undetectable by any other conventional NOESY experiment. Together, these experiments allow the exploration of additional chemical shift information and inter-residual proton distances important for high-resolution RNA secondary and tertiary structure determination.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , ARN/química , Aminas/química , Isótopos de Carbono/química , Conformación de Ácido NucleicoRESUMEN
Resonance assignment is a prerequisite for almost any NMR-based study of proteins. It can be very challenging in some cases, however, due to the nature of the protein under investigation. This is the case with intrinsically disordered proteins, for example, whose NMR spectra suffer from low chemical shifts dispersion and generally low resolution. For these systems, sequence specific assignment is highly time-consuming, so the prospect of using automatic strategies for their assignment is very attractive. In this article we present a new version of the automatic assignment program TSAR dedicated to intrinsically disordered proteins. In particular, we demonstrate how the automatic procedure can be improved by incorporating methods for amino acid recognition and information on chemical shifts in selected amino acids. The approach was tested in silico on 16 disordered proteins and experimentally on α-synuclein, with remarkably good results.
Asunto(s)
Aminoácidos/química , Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
In RNA secondary structure determination, it is essential to determine whether a nucleotide is base-paired and not. Base-pairing of nucleotides is mediated by hydrogen bonds. The NMR characterization of hydrogen bonds relies on experiments correlating the NMR resonances of exchangeable protons and can be best performed for structured parts of the RNA, where labile hydrogen atoms are protected from solvent exchange. Functionally important regions in RNA, however, frequently reveal increased dynamic disorder which often leads to NMR signals of exchangeable protons that are broadened beyond (1)H detection. Here, we develop (13)C direct detected experiments to observe all nucleotides in RNA irrespective of whether they are involved in hydrogen bonds or not. Exploiting the self-decoupling of scalar couplings due to the exchange process, the hydrogen bonding behavior of the hydrogen bond donor of each individual nucleotide can be determined. Furthermore, the adaption of HNN-COSY experiments for (13)C direct detection allows correlations of donor-acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed (13)C direct detected experiments therefore provide information about molecular sites not amenable by conventional proton-detected methods. Such information makes the RNA secondary structure determination by NMR more accurate and helps to validate secondary structure predictions based on bioinformatics.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Enlace de Hidrógeno , Conformación de Ácido Nucleico , ARN/química , Emparejamiento Base , Hidrógeno/química , Resonancia Magnética Nuclear BiomolecularRESUMEN
Intrinsically disordered proteins (IDPs) are functional proteins containing large fragments characterized by high local mobility. Bioinformatic studies have suggested that a significant fraction (more than 30%) of eukaryotic proteins has disordered regions of more than 50 amino acids in length. Hence, NMR methods for the characterization of local compactness and solvent accessibility in such highly disordered proteins are of high importance. Among the available approaches, the HET-SOFAST/BEST experiments (Schanda et al., 2006, Rennella et al., 2014) provide semi-quantitative information by monitoring longitudinal (1)H relaxation of amide protons under different initial conditions. However, when approaching physiological sample conditions, the potential of these amide (1)H detected experiments is reduced due to rapid amide proton solvent exchange. (13)C direct detection methods therefore provide a valuable alternative thanks to a higher chemical shift dispersion and their intrinsic insensitivity toward solvent exchange. Here we present two sets of (13)C-detected experiments, which indirectly measure (1)H(N) and (1)H(α) inversion recovery profiles. The experiments consist of an initial spin inversion-recovery block optimized for selective manipulation of different types of proton spins followed by a CON read-out scheme. The proposed experiments were tested on human α-synuclein and ubiquitin, two representative examples of unfolded and folded proteins.
Asunto(s)
Hidrógeno/química , Proteínas Intrínsecamente Desordenadas/química , Espectroscopía de Resonancia Magnética/métodos , Amidas/química , Radioisótopos de Carbono , Humanos , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Protones , Solventes , Ubiquitina/química , alfa-Sinucleína/químicaRESUMEN
Intrinsically disordered proteins (IDPs) are characterized by highly flexible solvent exposed backbones and can sample many different conformations. These properties confer them functional advantages, complementary to those of folded proteins, which need to be characterized to expand our view of how protein structural and dynamic features affect function beyond the static picture of a single well defined 3D structure that has influenced so much our way of thinking. NMR spectroscopy provides a unique tool for the atomic resolution characterization of highly flexible macromolecules in general and of IDPs in particular. The peculiar properties of IDPs however have profound effects on spectroscopic parameters. It is thus worth thinking about these aspects to make the best use of the great potential of NMR spectroscopy to contribute to this fascinating field of research. In particular, after many years of dealing with exclusively heteronuclear NMR experiments based on (13)C direct detection, we would like here to address their relevance when studying IDPs.