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Papers by Pavel Kadeřávek

Research paper thumbnail of Cross-correlated relaxation measurements under adiabatic sweeps: determination of local order in proteins

Journal of biomolecular NMR, Jan 28, 2015

Adiabatically swept pulses were originally designed for the purpose of broadband spin inversion. ... more Adiabatically swept pulses were originally designed for the purpose of broadband spin inversion. Later, unexpected advantages of their utilization were also found in other applications, such as refocusing to excite spin echoes, studies of chemical exchange or fragment-based drug design. Here, we present new experiments to characterize fast (ps-ns) protein dynamics, which benefit from little-known properties of adiabatic pulses. We developed a strategy for measuring cross-correlated cross-relaxation (CCCR) rates during adiabatic pulses. This experiment provides a linear combination of longitudinal and transverse CCCR rates, which is offset-independent across a typical amide [Formula: see text] spectrum. The pulse sequence can be recast to provide accurate transverse CCCR rates weighted by the populations of exchanging states. Sensitivity can be improved in systems in slow exchange. Finally, the experiments can be easily modified to yield residue-specific correlation times. The averag...

Research paper thumbnail of X-ray vs. NMR structure of N-terminal domain of δ-subunit of RNA polymerase

J Struct Biol, 2014

The crystal structure of the N-terminal domain of the RNA polymerase d subunit (Nd) from Bacillus... more The crystal structure of the N-terminal domain of the RNA polymerase d subunit (Nd) from Bacillus subtilis solved at a resolution of 2.0 Å is compared with the NMR structure determined previously. The molecule crystallizes in the space group C222(1) with a dimer in the asymmetric unit. Importantly, the X-ray structure exhibits significant differences from the lowest energy NMR structure. In addition to the overall structure differences, structurally important b sheets found in the NMR structure are not present in the crystal structure. We systematically investigated the cause of the discrepancies between the NMR and X-ray structures of Nd, addressing the pH dependence, presence of metal ions, and crystal packing forces. We convincingly showed that the crystal packing forces, together with the presence of Ni 2+ ions, are the main reason for such a difference. In summary, the study illustrates that the two structural approaches may give unequal results, which need to be interpreted with care to obtain reliable structural information in terms of biological relevance.

Research paper thumbnail of NMR Structure of the N-Terminal Domain of Capsid Protein from the Mason–Pfizer Monkey Virus

Journal of Molecular Biology, 2009

Research paper thumbnail of NMR 13 C-relaxation Study of Base and Sugar Dynamics in GCAA RNA Hairpin Tetraloop

Journal of Biomolecular Structure and Dynamics, 2007

Intramolecular dynamics of a 14-mer RNA hairpin including GCAA tetraloop was investigated by (13)... more Intramolecular dynamics of a 14-mer RNA hairpin including GCAA tetraloop was investigated by (13)C NMR relaxation. R(1) and R(1rho) relaxation rates were measured for all protonated base carbons as well as for C1' carbons of ribose sugars at several magnetic field strengths. The data has been interpreted in the framework of modelfree analysis [G. Lipari and A. Szabo. J Am Chem Soc 104, 4546-4559 (1982); G. Lipari and A. Szabo. J Am Chem Soc 104, 4559-4570 (1982)] characterizing the internal dynamics of the molecule by order parameters and correlation times for fast motions on picosecond to nanosecond time scale and by contributions of the chemical exchange. The fast dynamics reveals a rather rigid stem and a significantly more flexible loop. The cytosine and the last adenine bases in the loop as well as all the loop sugars exhibit a significant contribution of conformational equilibrium on microsecond to millisecond time scale. The high R(1rho) values detected on both base and sugar moieties of the loop indicate coordinated motions in this region. A semiquantitative analysis of the conformational equilibrium suggests the exchange rates on the order of 10(4) s(-1). The results are in general agreement with dynamics studies of GAAA loops by NMR relaxation and fluorescent spectroscopy and support the data on the GCAA loop dynamics obtained by MD simulations.

Research paper thumbnail of Spectral density mapping protocols for analysis of molecular motions in disordered proteins

Journal of Biomolecular NMR, 2014

Spectral density mapping represents the method of choice for investigations of molecular motions ... more Spectral density mapping represents the method of choice for investigations of molecular motions of intrinsically disordered proteins (IDPs). However, the current methodology has been developed for well-folded proteins. In order to find conditions for a reliable analysis of relaxation of IDPs, accuracy of the current reduced spectral density mapping protocols applied to IDPs was examined and new spectral density mapping methods employing cross-correlated relaxation rates have been designed. Various sources of possible systematic errors were analyzed theoretically and the presented approaches were tested on a partially disordered protein, delta subunit of bacterial RNA polymerase. Results showed that the proposed protocols provide unbiased description of molecular motions of IDPs and allow to separate slow exchange from fast dynamics.

Research paper thumbnail of Structural Study of the Partially Disordered Full-Length δ Subunit of RNA Polymerase from Bacillus subtilis

Research paper thumbnail of Corrigendum: Structural Study of the Partially Disordered Full-Length δ Subunit of RNA Polymerase from Bacillus subtilis

Research paper thumbnail of Soluble recombinant CD69 receptors optimized to have an exceptional physical and chemical stability display prolonged circulation and remain intact in the blood of mice

FEBS Journal, 2008

CD69, an earliest activation antigen of lymphocytes and a versatile leukocyte signaling molecule,... more CD69, an earliest activation antigen of lymphocytes and a versatile leukocyte signaling molecule, plays a key role in a large number of immune effector functions. This receptor is constitutively expressed at the surface of CD3 bright thymocytes, monocytes, neutrophils, epidermal Langerhans' cells and platelets, and appears very early upon the activation of T-lymphocytes, natural killer (NK) cells and some other cells of

Research paper thumbnail of Cross-correlated relaxation measurements under adiabatic sweeps: determination of local order in proteins

Journal of biomolecular NMR, Jan 28, 2015

Adiabatically swept pulses were originally designed for the purpose of broadband spin inversion. ... more Adiabatically swept pulses were originally designed for the purpose of broadband spin inversion. Later, unexpected advantages of their utilization were also found in other applications, such as refocusing to excite spin echoes, studies of chemical exchange or fragment-based drug design. Here, we present new experiments to characterize fast (ps-ns) protein dynamics, which benefit from little-known properties of adiabatic pulses. We developed a strategy for measuring cross-correlated cross-relaxation (CCCR) rates during adiabatic pulses. This experiment provides a linear combination of longitudinal and transverse CCCR rates, which is offset-independent across a typical amide [Formula: see text] spectrum. The pulse sequence can be recast to provide accurate transverse CCCR rates weighted by the populations of exchanging states. Sensitivity can be improved in systems in slow exchange. Finally, the experiments can be easily modified to yield residue-specific correlation times. The averag...

Research paper thumbnail of X-ray vs. NMR structure of N-terminal domain of δ-subunit of RNA polymerase

J Struct Biol, 2014

The crystal structure of the N-terminal domain of the RNA polymerase d subunit (Nd) from Bacillus... more The crystal structure of the N-terminal domain of the RNA polymerase d subunit (Nd) from Bacillus subtilis solved at a resolution of 2.0 Å is compared with the NMR structure determined previously. The molecule crystallizes in the space group C222(1) with a dimer in the asymmetric unit. Importantly, the X-ray structure exhibits significant differences from the lowest energy NMR structure. In addition to the overall structure differences, structurally important b sheets found in the NMR structure are not present in the crystal structure. We systematically investigated the cause of the discrepancies between the NMR and X-ray structures of Nd, addressing the pH dependence, presence of metal ions, and crystal packing forces. We convincingly showed that the crystal packing forces, together with the presence of Ni 2+ ions, are the main reason for such a difference. In summary, the study illustrates that the two structural approaches may give unequal results, which need to be interpreted with care to obtain reliable structural information in terms of biological relevance.

Research paper thumbnail of NMR Structure of the N-Terminal Domain of Capsid Protein from the Mason–Pfizer Monkey Virus

Journal of Molecular Biology, 2009

Research paper thumbnail of NMR 13 C-relaxation Study of Base and Sugar Dynamics in GCAA RNA Hairpin Tetraloop

Journal of Biomolecular Structure and Dynamics, 2007

Intramolecular dynamics of a 14-mer RNA hairpin including GCAA tetraloop was investigated by (13)... more Intramolecular dynamics of a 14-mer RNA hairpin including GCAA tetraloop was investigated by (13)C NMR relaxation. R(1) and R(1rho) relaxation rates were measured for all protonated base carbons as well as for C1' carbons of ribose sugars at several magnetic field strengths. The data has been interpreted in the framework of modelfree analysis [G. Lipari and A. Szabo. J Am Chem Soc 104, 4546-4559 (1982); G. Lipari and A. Szabo. J Am Chem Soc 104, 4559-4570 (1982)] characterizing the internal dynamics of the molecule by order parameters and correlation times for fast motions on picosecond to nanosecond time scale and by contributions of the chemical exchange. The fast dynamics reveals a rather rigid stem and a significantly more flexible loop. The cytosine and the last adenine bases in the loop as well as all the loop sugars exhibit a significant contribution of conformational equilibrium on microsecond to millisecond time scale. The high R(1rho) values detected on both base and sugar moieties of the loop indicate coordinated motions in this region. A semiquantitative analysis of the conformational equilibrium suggests the exchange rates on the order of 10(4) s(-1). The results are in general agreement with dynamics studies of GAAA loops by NMR relaxation and fluorescent spectroscopy and support the data on the GCAA loop dynamics obtained by MD simulations.

Research paper thumbnail of Spectral density mapping protocols for analysis of molecular motions in disordered proteins

Journal of Biomolecular NMR, 2014

Spectral density mapping represents the method of choice for investigations of molecular motions ... more Spectral density mapping represents the method of choice for investigations of molecular motions of intrinsically disordered proteins (IDPs). However, the current methodology has been developed for well-folded proteins. In order to find conditions for a reliable analysis of relaxation of IDPs, accuracy of the current reduced spectral density mapping protocols applied to IDPs was examined and new spectral density mapping methods employing cross-correlated relaxation rates have been designed. Various sources of possible systematic errors were analyzed theoretically and the presented approaches were tested on a partially disordered protein, delta subunit of bacterial RNA polymerase. Results showed that the proposed protocols provide unbiased description of molecular motions of IDPs and allow to separate slow exchange from fast dynamics.

Research paper thumbnail of Structural Study of the Partially Disordered Full-Length δ Subunit of RNA Polymerase from Bacillus subtilis

Research paper thumbnail of Corrigendum: Structural Study of the Partially Disordered Full-Length δ Subunit of RNA Polymerase from Bacillus subtilis

Research paper thumbnail of Soluble recombinant CD69 receptors optimized to have an exceptional physical and chemical stability display prolonged circulation and remain intact in the blood of mice

FEBS Journal, 2008

CD69, an earliest activation antigen of lymphocytes and a versatile leukocyte signaling molecule,... more CD69, an earliest activation antigen of lymphocytes and a versatile leukocyte signaling molecule, plays a key role in a large number of immune effector functions. This receptor is constitutively expressed at the surface of CD3 bright thymocytes, monocytes, neutrophils, epidermal Langerhans' cells and platelets, and appears very early upon the activation of T-lymphocytes, natural killer (NK) cells and some other cells of