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1H-15N HSQC
The Experts below are selected from a list of 12513 Experts worldwide ranked by ideXlab platform
Toshiyuki Kohno – One of the best experts on this subject based on the ideXlab platform.
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A novel way of amino acid-specific assignment in (1)H-(15)N HSQC spectra with a wheat germ cell-free protein synthesis system.
Journal of Biomolecular NMR, 2004Co-Authors: Eugene Hayato Morita, Rikou Tanaka, Yaeta Endo, Masato Shimizu, Tomio Ogasawara, Toshiyuki KohnoAbstract:For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized 15N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the 1H-15N HSQC spectra for native proteins and the corresponding ones for synthesized ones.
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A wheat germ cell-free system is a novel way to screen protein folding and function
Protein Science, 2003Co-Authors: Eugene Hayato Morita, Tatsuya Sawasaki, Rikou Tanaka, Yaeta Endo, Toshiyuki KohnoAbstract:For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two 15N-labeled proteins and obtained 1H-15N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and 1H-15N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell-free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell-free protein synthesis system to high-throughput protein structural analysis.
C. Griesinger – One of the best experts on this subject based on the ideXlab platform.
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Determination of ^3J(H
_infi
^supN
,C
_infi
^sup′
) coupling constants in proteins with the C′-FIDS methodJournal of Biomolecular NMR, 1995Co-Authors: A. Rexroth, S. Szalma, R. Weisemann, W. Bermel, H. Schwalbe, C. GriesingerAbstract:We introduce the C′-FIDS-^1H,^15N-HSQC experiment, a new method for the determination of ^3J(H _infi ^supN ,C _infi ^sup′ ) coupling constants in proteins, yielding information about the torsional angle ϕ. It relies on the ^1H,^15N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three ^1H,^15N-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t_1 and t_2, a second experiment in which they are decoupled exclusively during t_1 and a third one in which they are coupled in t_1 as well as t_2. The last experiment yields an E.COSY-type pattern from which the ^2J(H _infi ^supN ,C _infi-1 ^sup′ ) and ^1J(N_i,C _infi-1 ^sup′ ) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the ^2J(H _infi ^supN ,C _infi-1 ^sup′ ) coupling, the ^3J(H _infi ^supN ,C _infi ^sup′ ) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for ^nJ(H^N,C′) couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.
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Determination of (3)J(H (infi) (supN) ,C (infi) (sup’) ) coupling constants in proteins with the C’-FIDS method.
Journal of Biomolecular NMR, 1995Co-Authors: A. Rexroth, S. Szalma, R. Weisemann, W. Bermel, H. Schwalbe, C. GriesingerAbstract:We introduce the C′-FIDS-1H,15N-HSQC experiment, a new method for the determination of 3J(H
infi
supN
,C
infi
sup′
) coupling constants in proteins, yielding information about the torsional angle ϕ. It relies on the 1H,15N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three 1H,15N-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t1 and t2, a second experiment in which they are decoupled exclusively during t1 and a third one in which they are coupled in t1 as well as t2. The last experiment yields an E.COSY-type pattern from which the 2J(H
infi
supN
,C
infi-1
sup′
) and 1J(Ni,C
infi-1
sup′
) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the 2J(H
infi
supN
,C
infi-1
sup′
) coupling, the 3J(H
infi
supN
,C
infi
sup′
) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for nJ(HN,C′) couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.
David S Libich – One of the best experts on this subject based on the ideXlab platform.
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solution nmr and cd spectroscopy of an intrinsically disordered peripheral membrane protein evaluation of aqueous and membrane mimetic solvent conditions for studying the conformational adaptability of the 18 5 kda isoform of myelin basic protein mbp
European Biophysics Journal, 2008Co-Authors: David S Libich, George HarauzAbstract:The stability and secondary structure propensity of recombinant murine 18.5 kDa myelin basic protein (rmMBP, 176 residues) was assessed using circular dichroic and nuclear magnetic resonance spectroscopy (1H–15N HSQC experiments) to determine the optimal sample conditions for further NMR studies (i.e., resonance assignments and protein-protein interactions). Six solvent conditions were selected based on their ability to stabilise the protein, and their tractability to currently standard solution NMR methodology. Selected solvent conditions were further characterised as functions of concentration, temperature, and pH. The results of these trials indicated that 30% TFE-d2 in H2O (v/v), pH 6.5 at 300 K, and 100 mM KCl, pH 6.5 at 277 K were the best conditions to use for future solution NMR studies of MBP. Micelles of DPC were found to be inappropriate for backbone resonance assignments of rmMBP in this instance.
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Solution NMR and CD spectroscopy of an intrinsically disordered, peripheral membrane protein: evaluation of aqueous and membrane-mimetic solvent conditions for studying the conformational adaptability of the 18.5 kDa isoform of myelin basic protein (
European Biophysics Journal, 2008Co-Authors: David S Libich, George HarauzAbstract:The stability and secondary structure propensity of recombinant murine 18.5 kDa myelin basic protein (rmMBP, 176 residues) was assessed using circular dichroic and nuclear magnetic resonance spectroscopy (1H–15N HSQC experiments) to determine the optimal sample conditions for further NMR studies (i.e., resonance assignments and protein-protein interactions). Six solvent conditions were selected based on their ability to stabilise the protein, and their tractability to currently standard solution NMR methodology. Selected solvent conditions were further characterised as functions of concentration, temperature, and pH. The results of these trials indicated that 30% TFE-d2 in H2O (v/v), pH 6.5 at 300 K, and 100 mM KCl, pH 6.5 at 277 K were the best conditions to use for future solution NMR studies of MBP. Micelles of DPC were found to be inappropriate for backbone resonance assignments of rmMBP in this instance.