NMR determined Rotational correlation time - Revision history

Alex at 22:43, 13 October 2011

2011-10-13T22:43:05Z

← Older revision		Revision as of 22:43, 13 October 2011
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	:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,		:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,

	where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in ref.<ref><pubmed>2690953</pubmed>~~<doi>10.1021/bi00449a003</doi>~~		where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in ref.<ref><pubmed>2690953</pubmed></ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.
	</ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.

	<br> Average <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for a given protein can be measured using 1D <sup>15</sup>N-edited relaxation experiments <ref><pubmed>7514039</pubmed></ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide <sup>1</sup>H region (typically 10.5 to 8.5 ppm) and the results are used to fit an exponential decay as a function of delay time. One then computes the correlation time using Eq. 3, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T<sub>1</sub>/T<sub>2</sub> method is suitable for proteins with molecular weight of up to MW ≈ 25 kDa. Accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes difficult for larger proteins and cross-correlated relaxation rates are measured instead.		<br> Average <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for a given protein can be measured using 1D <sup>15</sup>N-edited relaxation experiments <ref><pubmed>7514039</pubmed></ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide <sup>1</sup>H region (typically 10.5 to 8.5 ppm) and the results are used to fit an exponential decay as a function of delay time. One then computes the correlation time using Eq. 3, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T<sub>1</sub>/T<sub>2</sub> method is suitable for proteins with molecular weight of up to MW ≈ 25 kDa. Accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes difficult for larger proteins and cross-correlated relaxation rates are measured instead.

Alex at 22:42, 13 October 2011

2011-10-13T22:42:23Z

← Older revision		Revision as of 22:42, 13 October 2011
Line 15:		Line 15:
	:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,		:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,

	where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in ref.<ref><pubmed>2690953</pubmed></ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.		where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in ref.<ref><pubmed>2690953</pubmed><doi>10.1021/bi00449a003</doi>
			</ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.

	<br> Average <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for a given protein can be measured using 1D <sup>15</sup>N-edited relaxation experiments <ref><pubmed>7514039</pubmed></ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide <sup>1</sup>H region (typically 10.5 to 8.5 ppm) and the results are used to fit an exponential decay as a function of delay time. One then computes the correlation time using Eq. 3, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T<sub>1</sub>/T<sub>2</sub> method is suitable for proteins with molecular weight of up to MW ≈ 25 kDa. Accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes difficult for larger proteins and cross-correlated relaxation rates are measured instead.		<br> Average <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for a given protein can be measured using 1D <sup>15</sup>N-edited relaxation experiments <ref><pubmed>7514039</pubmed></ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide <sup>1</sup>H region (typically 10.5 to 8.5 ppm) and the results are used to fit an exponential decay as a function of delay time. One then computes the correlation time using Eq. 3, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T<sub>1</sub>/T<sub>2</sub> method is suitable for proteins with molecular weight of up to MW ≈ 25 kDa. Accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes difficult for larger proteins and cross-correlated relaxation rates are measured instead.

Alex at 22:36, 13 October 2011

2011-10-13T22:36:46Z

← Older revision		Revision as of 22:36, 13 October 2011
Line 15:		Line 15:
	:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,		:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,

	where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in ref.<ref>~~Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by &lt;sup&gt;15&lt;~~/~~sup&gt;N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. ''Biochemistry'', '''28''', 8972-8979.~~</ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.		where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in ref.<ref><pubmed>2690953</pubmed></ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.

	<br> Average <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for a given protein can be measured using 1D <sup>15</sup>N-edited relaxation experiments <ref>Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay, L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by &lt;sup&gt;15&lt;/~~sup&gt;N NMR relaxation. ''Biochemistry'', '''33''', 5984-6003.~~</ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide <sup>1</sup>H region (typically 10.5 to 8.5 ppm) and the results are used to fit an exponential decay as a function of delay time. One then computes the correlation time using Eq. 3, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T<sub>1</sub>/T<sub>2</sub> method is suitable for proteins with molecular weight of up to MW ≈ 25 kDa. Accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes difficult for larger proteins and cross-correlated relaxation rates are measured instead.		<br> Average <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for a given protein can be measured using 1D <sup>15</sup>N-edited relaxation experiments <ref><pubmed>7514039</pubmed></ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide <sup>1</sup>H region (typically 10.5 to 8.5 ppm) and the results are used to fit an exponential decay as a function of delay time. One then computes the correlation time using Eq. 3, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T<sub>1</sub>/T<sub>2</sub> method is suitable for proteins with molecular weight of up to MW ≈ 25 kDa. Accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes difficult for larger proteins and cross-correlated relaxation rates are measured instead.

	<br>		<br>

Jma at 17:04, 5 October 2011

2011-10-05T17:04:24Z

Show changes

Alex at 17:57, 4 January 2010

2010-01-04T17:57:19Z

← Older revision		Revision as of 17:57, 4 January 2010
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	Brownian rotation diffusion of a particle in solution has a characteristic time constant called rotational correlation time (τ<sub>c</sub>). It is the time it takes the particle to rotate by one radian and it depends on the particle size. For globular proteins a spherical approximation can be used and the rotational correlation time is given by Stoke's law		[[Image:T1wiki fig5.png\|right\|400px]] Brownian rotation diffusion of a particle in solution has a characteristic time constant called rotational correlation time (τ<sub>c</sub>). It is the time it takes the particle to rotate by one radian and it depends on the particle size. For globular proteins a spherical approximation can be used and the rotational correlation time is given by Stoke's law

	:<math>\tau_c=\frac{4\pi\eta r^3}{3kT}</math>,		:<math>\tau_c=\frac{4\pi\eta r^3}{3kT}</math>,
Line 8:		Line 8:

	where <math>\rho</math> is the average density for proteins (1.37 g/cm3), <math>N_a</math> is the Avogadro's number and <math>r_w</math> is the hydration radius (1.6 to 3.2 A)<ref>Cavanagh J., Fairbrother W.J., Palmer, A.G, Rance M., Skelton N.J. (2007) Protein NMR Spectroscopy: Principles and Practice, p21, Elsevier</ref>. As a general rule of thumb, the τ<sub>c</sub> of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kDa.		where <math>\rho</math> is the average density for proteins (1.37 g/cm3), <math>N_a</math> is the Avogadro's number and <math>r_w</math> is the hydration radius (1.6 to 3.2 A)<ref>Cavanagh J., Fairbrother W.J., Palmer, A.G, Rance M., Skelton N.J. (2007) Protein NMR Spectroscopy: Principles and Practice, p21, Elsevier</ref>. As a general rule of thumb, the τ<sub>c</sub> of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kDa.



	For rigid protein molecules, in the limit of slow molecular motion (τ<sub>c</sub> >> 0.5 ns) and high magnetic field (500 MHz or greater), a closed-form solution for τ<sub>c</sub> as a function of the ratio of the longitudinal (T<sub>1</sub>) and transverse (T<sub>2</sub>) <sup>15</sup>N relaxation times exists:		For rigid protein molecules, in the limit of slow molecular motion (τ<sub>c</sub> >> 0.5 ns) and high magnetic field (500 MHz or greater), a closed-form solution for τ<sub>c</sub> as a function of the ratio of the longitudinal (T<sub>1</sub>) and transverse (T<sub>2</sub>) <sup>15</sup>N relaxation times exists:
Line 13:		Line 15:
	:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,		:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,

	where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in <ref>Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15</sup>N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. ''Biochemistry'', '''28''', 8972-8979.</ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.		where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in ref.<ref>Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15</sup>N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. ''Biochemistry'', '''28''', 8972-8979.</ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.

TheresaRamelot at 15:25, 17 December 2009

2009-12-17T15:25:24Z

← Older revision		Revision as of 15:25, 17 December 2009
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	== <span class="mw-headline">'''References''' </span> ==		== <span class="mw-headline">'''References''' </span> ==

	<references/>		<references />

	~~-- JimAramini - 10 Nov 2009~~

Alex at 18:48, 16 December 2009

2009-12-16T18:48:32Z

← Older revision		Revision as of 18:48, 16 December 2009
Line 1:		Line 1:
	Brownian rotation diffusion of a particle in solution has a characteristic time constant called rotational correlation time (τ<sub>c</sub>). It is the time it takes the particle to rotate by one radian and it depends on the particle size. For globular proteins a spherical approximation can be used and the rotational correlation time is given by Stoke's law		Brownian rotation diffusion of a particle in solution has a characteristic time constant called rotational correlation time (τ<sub>c</sub>). It is the time it takes the particle to rotate by one radian and it depends on the particle size. For globular proteins a spherical approximation can be used and the rotational correlation time is given by Stoke's law

	<math>\tau_c=\frac{4\pi\eta r^3}{3kT}</math>,		:<math>\tau_c=\frac{4\pi\eta r^3}{3kT}</math>,

	where <math>\eta</math> is the viscosity of the solvent, <math>r</math> is the effective hydrodynamic radius of the protein molecule, <math>k</math> is the Boltzmann constant and <math>T</math> is the temperature. The hydrodynamic radius can be estimated from the molecular weight of the protein (M) as		where <math>\eta</math> is the viscosity of the solvent, <math>r</math> is the effective hydrodynamic radius of the protein molecule, <math>k</math> is the Boltzmann constant and <math>T</math> is the temperature. The hydrodynamic radius can be estimated from the molecular weight of the protein (M) as

	<math>r\approx\sqrt[3]{\frac{3M}{4\pi\rho N_a}}+r_w</math>,		:<math>r\approx\sqrt[3]{\frac{3M}{4\pi\rho N_a}}+r_w</math>,

	where <math>\rho</math> is the average density for proteins (1.37 g/cm3), <math>N_a</math> is the Avogadro's number and <math>r_w</math> is the hydration radius (1.6 to 3.2 A)<ref>Cavanagh J., Fairbrother W.J., Palmer, A.G, Rance M., Skelton N.J. (2007) Protein NMR Spectroscopy: Principles and Practice, p21, Elsevier</ref>. As a general rule of thumb, the τ<sub>c</sub> of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kDa.		where <math>\rho</math> is the average density for proteins (1.37 g/cm3), <math>N_a</math> is the Avogadro's number and <math>r_w</math> is the hydration radius (1.6 to 3.2 A)<ref>Cavanagh J., Fairbrother W.J., Palmer, A.G, Rance M., Skelton N.J. (2007) Protein NMR Spectroscopy: Principles and Practice, p21, Elsevier</ref>. As a general rule of thumb, the τ<sub>c</sub> of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kDa.
Line 11:		Line 11:
	For rigid protein molecules, in the limit of slow molecular motion (τ<sub>c</sub> >> 0.5 ns) and high magnetic field (500 MHz or greater), a closed-form solution for τ<sub>c</sub> as a function of the ratio of the longitudinal (T<sub>1</sub>) and transverse (T<sub>2</sub>) <sup>15</sup>N relaxation times exists:		For rigid protein molecules, in the limit of slow molecular motion (τ<sub>c</sub> >> 0.5 ns) and high magnetic field (500 MHz or greater), a closed-form solution for τ<sub>c</sub> as a function of the ratio of the longitudinal (T<sub>1</sub>) and transverse (T<sub>2</sub>) <sup>15</sup>N relaxation times exists:

	<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,		:<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,

	where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in <ref>Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15</sup>N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. ''Biochemistry'', '''28''', 8972-8979.</ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.		where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in <ref>Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15</sup>N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. ''Biochemistry'', '''28''', 8972-8979.</ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.


	Average <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for a given protein can be measured using 1D <sup>15</sup>N-edited relaxation experiments <ref>Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay, L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by <sup>15</sup>N NMR relaxation. ''Biochemistry'', '''33''', 5984-6003.</ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide <sup>1</sup>H region (typically 10.5 to 8.5 ppm) and the results are used to fit an exponential decay as a function of delay time. One then computes the correlation time using Eq. 3, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The ~~T1-over-T2 approach~~ is ~~reliable~~ for proteins up to MW ≈ 25 kDa~~, where accurate~~ measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes ~~problematic~~.		Average <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for a given protein can be measured using 1D <sup>15</sup>N-edited relaxation experiments <ref>Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay, L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by <sup>15</sup>N NMR relaxation. ''Biochemistry'', '''33''', 5984-6003.</ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide <sup>1</sup>H region (typically 10.5 to 8.5 ppm) and the results are used to fit an exponential decay as a function of delay time. One then computes the correlation time using Eq. 3, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T<sub>1</sub>/T<sub>2</sub> method is suitable for proteins with molecular weight of up to MW ≈ 25 kDa. Accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes difficult for larger proteins and cross-correlated relaxation rates are measured instead.

	== <span class="mw-headline">'''Protocols for Bruker and Varian NMR Instruments'''</span> ==		== <span class="mw-headline">'''Protocols for Bruker and Varian NMR Instruments'''</span> ==

Alex at 18:23, 16 December 2009

2009-12-16T18:23:37Z

← Older revision		Revision as of 18:23, 16 December 2009
Line 1:		Line 1:
	== '~~''Introduction''' ==~~		Brownian rotation diffusion of a particle in solution has a characteristic time constant called rotational correlation time (τ<sub>c</sub>). It is the time it takes the particle to rotate by one radian and it depends on the particle size. For globular proteins a spherical approximation can be used and the rotational correlation time is given by Stoke's law

	~~The rotational correlation time of a protein in solution is the time for a protein to rotate one radian. In the limit of slow molecular motion (τ~~<~~sub~~>c</~~sub~~> ~~>> 0.5 ns)~~, the correlation time of a protein is related to the ratio of the longitudinal (T<sub>1</sub>) and transverse (T<sub>2</sub>) <sup>15</sup>N relaxation times, and nuclear frequency (ν<sub>N</sub>) according to Eq. 1, which is derived from Eq. 8 in Kay et al. (Ref. 1) by considering only J(0) and J(ω) spectral densities and neglecting higher frequency terms.          [[Image:Tc eq1.png\|center\|250x50px]]		<math>\tau_c=\frac{4\pi\eta r^3}{3kT}</math>,

	<br>		where <math>\eta</math> is the viscosity of the solvent, <math>r</math> is the effective hydrodynamic radius of the protein molecule, <math>k</math> is the Boltzmann constant and <math>T</math> is the temperature. The hydrodynamic radius can be estimated from the molecular weight of the protein (M) as

	~~In practice~~, ~~global~~ <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for ~~an unknown~~ protein ~~target~~ can be ~~obtained on Bruker or Varian NMR sytems~~ using 1D <sup>15</sup>N-edited relaxation experiments (~~Ref~~. 2), ~~by fitting~~ integrated ~~signal in~~ the backbone amide <sup>1</sup>H region of the ~~spectrum~~ as a function of delay time ~~to an exponential decay~~. One then computes the correlation time using Eq. 1, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size.~~  As a general rule of thumb, the τ<sub>c</sub> of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kiloDaltons. This~~ approach is reliable up to MW ≈ 25 kDa, where accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes problematic.~~ <br>~~		<math>r\approx\sqrt[3]{\frac{3M}{4\pi\rho N_a}}+r_w</math>,

			where <math>\rho</math> is the average density for proteins (1.37 g/cm3), <math>N_a</math> is the Avogadro's number and <math>r_w</math> is the hydration radius (1.6 to 3.2 A)<ref>Cavanagh J., Fairbrother W.J., Palmer, A.G, Rance M., Skelton N.J. (2007) Protein NMR Spectroscopy: Principles and Practice, p21, Elsevier</ref>. As a general rule of thumb, the τ<sub>c</sub> of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kDa.

			For rigid protein molecules, in the limit of slow molecular motion (τ<sub>c</sub> >> 0.5 ns) and high magnetic field (500 MHz or greater), a closed-form solution for τ<sub>c</sub> as a function of the ratio of the longitudinal (T<sub>1</sub>) and transverse (T<sub>2</sub>) <sup>15</sup>N relaxation times exists:

			<math>\tau_c\approx\frac{1}{4\pi\nu_N}\sqrt{6\frac{T_1}{T_2}-7}</math>,

			where ν<sub>N</sub> is the <sup>15</sup>N resonance frequency (in Hz). This equation is derived from Eq. 8 in <ref>Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15</sup>N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease. ''Biochemistry'', '''28''', 8972-8979.</ref> by considering only J(0) and J(ωN) spectral density terms and neglecting higher frequency terms.


			Average <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for a given protein can be measured using 1D <sup>15</sup>N-edited relaxation experiments <ref>Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay, L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by <sup>15</sup>N NMR relaxation. ''Biochemistry'', '''33''', 5984-6003.</ref>. To minimize contributions from unfolded segments each 1D spectum is integrated over the downfield backbone amide <sup>1</sup>H region (typically 10.5 to 8.5 ppm) and the results are used to fit an exponential decay as a function of delay time. One then computes the correlation time using Eq. 3, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size. The T1-over-T2 approach is reliable for proteins up to MW ≈ 25 kDa, where accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes problematic.

	== <span class="mw-headline">'''Protocols for Bruker and Varian NMR Instruments'''</span> ==		== <span class="mw-headline">'''Protocols for Bruker and Varian NMR Instruments'''</span> ==
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	== <span class="mw-headline">'''References''' </span> ==		== <span class="mw-headline">'''References''' </span> ==

	~~1.    Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15~~</sup>N inverse detected heteronuclear NMR spectroscopy:  Application to staphylococcal nuclease. ''Biochemistry 28'', 8972-8979.<br>2.    Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay, L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by <sup>15</sup>N NMR relaxation. ''Biochemistry 33'', 5984-6003.<br>		<references/>

	~~<br~~>

	-- JimAramini - 10 Nov 2009		-- JimAramini - 10 Nov 2009

TheresaRamelot: Created page with '== '''Introduction''' == The rotational correlation time of a protein in solution is the time for a protein to rotate one radian. In the limit of slow molecular motion (τ_…'

2009-11-16T17:44:34Z

Created page with '== '''Introduction''' == The rotational correlation time of a protein in solution is the time for a protein to rotate one radian. In the limit of slow molecular motion (τ…'

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== '''Introduction''' ==

The rotational correlation time of a protein in solution is the time for a protein to rotate one radian. In the limit of slow molecular motion (τc >> 0.5 ns), the correlation time of a protein is related to the ratio of the longitudinal (T1) and transverse (T2) 15N relaxation times, and nuclear frequency (νN) according to Eq. 1, which is derived from Eq. 8 in Kay et al. (Ref. 1) by considering only J(0) and J(ω) spectral densities and neglecting higher frequency terms.          [[Image:Tc eq1.png|center|250x50px]]

 

In practice, global 15N T1 and T2 relaxation times for an unknown protein target can be obtained on Bruker or Varian NMR sytems using 1D 15N-edited relaxation experiments (Ref. 2), by fitting integrated signal in the backbone amide 1H region of the spectrum as a function of delay time to an exponential decay. One then computes the correlation time using Eq. 1, and compares it to a standard curve of τc vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size.  As a general rule of thumb, the τc of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kiloDaltons. This approach is reliable up to MW ≈ 25 kDa, where accurate measurement of the diminishing 15N T2 becomes problematic.  

== '''Protocols for Bruker and Varian NMR Instruments''' ==

*[[Measuring_15N_T1_and_T2_Relaxation_Times_on_Bruker_Instruments|Bruker protocol]]
*[[Estimation_of_rotational_correlation_time|Varian protocol]]
 
== '''References''' ==

1.    Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy:  Application to staphylococcal nuclease. ''Biochemistry 28'', 8972-8979. 2.    Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay, L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. ''Biochemistry 33'', 5984-6003. 

 

-- JimAramini - 10 Nov 2009