PCA-based denoising usually implies either discarding a number of high-index principal components (PCs) of a data matrix or their attenuation according to a regularization model. This work introduces an alternative, model-free, approach to high-index PC attenuation that seeks to average values of PC vectors as if they were expected from noise perturbation of data. According to the perturbation theory, the average PCs are attenuated versions of the clean PCs of noiseless data - the higher the noise-related content in a PC vector, the lower is its average's norm. This enables a regularization of the PC expansion of data where the PC terms are self-adapted to their noise content. To approximate the average PC vectors, the data matrix is randomly sampled several times to obtain numerous pseudo-random PC sets. The PCs of same ranks are then used to reconstruct the full-data PCs of that rank. A numerical algorithm of the reconstruction and its implementation in Python are provided. The proposed automatic adaptation to data offers a convenient solution for those who face with a problem of scaling or discarding PCs in PCA-based denoising. Questions of optimal sampling schedule and sampling amount remain issues that future work must address.

• Klíčová slova
• Non-uniform sampling, Principal component analysis, Signal denoising,
• Publikační typ
• časopisecké články 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

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarization-selective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

• Klíčová slova
• 3D NMR, BEST-HNCO, Direct 13C detection, Dissolution-dynamic nuclear polarization (D-DNP), Hyperpolarization, Non-uniform sampling,
• MeSH
• lidé MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• osteopontin chemie   MeSH
• ubikvitin chemie   MeSH
• vnitřně neuspořádané proteiny chemie   MeSH
• voda chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• osteopontin MeSH
• SPP1 protein, human MeSH Prohlížeč
• ubikvitin MeSH
• vnitřně neuspořádané proteiny MeSH
• voda MeSH

Proteins, which, in their native conditions, sample a multitude of distinct conformational states characterized by high spatiotemporal heterogeneity, most often termed as intrinsically disordered proteins (IDPs), have become a target of broad interest over the past 15years. With the growing evidence of their important roles in fundamental cellular processes, there is an urgent need to characterize the conformational behavior of IDPs at the highest possible level. The unique feature of NMR spectroscopy in the context of IDPs is its ability to supply details of their structural and temporal alterations at atomic-level resolution. Here, we briefly review recently proposed NMR-based strategies to characterize transient states populated by IDPs and summarize the latest achievements and future prospects in methodological development. Because low chemical shift dispersion represents the major obstacle encountered when studying IDPs by nuclear magnetic resonance, particular attention is paid to techniques allowing one to approach the physical limits of attainable resolution.

• Klíčová slova
• IDPRs, IDPs, Multi-dimensional NMR, NUS, Non-uniform sampling, PRE, RDC, RNAP δ-subunit,
• MeSH
• algoritmy MeSH
• konformace proteinů MeSH
• lidé MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• proteiny chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• proteiny MeSH

Human tyrosine hydroxylase 1 (hTH1) activity is regulated by phosphorylation of its regulatory domain (RD-hTH1) and by an interaction with the 14-3-3 protein. The RD-hTH1 is composed of a structured region (66-169) preceded by an intrinsically disordered protein region (IDP, hTH1_65) containing two phosphorylation sites (S19 and S40) which are highly relevant for its increase in activity. The NMR signals of the IDP region in the non-phosphorylated, singly phosphorylated (pS40) and doubly phosphorylated states (pS19_pS40) were assigned by non-uniformly sampled spectra with increased dimensionality (5D). The structural changes induced by phosphorylation were analyzed by means of secondary structure propensities. The phosphorylation kinetics of the S40 and S19 by kinases PKA and PRAK respectively were monitored by non-uniformly sampled time-resolved NMR spectroscopy followed by their quantitative analysis.

• Klíčová slova
• Human tyrosine hydroxylase, IDP, Kinetics, NMR, Non-uniform sampling, Phosphorylation, SSP, Time-resolved NMR,
• MeSH
• fosforylace MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie metody   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteinové domény MeSH
• sekundární struktura proteinů MeSH
• tyrosin-3-monooxygenasa chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• intracelulární signální peptidy a proteiny MeSH
• MAP-kinase-activated kinase 5 MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• proteinkinasy závislé na cyklickém AMP MeSH
• tyrosin-3-monooxygenasa MeSH

Improving our understanding of nanosecond motions in disordered proteins requires the enhanced sampling of the spectral density function obtained from relaxation at low magnetic fields. High-resolution relaxometry and two-field NMR measurements of relaxation have, so far, only been based on the recording of one- or two-dimensional spectra, which provide insufficient resolution for challenging disordered proteins. Here, we introduce a 3D-HNCO-based two-field NMR experiment for measurements of protein backbone 15 N amide longitudinal relaxation rates. The experiment provides accurate longitudinal relaxation rates at low field (0.33 T in our case) preserving the resolution and sensitivity typical for high-field NMR spectroscopy. Radiofrequency pulses applied on six different radiofrequency channels are used to manipulate the spin system at both fields. The experiment was demonstrated on the C-terminal domain of δ subunit of RNA polymerase from Bacillus subtilis, a protein with highly repetitive amino-acid sequence and very low dispersion of backbone chemical shifts.

• Klíčová slova
• Dynamics, High-resolution relaxometry, Intrinsically disordered proteins, Non-uniform sampling, Nuclear magnetic resonance, Relaxation,
• MeSH
• Bacillus subtilis enzymologie   MeSH
• bakteriální proteiny chemie   MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• rekombinantní proteiny chemie   MeSH
• vnitřně neuspořádané proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA řízené RNA-polymerasy MeSH
• rekombinantní proteiny MeSH
• vnitřně neuspořádané proteiny MeSH

A microfluidic-based digital polymerase chain reaction (dPCR) chip requires precise temperature control as well as uniform temperature distribution to ensure PCR efficiency. However, measuring local temperature and its distribution over thousands of μL/nL-volume samples with minimum disturbance is challenging. Here, we present a method of non-contact localized temperature measurement for determination of the non-uniformity of temperature distribution over a dPCR chip. We filled the dPCR chip with a PCR solution containing amplified DNA fragments with a known melting temperature (T M). We then captured fluorescent images of the chip when it was heated from 70 to 99 °C, plotted the fluorescence intensity of each partition as a function of temperature, and calculated measured T M values from each partition. Finally, we created a 3-D map of the dPCR chip with the measured T M as the parameter. Even when the actual T M of the PCR solution was constant, the measured T M value varied between locations due to temperature non-uniformity in the dPCR chip. The method described here thereby characterized the distribution of temperature non-uniformity using a PCR solution with known T M as a temperature sensor. Among the non-contact temperature measurement methods, the proposed T M-based method can determine the temperature distribution within the chip, instead of only at the chip surface. The method also does not suffer from the undesirable photobleaching effect of fluorescein-based temperature measurement method. Temperature determination over the dPCR chip based on T M allowed us to calibrate the temperature sensor and improve the dPCR configuration and precision. This method is also suitable for determining the temperature uniformity of other microarray systems where there is no physical access to the system and thus direct temperature measurement is not possible.

• Publikační typ
• časopisecké články MeSH

PURPOSE: Since the number of cancer patients treated by proton irradiation has increased in the last few years, it seems appropriate to study dose-dependent effects of proton irradiation on mammalian tissues in more detail. MATERIALS AND METHODS: Tissue samples of normal skin of mouse and swine, of a human tumour model xenograph, and of normal skin and a skin tumour (basal cell carcinoma) of a human patient of about 1 mm thickness were irradiated by 24 MeV protons (uniform delivered doses of 1, 7 and 50 Gy: skin of mouse and a human tumour model xenograph, and 0.5, 5 and 50 Gy: swine and human skin). Raman spectra of non-irradiated and irradiated samples were recorded and analysed. RESULTS: Amide I, P=O and C-O bond vibrations and aromatics were sensitive to the proton irradiation dose. In the C-H stretching region, the irradiation-mediated change of Raman spectra was significant only in the case of the skin tumour. CONCLUSIONS: It has been shown that Raman spectroscopy is suited to assess the radiation damage done to biological samples by protons. Proteins of the human skin tumour seem to be more sensitive to proton irradiation than proteins of normal human skin.

• MeSH
• kůže účinky záření   MeSH
• lidé MeSH
• myši MeSH
• prasata MeSH
• protony * MeSH
• Ramanova spektroskopie * MeSH
• transplantace heterologní MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protony * MeSH

A pair of 4D NMR experiments for the backbone assignment of disordered proteins is presented. The experiments exploit (13)C direct detection and non-uniform sampling of the indirectly detected dimensions, and provide correlations of the aliphatic proton (H(α), and H(β)) and carbon (C(α), C(β)) resonance frequencies to the protein backbone. Thus, all the chemical shifts regularly used to map the transient secondary structure motifs in the intrinsically disordered proteins (H(α), C(α), C(β), C', and N) can be extracted from each spectrum. Compared to the commonly used assignment strategy based on matching the C(α) and C(β) chemical shifts, inclusion of the H(α) and H(β) provides up to three extra resonance frequencies that decrease the chance of ambiguous assignment. The experiments were successfully applied to the original assignment of a 12.8 kDa intrinsically disordered protein having a high content of proline residues (26 %) in the sequence.

• MeSH
• izotopy uhlíku chemie   MeSH
• molekulární sekvence - údaje MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• prolin chemie   MeSH
• proteiny chemie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• izotopy uhlíku MeSH
• prolin MeSH
• proteiny MeSH

Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of (13)C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.

• MeSH
• Bacillus subtilis enzymologie   MeSH
• bakteriální proteiny chemie   MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• izotopy uhlíku MeSH
• konformace proteinů MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• proteiny chemie   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA řízené RNA-polymerasy MeSH
• izotopy uhlíku MeSH
• proteiny MeSH