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1.
Chemphyschem ; 21(3): 220-231, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31805217

RESUMO

We provide an experimental and computational framework for 2 H quadrupolar chemical exchange saturation transfer NMR experiments (Q-CEST) under static solid-state conditions for the quantification of dynamics on µs-ms timescales. Simulations using simple 2-site exchange models provide insights into the relation between spin dynamics and motions. Biological applications focus on two sites of amyloid-ß fibrils in the 3-fold symmetric polymorph. The first site, the methyl group of A2 of the disordered N-terminal domain, undergoes diffusive motions and conformational exchange due to transient interactions. Earlier 2 H rotating frame relaxation and quadrupolar CPMG measurements are combined with the Q-CEST approach to characterize the multiple conformational states of the domain. The second site, the methyl group of M35, spans the water-accessible cavity inside the fibrils' core and undergoes extensive rotameric exchange. Q-CEST permits us to refine the rotameric exchange model for this site and allows the more precise determination of populations and rotameric exchange rate constants than line shape analysis.


Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Deutério , Modelos Químicos , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética/métodos
2.
Nat Commun ; 10(1): 4922, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664028

RESUMO

Isotopically labeled methyl groups provide NMR probes in large, otherwise deuterated proteins. However, the resonance assignment constitutes a bottleneck for broader applicability of methyl-based NMR. Here, we present the automated MethylFLYA method for the assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA is applied to five proteins (28-358 kDa) comprising a total of 708 isotope-labeled methyl groups, of which 612 contribute NOESY cross peaks. MethylFLYA confidently assigns 488 methyl groups, i.e. 80% of those with NOESY data. Of these, 459 agree with the reference, 6 were different, and 23 were without reference assignment. MethylFLYA assigns significantly more methyl groups than alternative algorithms, has an average error rate of 1%, modest runtimes of 0.4-1.2 h, and can handle arbitrary isotope labeling patterns and data from other types of NMR spectra.


Assuntos
Automação/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Algoritmos , Metilação , Modelos Moleculares , Peso Molecular , Software
3.
PLoS Pathog ; 15(10): e1008117, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31644574

RESUMO

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Priônicas/metabolismo , Príons/metabolismo , Animais , Arvicolinae , Sistema Nervoso Central/patologia , Sulfato de Dextrana/farmacologia , Modelos Animais de Doenças , Camundongos Transgênicos , Doenças Priônicas/patologia , Estrutura Terciária de Proteína , Deficiências na Proteostase/patologia
4.
J Phys Chem Lett ; 10(19): 5917-5922, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31509419

RESUMO

Nuclear magnetic relaxation provides invaluable quantitative site-specific information on the dynamics of complex systems. Determining dynamics on nanosecond time scales requires relaxation measurements at low magnetic fields incompatible with high-resolution NMR. Here, we use a two-field NMR spectrometer to measure carbon-13 transverse and longitudinal relaxation rates at a field as low as 0.33 T (proton Larmor frequency 14 MHz) in specifically labeled side chains of the protein ubiquitin. The use of radiofrequency pulses enhances the accuracy of measurements as compared to high-resolution relaxometry approaches, where the sample is moved in the stray field of the superconducting magnet. Importantly, we demonstrate that accurate measurements at a single low magnetic field provide enough information to characterize complex motions on low nanosecond time scales, which opens a new window for the determination of site-specific nanosecond motions in complex systems such as proteins.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Isótopos de Carbono , Cinética , Campos Magnéticos , Movimento (Física) , Prótons , Ubiquitina/química
5.
Mar Drugs ; 17(9)2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31480659

RESUMO

In an effort to discover new bioactive anti-tumor lead compounds, a specific tyrosine phosphatase CDC25B and an Erb family receptor EGFR were selected as drug screening targets. This work led to the investigation of the soft coral-derived fungus Talaromyces verruculosus and identification of two new oligophenalenone dimers, verruculosins A-B (1-2), along with three known analogues, bacillisporin F (3), duclauxin (4), and xenoclauxin (5). Compound 1 was the first structure of the oligophenalenone dimer possessing a unique octacyclic skeleton. The detailed structures and absolute configurations of the new compounds were elucidated on the basis of spectroscopic data, X-ray crystallography, optical rotation, Electronic Circular Dichroism (ECD) analysis, and nuclear magnetic resonance (NMR) calculations. Among which, compounds 1, 3, and 5 exhibited modest inhibitory activity against CDC25B with IC50 values of 0.38 ± 0.03, 0.40 ± 0.02, and 0.26 ± 0.06 µM, respectively.


Assuntos
Fungos/química , Fenalenos/química , Talaromyces/química , Dicroísmo Circular/métodos , Carvão Mineral , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos
6.
Nat Methods ; 16(8): 743-749, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31363225

RESUMO

Cellular behavior is controlled by the interplay of diverse biomolecules. Most experimental methods, however, can only monitor a single molecule class or reaction type at a time. We developed an in vitro nuclear magnetic resonance spectroscopy (NMR) approach, which permitted dynamic quantification of an entire 'heterotypic' network-simultaneously monitoring three distinct molecule classes (metabolites, proteins and RNA) and all elementary reaction types (bimolecular interactions, catalysis, unimolecular changes). Focusing on an eight-reaction co-transcriptional RNA folding network, in a single sample we recorded over 35 time points with over 170 observables each, and accurately determined five core reaction constants in multiplex. This reconstruction revealed unexpected cross-talk between the different reactions. We further observed dynamic phase-separation in a system of five distinct RNA-binding domains in the course of the RNA transcription reaction. Our Systems NMR approach provides a deeper understanding of biological network dynamics by combining the dynamic resolution of biochemical assays and the multiplexing ability of 'omics'.


Assuntos
Redes Reguladoras de Genes , Metaboloma , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/análise , RNA/análise , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Conformação Proteica , Proteínas/química , RNA/química , Dobramento de RNA
7.
Chem Commun (Camb) ; 55(66): 9761-9764, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31355386

RESUMO

The effect of ions on the structure and dynamics of a spider silk protein is elucidated. Chaotropic ions prevent intra- and inter-molecular interactions on the repetitive domain, which are required to maintain the solubility, while kosmotropic ions promote hydrogen bond interactions in the glycine-rich region, which are a prerequisite for ß-sheet formation.


Assuntos
Conformação Proteica em Folha beta , Seda/química , Animais , Cloretos/química , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/química , Sódio/química , Solubilidade , Aranhas
8.
Int J Mol Sci ; 20(15)2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31357624

RESUMO

The divisome is a large protein complex that regulates bacterial cell division and therefore represents an attractive target for novel antibacterial drugs. In this study, we report on the ligandability of FtsQ, which is considered a key component of the divisome. For this, the soluble periplasmic domain of Escherichia coli FtsQ was immobilized and used to screen a library of 1501 low molecular weight (< 300 Da), synthetic compounds for those that interact with the protein. A primary screen was performed using target immobilized NMR screening (TINS) and yielded 72 hits. Subsequently, these hits were validated in an orthogonal assay. At first, we aimed to do this using surface plasmon resonance (SPR), but the lack of positive control hampered optimization of the experiment. Alternatively, a two-dimensional heteronuclear single quantum coherence (HSQC) NMR spectrum of FtsQ was obtained and used to validate these hits by chemical shift perturbation (CSP) experiments. This resulted in the identification of three fragments with weak affinity for the periplasmic domain of FtsQ, arguing that the ligandability of FtsQ is low. While this indicates that developing high affinity ligands for FtsQ is far from straightforward, the identified hit fragments can help to further interrogate FtsQ interactions.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Divisão Celular , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Relação Estrutura-Atividade
9.
J Chem Phys ; 151(3): 034102, 2019 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-31325945

RESUMO

Nuclear magnetic resonance (NMR) is sensitive to dynamics on a wide range of correlation times. Recently, we have shown that analysis of relaxation rates via fitting to a correlation function with a small number of exponential terms could yield a biased characterization of molecular motion in solid-state NMR due to limited sensitivity of experimental data to certain ranges of correlation times. We introduced an alternative approach based on "detectors" in solid-state NMR, for which detector responses characterize motion for a range of correlation times and reduce potential bias resulting from the use of simple models for the motional correlation functions. Here, we show that similar bias can occur in the analysis of solution-state NMR relaxation data. We have thus adapted the detector approach to solution-state NMR, specifically separating overall tumbling motion from internal motions and accounting for contributions of chemical exchange to transverse relaxation. We demonstrate that internal protein motions can be described with detectors when the overall motion and the internal motions are statistically independent. We illustrate the detector analysis on ubiquitin with typical relaxation data sets recorded at a single high magnetic field or at multiple high magnetic fields and compare with results of model-free analysis. We also compare our methodology to LeMaster's method of dynamics analysis.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Termodinâmica
10.
Carbohydr Polym ; 220: 176-184, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31196538

RESUMO

A new glycosaminoglycan (LF-GAG) was purified from the slug Limacus flavus. Its unique chemical structure and heparanase inhibitory activity were studied in this work. The native LF-GAG was composed of L-iduronic acid (L-IdoA) and N-acetyl-D-glucosamine (D-GlcNAc), with a Mw of 22,700 Da. To elucidate the precise structure and structure-activity relationship, its deacetylation-deaminative depolymerized product (dLF-GAG) was prepared, and from which four oligosaccharides were purified. Combining the NMR spectral analysis of LF-GAG and its derived oligosaccharides, the structure of LF-GAG was deduced to be -4)-L-IdoA2R-(α1,4)-D-GlcNAc-(α1-, in which R was -OH (˜80%) or -OSO3- (˜20%). Bioactivity assays showed that LF-GAG could potently inhibit human heparanase (IC50, 0.10 µM). dLF-GAG and LF-3 were less potent but also active for heparanase inhibition. Structure-activity relationship analysis indicated that the chain length and sulfate substitution of LF-GAG are essential for its heparanase inhibitory activity.


Assuntos
Acetilglucosamina/química , Gastrópodes/metabolismo , Glucuronidase/antagonistas & inibidores , Glicosaminoglicanos , Ácido Idurônico/química , Animais , Glicosaminoglicanos/química , Glicosaminoglicanos/isolamento & purificação , Glicosaminoglicanos/farmacologia , Ressonância Magnética Nuclear Biomolecular/métodos
11.
Chem Commun (Camb) ; 55(55): 7899-7902, 2019 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-31199417

RESUMO

Fast-magic-angle-spinning solid-state NMR is a developing technique for determination of protein structure and dynamics. Proton-proton correlations usually lead to rough distance restraints, a serious hurdle towards high-resolution structures. Analogous to the "eNOE" concept in solution, an integrative approach for more accurate restraints enables improved structural accuracy with minimal analytical effort.


Assuntos
Anidrase Carbônica II/química , Espectrina/química , Animais , Galinhas , Humanos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Domínios de Homologia de src
12.
J Biomol NMR ; 73(5): 223-227, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31165320

RESUMO

Staphylococcus aureus hibernation promoting factor (SaHPF) is a 22,2 kDa protein which plays a crucial role in 100S Staphylococcus aureus ribosome formation during stress. SaHPF consists of N-terminal domain (NTD) that prevents proteins synthesis by binding to the 30S subunit at the P- and A-sites, connected through a flexible linker with a C-terminal domain (CTD) that keeps ribosomes in 100S form via homodimerization. Recently obtained 100S ribosome structure of S. aureus by cryo-EM shown that SaHPF-NTD bound to the ribosome active sites, however due to the absence of SaHPF-NTD structure it was modeled by homology with the E. coli hibernation factors HPF and YfiA. In present paper we have determined the solution structure of SaHPF-NTD by high-resolution NMR spectroscopy which allows us to increase structural knowledge about HPF structure from S. aureus.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo
13.
J Biomol NMR ; 73(5): 213-222, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31165321

RESUMO

Various methods for understanding the structural and dynamic properties of proteins rely on the analysis of their NMR chemical shifts. These methods require the initial assignment of NMR signals to particular atoms in the sequence of the protein, a step that can be very time-consuming. The probabilistic interaction network of evidence (PINE) algorithm for automated assignment of backbone and side chain chemical shifts utilizes a Bayesian probabilistic network model that analyzes sequence data and peak lists from multiple NMR experiments. PINE, which is one of the most popular and reliable automated chemical shift assignment algorithms, has been available to the protein NMR community for longer than a decade. We announce here a new web server version of PINE, called Integrative PINE (I-PINE), which supports more types of NMR experiments than PINE (including three-dimensional nuclear Overhauser enhancement and four-dimensional J-coupling experiments) along with more comprehensive visualization of chemical shift based analysis of protein structure and dynamics. The I-PINE server is freely accessible at http://i-pine.nmrfam.wisc.edu . Help pages and tutorial including browser capability are available at: http://i-pine.nmrfam.wisc.edu/instruction.html . Sample data that can be used for testing the web server are available at: http://i-pine.nmrfam.wisc.edu/examples.html .


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Algoritmos , Proteínas/análise
14.
Nat Commun ; 10(1): 2511, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175284

RESUMO

Chemical shifts (CS) are determined from NMR experiments and represent the resonance frequency of the spin of atoms in a magnetic field. They contain a mixture of information, encompassing the in-solution conformations a protein adopts, as well as the movements it performs. Due to their intrinsically multi-faceted nature, CS are difficult to interpret and visualize. Classical approaches for the analysis of CS aim to extract specific protein-related properties, thus discarding a large amount of information that cannot be directly linked to structural features of the protein. Here we propose an autoencoder-based method, called ShiftCrypt, that provides a way to analyze, compare and interpret CS in their native, multidimensional space. We show that ShiftCrypt conserves information about the most common structural features. In addition, it can be used to identify hidden similarities between diverse proteins and peptides, and differences between the same protein in two different binding states.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/ultraestrutura , Aminoácidos , Fenômenos Biofísicos , Imagem por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Secundária de Proteína
15.
Chem Commun (Camb) ; 55(41): 5777-5780, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31041432

RESUMO

Investigating the interplay in a minimal redox complex of cytochrome-P450 and its reductase is crucial for understanding cytochrome-P450's enzymatic activity. Probing the hotspots of dynamic structural interactions using NMR revealed the engagement of loop residues from P450-reductase to be responsible for the enhanced affinity of CYP450 towards its obligate redox partner.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Mapas de Interação de Proteínas , Animais , Sistema Enzimático do Citocromo P-450/química , Humanos , Modelos Moleculares , NADPH-Ferri-Hemoproteína Redutase/química , Ressonância Magnética Nuclear Biomolecular/métodos , Oxirredução , Mapeamento de Interação de Proteínas/métodos , Coelhos
16.
J Biomol NMR ; 73(3-4): 183-189, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31041647

RESUMO

The physiological role of proteins is frequently linked to interactions with non-protein ligands or posttranslational modifications. Structural characterization of these complexes or modified proteins by NMR may be difficult as the ligands are usually not available in an isotope-labeled form and NMR spectra may suffer from signal overlap. Here, we present an optimized approach that uses specific NMR isotope-labeling schemes for overcoming both hurdles. This approach enabled the high-resolution structure determination of the farnesylated C-terminal domain of the peroxisomal protein PEX19. The approach combines specific 13C, 15N and 2H isotope labeling with tailored NMR experiments to (i) unambiguously identify the NMR frequencies and the stereochemistry of the unlabeled 15-carbon isoprenoid, (ii) resolve the NMR signals of protein methyl groups that contact the farnesyl moiety and (iii) enable the unambiguous assignment of a large number of protein-farnesyl NOEs. Protein deuteration was combined with selective isotope-labeling and protonation of amino acids and methyl groups to resolve ambiguities for key residues that contact the farnesyl group. Sidechain-labeling of leucines, isoleucines, methionines, and phenylalanines, reduced spectral overlap, facilitated assignments and yielded high quality NOE correlations to the unlabeled farnesyl. This approach was crucial to enable the first NMR structure of a farnesylated protein. The approach is readily applicable for NMR structural analysis of a wide range of protein-ligand complexes, where isotope-labeling of ligands is not well feasible.


Assuntos
Marcação por Isótopo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas/química , Ligantes , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação Proteica
17.
J Biomol NMR ; 73(3-4): 105-116, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31041648

RESUMO

Spectral resolution remains one of the most significant limitations in the NMR study of biomolecules. We present the srNOESY (super resolution nuclear overhauser effect spectroscopy) experiment, which enhances the resolution of NOESY cross-peaks at the expense of the diagonal peak line-width. We studied two proteins, ubiquitin and the influenza hemagglutinin fusion peptide in bicelles, and we achieved average resolution enhancements of 21-47% and individual peak enhancements as large as ca. 450%. New peaks were observed over the conventional NOESY experiment in both proteins as a result of these improvements, and the final structures generated from the calculated restraints matched published models. We discuss the impact of the experimental parameters, spin diffusion and the information content of the srNOESY lineshape.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Algoritmos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Modelos Teóricos , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/genética
18.
J Biomol NMR ; 73(3-4): 191-198, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31041649

RESUMO

Residual dipolar couplings (RDCs) provide both structural and dynamical information useful in the characterization of biological macromolecules. While most data come from the interaction of simple pairs of directly bonded spin-1/2 nuclei (1H-15N, 1H-13C, 1H-1H), it is possible to acquire data from interactions among the multiple spins of 13C-labeled methyl groups (1H3-13C). This is especially important because of the advantages that observation of 13C-labeled methyl groups offers in working with very large molecules. Here we consider some of the options for measurement of methyl RDCs in large and often fully protonated proteins and arrive at a pulse sequence that exploits both J-modulation and direct detection of 13C. Its utility is illustrated by application to a fully protonated two domain fragment from the mammalian glycoprotein, Robo1, 13C-methyl-labeled in all valines.


Assuntos
Carbono/química , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Isótopos de Carbono/química , Glicosilação , Espectroscopia de Ressonância Magnética , Metilação , Ressonância Magnética Nuclear Biomolecular/métodos
19.
J Chem Theory Comput ; 15(6): 3844-3853, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31042036

RESUMO

Configurational entropy change is a central constituent of the free energy change in noncovalent interactions between biomolecules. Due to both experimental and computational limitations, however, the impact of individual contributions to configurational entropy change remains underexplored. Here, we develop a novel, fully analytical framework to dissect the configurational entropy change of binding into contributions coming from molecular internal and external degrees of freedom. Importantly, this framework accounts for all coupled and uncoupled contributions in the absence of an external field. We employ our parallel implementation of the maximum information spanning tree algorithm to provide a comprehensive numerical analysis of the importance of the individual contributions to configurational entropy change on an extensive set of molecular dynamics simulations of protein binding processes. Contrary to commonly accepted assumptions, we show that different coupling terms contribute significantly to the overall configurational entropy change. Finally, while the magnitude of individual terms may be largely unpredictable a priori, the total configurational entropy change can be well approximated by rescaling the sum of uncoupled contributions from internal degrees of freedom only, providing support for NMR-based approaches for configurational entropy change estimation.


Assuntos
Entropia , Proteínas/química , Algoritmos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica
20.
J Biomol NMR ; 73(5): 245-260, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31089943

RESUMO

Side chains possess a broader conformational space (compared to the backbone) and are directly affected by intra- and intermolecular interactions, hence their dynamics and the corresponding NMR relaxation data are more sensitive and informative. Nevertheless, transverse relaxation in [Formula: see text] ([Formula: see text] or [Formula: see text]) spin systems is predominantly non-measurable in uniformly [Formula: see text]-labeled proteins due to cross-correlation effects. In the present publication, we propose a number of pulse sequences for accurate and precise measurement of the dipole-dipole transverse cross-correlated relaxation rate [Formula: see text], which, similarly to [Formula: see text] measurements, provides information about the amplitudes of intramolecular dynamics. The suggested approach has allowed us to circumvent a number of obstacles that were limiting earlier applications of [Formula: see text]: (1) impossibility of transmission of the central component of the triplet of [Formula: see text] group to [Formula: see text]-acquisition via INEPT has been solved by transmission of the averaged signal of "inner" and "outer" components of the triplet; (2) direct recording of the entire triplets resulting in substantial overlap of side chain signals has been replaced by recording of individual singlets with the use of [Formula: see text]-modulated approach and constant-time evolution; (3) low sensitivity has been enhanced via proton acquisition which required special attention to a zero-quantum coherence evolution. The proposed method expands the set of "dynamics sensors" covering protein side chains and substantially improves the quality and the level of detail of experimental data describing dynamic processes in proteins and protein complexes.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química
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