Fast multidimensional NMR with a time resolution of a few seconds provides a new tool for high throughput screening and site-resolved real-time studies of kinetic molecular processes by NMR. Recently we have demonstrated the feasibility to record protein 1H-15N correlation spectra in a few seconds of acquisition time using a new SOFAST-HMQC experiment (Schanda and Brutscher (2005) J. Am. Chem. Soc. 127, 8014). Here, we investigate in detail the performance of SOFAST-HMQC to record 1H-15N and 1H-13C correlation spectra of proteins of different size and at different magnetic field strengths. Compared to standard 1H-15N correlation experiments SOFAST-HMQC provides a significant gain in sensitivity, especially for fast repetition rates. Guidelines are provided on how to set up SOFAST-HMQC experiments for a given protein sample. In addition, an alternative pulse scheme, IPAP-SOFAST-HMQC is presented that allows application on NMR spectrometers equipped with cryogenic probes, and fast measurement of one-bond 1H-13C and 1H-15N scalar and residual dipolar coupling constants.

No abstract

The speed of multidimensional NMR spectroscopy can be significantly increased by drastically shortening the customary relaxation delay between scans. The consequent loss of longitudinal magnetization can be retrieved if 'new' polarization is transferred from nearby spins. For correlation spectroscopy involving heteronuclei (X=13C or 15N), protons not directly bound to X can repeatedly transfer polarization to the directly bound protons through Hartmann-Hahn mixing. An order of magnitude increase in speed has been observed for the 600 MHz two-dimensional HMQC spectra of amikacin and strychnine using this technique, and it also reduces the noisy F1 ridges that degrade many heteronuclear correlation spectra recorded with short recovery times.

Two-dimensional NMR spectroscopy can be speeded up by orders of magnitude by severely restricting the number of sampling operations in the evolution dimension-we demonstrate that just a single measurement may suffice. The frequencies evolving in the indirect dimension (t 1 ) are deduced from the amplitudes of the signals acquired in the direct dimension (t 2 ). Prior measurements of the one-dimensional spectra are required. Results are presented for the two-dimensional 13 C-HSQC spectrum of 2-ethylindanone recorded at a single fixed setting of the evolution time, demonstrating a speed advantage of 120. The method can be extended to multidimensional spectra, with correspondingly greater gains in speed. Copyright There have been many recent attempts to speed up multidimensional NMR spectroscopy by severely limiting the number of sampling operations in the evolution dimensions either by the filter diagonalization method, 1 G-matrix Fourier transform, 2 Hadamard encoding, 3 projection-reconstruction, 4 or simply by sparse sampling. 5 We propose a new method that employs minimal sampling of evolution space, employing in the limit only a single value of t 1 . It relies on prior knowledge of the relevant chemical shifts from one-dimensional spectra. The method is quite general, but for clarity of presentation a simple two-dimensional example is described here. Suppose that 13 C spins are allowed to evolve during t 1 with direct proton detection during t 2 , as in the standard HSQC scheme. It is well known that the 13 C frequencies are encoded as amplitude modulation of the coupled proton signals. The proposed speed advantage stems from the realization that all the essential information can be obtained at a single, fixed value of the evolution time t 1 Ł . Contrary to common belief, there is no need for step-by-step exploration of an entire array of t 1 values. This translates into a reduction in total experiment time of 1 or 2 orders of magnitude for a two-dimensional spectrum, and much more if additional dimensions are involved.The proton free induction signals are acquired with broadband 13 C decoupling, giving, after Fourier transformation, a single peak from each chemically distinct proton site. We assume there is an adequate signal-to-noise ratio in the proton spectrum. As in the standard HSQC sequence Ł Correspondence to: Ray Freeman, Jesus College, Cambridge CB5 8BL, UK. E-mail: rf110@hermes.cam.ac.uk (with refocused scalar coupling during t 1 ), the phase of the 13 C coherence transfer pulse is alternated between 0°and 90°, generating the proton amplitudes A 0 and A 90 . These are used to define a phase angle H D arctan A 0 /A 90 , which in turn determines the correlated 13 C frequency in the indirect dimension:C D H / 2 t 1 Ł Hz on the basis that H D 0 at t 1 D 0. The fixed evolution delay t 1 Ł is normally a relatively short value. Allowance is made (via the Bloch equations) for any small offsetdependent phase shifts due to off-resonance nutation during the radiofrequency pulses.The next step is to measure...

The acquisition of multidimensional NMR spectra can be speeded up by a large factor by a projection-reconstruction method related to a technique used in X-ray scanners. The information from a small number of plane projections is used to recreate the full multidimensional spectrum in the familiar format. Projections at any desired angle of incidence are obtained by Fourier transformation of time-domain signals acquired when two or more evolution intervals are incremented simultaneously at different rates. The new technique relies on an established Fourier transform theorem that relates time-domain sections to frequency-domain projections. Recent developments in NMR instrumentation, such as increased resolution and sensitivity, make fast methods for data gathering much more practical for protein and RNA research. Hypercomplex Fourier transformation generates projections in symmetrically related pairs that provide two independent "views" of the spectrum. A new reconstruction algorithm is proposed, based on the inverse Radon transform. Examples are presented of three- and four-dimensional NMR spectra of nuclease A inhibitor reconstructed by this technique with significant savings in measurement time.