Electricity: measuring and testing – Particle precession resonance – Using a nuclear resonance spectrometer system
Reexamination Certificate
2000-01-19
2002-05-28
Patidar, Jay (Department: 2862)
Electricity: measuring and testing
Particle precession resonance
Using a nuclear resonance spectrometer system
C324S309000
Reexamination Certificate
active
06396267
ABSTRACT:
This application claims Paris convention priority of European Patent Application No. 99 103 142.8 filed on Feb. 18, 1999, the complete disclosure of which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
The invention refers to a method for performing polarization transfer in a nuclear magnetic resonance (=NMR) experiment with spin systems of large molecules, especially biological macromolecules in solution, comprising at least two kinds of spin ½ nuclei I and S being coupled to each other, the spin system being subjected to a homogeneous magnetic field B
0
, being irradiated by a sequence of radio frequency (=rf) pulses comprising a first 90° pulse exciting the spins of the nuclei I and after a delay time a further 90° pulse exciting the spins of the nuclei S.
Such a method is used in the INEPT-type experiments published by Morris and Freeman, J. Am. Chem. Soc. 101, (1979) p. 760-762, describing magnetisation transfer via spin-spin couplings.
For the study of large biological macromolecules, the INEPT sequence is nowadays widely used as transfer element for heteronuclear NMR experiments. However, for molecular weights beyond 100000, the transfer time becomes a limiting factor and the INEPT sequence will fail to yield good results.
It is therefore an object of the present invention, to improve the INEPT method and provide a novel polarization transfer element which can be used as a “building block” for a great variety of complex NMR experiments including macromolecules with molecular weights far beyond 100000 and yielding higher sensitivity in comparison with methods according to the state of the art.
SUMMARY OF THE INVENTION
The objects of the present invention are achieved in that the sequence of rf pulses is chosen such that there is no inversion pulse acting on the spins of the nuclei S during a time period T between the first 90° pulse exciting the spins of the nuclei I and either the further 90° pulse exciting the spins of the nuclei S or a second 90° pulse acting on the spins of the nuclei I, and that the length of the time period T is chosen such that
d/dT
[{square root over (sin
h
+L (
R
C
T
+L )
2
+L +sin(&pgr;
J
IS
T
+L )
2
+L )} exp(−
R
I
T
)]
is minimized, where
R
C
is the transverse cross-correlation-relaxation rate of nuclei I,
R
I
is the total transverse relaxation rate of nuclei I and J
IS
is the scalar coupling constant between nuclei I and S.
Thus, the main features of the INEPT method transferring magnetization via spin-spin couplings can be combined with the advantages of cross-correlated relaxation-induced polarization transfer. This combination can be mainly achieved by the omission of the refocussing and inversion pulses during the time period T. This is, at the first glance, surprising because of the usual idea that those pulses are in any case necessary for the detection of magnetization after the application of the rf pulse sequence since during the time period T the magnetization components disperse and are hence attenuated to a large degree. However, the method according to the present invention has turned out to work anyway with large molecules, since there seems to be still enough magnetization despite the omission of a refocussing mechanism, because the inventional rf sequence more than compensates the mentioned signal losses.
In a preferred variant of the inventional method, a magnetic field gradient G
1
is applied within the time period T, allowing to eliminate artefacts.
In another preferred variant of the invention a 180° pulse acting on the nuclei I is irradiated in the middle of the time period T, thus refocussing the magnetization due to the chemical shift and selecting only the magnetization transfer by cross-correlated relaxation.
An improved version of this variant is characterized in that a magnetic field gradient G
1
is applied within the time period T/2 before the 180° pulse and another magnetic field gradient G
1
is applied within a time period T/2 after the 180° pulse, whereby artefacts can be efficiently eliminated.
In order to obtain single quantum coherence, in another variant of the inventional method the further 90° pulse exciting the spins of nuclei S is irradiated at the same time as the second 90° pulse acting on the spins of the nuclei I.
Alternatively, the inventional method can be performed such that the further 90° pulse exciting the spins of nuclei S is following up the second 90° pulse acting on the spins of the nuclei I after a time delay.
In an improved version of this variant, a magnetic field gradient G
2
being applied within the delay time between the second 90° pulse acting on the spins of the nuclei I and the further 90° pulse exciting the spins of nuclei S. This leads to the elimination of magnetization components at the end of the sequence, which are of no interest in the experiment.
In another alternative variant of the inventional method, the further 90° pulse exciting the spins of nuclei S is irradiated after the time period T following up the first 90° pulse exciting the spins of nuclei I, and the second 90° pulse acting on the spins of the nuclei I is being omitted, thus allowing to obtain zero and double quantum coherence.
Another preferred variant of the method according to the present invention is characterized in that the sequence of rf pulses comprises a 180° pulse acting on the nuclei I irradiated at the same time as the further 90° pulse exciting the spins of nuclei S after the time period T following up the first 90° pulse exciting the spins of nuclei I and that the second 90° pulse acting on the spins of nuclei I is irradiated after a second time period T following up the 180° pulse acting on the nuclei I. This allows refocussing the evolution of the magnetization due to the chemical shift.
In an improved version of this variant, a magnetic field gradient G
1
is applied within the first time period T and another magnetic field gradient G
1
is applied within the second time period T, thereby eliminating artefacts.
In a preferred variant, at the beginning of the experiment before the irradiation of the first 90° pulse exciting the spins of nuclei I a 90° pulse acting on the spins of the nuclei S is irradiated followed up by the application of a magnetic field the S-magnetization is excluded from the further evolution of spins in the system under observation.
It can be of advantage to the inventional method, when the sequence of rf pulses comprises a part adapted to suppress NMR signals of a solvent.
Also may it be of advantage, when the sequence of rf pulses comprises a part adapted to maintain the magnetization of a solvent along the homogeneous magnetic field B
0
.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In common multidimensional NMR experiments for studies of biological macromolecules in solution, magnetization transfers via spin-spin couplings (INEPT) are key elements of the pulse schemes. For molecular weights beyond 100'000, transverse relaxation during the transfer time may become a limiting factor. This invention presents a novel transfer technique for work with big molecules, called CRINEPT, which largely eliminates the size limitation of INEPT transfers with the use of cross-correlated relaxation-induced polarization transfer. The rate of polarization transfer by cross-correlated relaxation is inversely proportional to the rotational correlation time, so that it becomes a highly efficient transfer mechanism for solution NMR with very high molecular weights. As a first implementation, [
15
N,
1
H]-correlation experiments were designed that make use of cross-correlation between dipole-dipole coupling and chemical shift anisotropy of the
15
N-
1
H-moieties for both CRINEPT and TROSY. When compared with INEPT-based [
15
N,
1
H]-TROSY these new experiments yielded up to three-fold signal enhancement for amide groups of a 110000 MW protein in aqueous solution at 4° C., which has a rotational correlation time of about 70 ns. CRINEPT opens new avenues for solution NMR with supramolecular st
Pervushin Konstantin
Riek Roland
Wider Gerhard
Wuethrich Kurt
Bruker AG
Patidar Jay
Shrivastav Brij B.
Vincent Paul
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