Electricity: measuring and testing – Particle precession resonance – Using a nuclear resonance spectrometer system
Reexamination Certificate
2003-07-11
2004-12-14
Gutierrez, Diego (Department: 2859)
Electricity: measuring and testing
Particle precession resonance
Using a nuclear resonance spectrometer system
C324S310000
Reexamination Certificate
active
06831459
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of using G-matrix Fourier transformation nuclear magnetic resonance (GFT NMR) spectroscopy for rapidly obtaining and connecting precise chemical shift values and determining the structure of proteins and other molecules.
BACKGROUND OF THE INVENTION
Nuclear magnetic resonance (NMR) (Ernst et al.,
Principles of Nuclear Magnetic Resonance in One and Two Dimensions
, Clarendon, Oxford (1987); Wüthrich,
NMR of Proteins and Nucleic Acids
, Wiley, New York (1986); Cavanagh et al.,
Protein NMR Spectroscopy
, Academic Press, San Diego (1996))-based structural studies rely on two broad classes of experimental radio-frequency pulse schemes for recording two-dimensional (2D) (Ernst et al.,
Principles of Nuclear Magnetic Resonance in One and Two Dimensions
, Clarendon, Oxford (1987)), three-dimensional (3D) (Oschkinat et al.,
Nature
, 332:374-376 (1988)), or four-dimensional (4D) (Kay et al.,
Science
, 249:411-414 (1990)) Fourier transformation (FT) NMR spectra. Correlation spectroscopy (COSY) delineates exclusively scalar coupling connectivities to measure chemical shifts, and (heteronuclear resolved)
1
H,
1
H-nuclear Overhauser enhancement spectroscopy (NOESY) reveals the strength of through-space dipolar couplings of
1
H spins to estimate distances (Ernst et al.,
Principles of Nuclear Magnetic Resonance in One and Two Dimensions
, Clarendon, Oxford (1987); Wüthrich,
NMR of Proteins and Nucleic Acids
, Wiley, New York (1986)). NMR spectra need to exhibit (i) signal-to-noise (S/N) ratios warranting reliable data interpretation, (ii) digital resolutions ensuring adequate precision for the measurement of NMR parameters such as chemical shifts, and (iii) a dimensionality at which a sufficient number of NMR parameters is correlated (Ernst et al.,
Principles of Nuclear Magnetic Resonance in One and Two Dimensions
, Clarendon, Oxford (1987); Cavanagh et al.,
Protein NMR Spectroscopy
, Academic Press, San Diego (1996)). While increased intensity of NOESY peaks ensures their more accurate integration (which, in turn, may translate into increased accuracy of the NMR structure), the mere identification of COSY peaks suffices to obtain the desired chemical shifts. Hence, COSY peak signal-to noise ratios larger that ~3:1 reflect, in essence, inappropriately long measurement times. Moreover, the total number of peaks in COSY grows only linearly with the number of spins involved and is, for a defined magnetization transfer pathway, “independent” of the dimensionality N. Thus, a minimal “target dimensionality” N
t
at which most of the COSY peaks detected for a given molecule are resolved can be defined. Further increased dimensionality does not aim at resolving peak overlap but at increasing the number of correlations obtained in a single data set. This eliminates ambiguities when several multidimensional NMR spectra are combined for resonance assignment, for example, when using
1
H,
13
C,
15
N triple-resonance NMR to assign protein resonances (Cavanagh et al.,
Protein NMR Spectroscopy
, Academic Press, San Diego (1996)).
An increase in dimensionality is, however, limited by the need to independently sample the indirect dimensions, because this leads to longer measurement times. Although the measurement time can be somewhat reduced by aliasing signals (Cavanagh et al.,
Protein NMR Spectroscopy
, Academic Press, San Diego (1996)) or accepting a lower digital resolution in the indirect dimensions, high dimensionality often prevents one from tuning the measurement time to a value that ensures to obtain sufficient, but not unnecessarily large S/N ratios.
In view of these considerations, “sampling” and “sensitivity limited” data collection regimes are defined (Szyperski et al.,
Proc. Natl. Acad. Sci. USA
, 99:8009-8014 (2002)), depending on whether the sampling of the indirect dimensions or the sensitivity of the FT NMR experiment determines the minimal measurement time. In the sensitivity limited regime, long measurement times are required to achieve sufficient S/N ratios, so that the sampling of indirect dimensions is not necessarily constraining the adjustment of the measurement time. In the sampling limited regime, some or even most of the instrument time is invested for sampling, which yields excessively large S/N ratios. In view of the ever increasing sensitivity of NMR instrumentation, new methodology to avoid the sampling limited regime is needed. (Szyperski et al.,
Proc. Natl. Acad. Sci. USA
, 99:8009-8014 (2002)).
In general, phase-sensitive acquisition of an N-dimensional (ND) FT NMR experiment (Ernst et al.,
Principles of Nuclear Magnetic Resonance in One and Two Dimensions
, Clarendon, Oxford (1987); Cavanagh et al.,
Protein NMR Spectroscopy
, Academic Press, San Diego (1996)) requires sampling of N−1 indirect dimensions with n
1
×n
2
. . . n
N−1
complex points representing
n
FID
=
2
N
-
1
·
∏
j
=
1
N
-
1
n
j
free induction decays (FIDs). The resulting steep increase of the minimal measurement time, T
m
, with dimensionality prevents one from recording five- or higher-dimensional FT NMR spectra: acquiring 16 complex points in each indirect dimension (with one scan per FID each second) yields T
m
(3D)=0.5 hour, T
m
(4D)=9.1 hours, T
m
(5D)=12 days, and T
m
(6D)=1.1 years.
Thus, higher-dimensional FT NMR spectroscopy suffers from two major drawbacks: (i) The minimal measurement time of an ND FT NMR experiment, which is constrained by the need to sample N−1 indirect dimensions, may exceed by far the measurement time required to achieve sufficient signal-to-noise ratios. (ii) The low resolution in the indirect dimensions severely limits the precision of the indirect chemical shift measurements.
The present invention is directed to overcoming the deficiencies in the art.
SUMMARY OF THE INVENTION
The present invention relates to a method of conducting a (N,N−K) dimensional (D) G-matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiment, where N is the dimensionality of an N-dimensional (ND) Fourier transformation (FT) NMR experiment and K is the desired reduction in dimensionality relative to N. The method involves providing a sample and applying radiofrequency pulses for the ND FT NMR experiment to the sample. Then, m indirect chemical shift evolution periods of the ND FT NMR experiment are selected, where m equals K+1, and the m indirect chemical shift evolution periods are jointly sampled. Next, NMR signals detected in a direct dimension are independently cosine and sine modulated to generate (N−K)D basic NMR spectra containing frequency domain signals with 2
K
chemical shift multiplet components, thereby enabling phase-sensitive sampling of all jointly sampled m indirect chemical shift evolution periods. Finally, the (N−K) D basic NMR spectra are transformed into (N−K) D phase-sensitively edited basic NMR spectra, where the 2
K
chemical shift multiplet components of the (N−K) D basic NMR spectra are edited to yield (N−K) D phase-sensitively edited basic NMR spectra having individual chemical shift multiplet components.
Another aspect of the present invention relates to a method for sequentially assigning chemical shift values of an &agr;-proton,
1
H
&agr;
, an &agr;-carbon,
13
C
&agr;
, a polypeptide backbone carbonyl carbon,
13
C′, a polypeptide backbone amide nitrogen,
15
N, and a polypeptide backbone amide proton,
1
H
N
, of a protein molecule. The method involves providing a protein sample and conducting a set of G matrix Fourier transformation (GFT) nuclear magnetic resonance (NMR) experiments on the protein sample including: (1) a (5,2)D [
HACACON
HN] GFT NMR experiment to measure and connect the chemical shift values of the &agr;-proton of amino acid residue i−1,
1
H
&agr;
i−1
, the &agr;-carbon of amino acid residue i−1,
13
C
&agr;
i−1
, the polypeptide backbone carbonyl carbon of amino acid residue i&min
Atreya Hanudatta S.
Kim Seho
Szyperski Thomas A.
Nixon & Peabody LLP
The Research Foundation of State University of New York
Vargas Dixomara
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