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Request for a 900 MHz Cryoprobe with Platform

English title Request for a 900 MHz Cryoprobe with Platform
Applicant Riek Roland
Number 121270
Funding scheme R'EQUIP
Research institution Laboratorium für Physikalische Chemie D-CHAB ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Physical Chemistry
Start/End 01.09.2008 - 31.08.2009
Approved amount 260'930.00
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All Disciplines (3)

Discipline
Physical Chemistry
Molecular Biology
Biophysics

Keywords (7)

Structural Biology; NMR; Biophysics; cryoprobe; Nuclear Magnetic Resonance Spectroscopy; membrane proteins; amyloid proteins

Lay Summary (English)

Lead
Lay summary
Nuclear Magnetic Resonance Spectroscopy (NMR) is one of the principal experimental techniques in structural biology, with abilities to determine atomic resolution structures as well as to investigate dynamic features and intermolecular interactions of biological macromolecules such as proteins, enzymes, RNA and DNA at near-physiological solution conditions. The proposal requests the purchase of a low temperature detection probe (cryoprobe) that enhances the signal-to-noise two- to three-fold when compared with conventional equipment. On the one hand this signal gain enhances the capacity of the prestigious 900 MHz NMR spectrometer by four fold in measuring time making it available for more projects. On the other hand, a signal gain of two to three makes it possible to study many classes of biologically relevant proteins, which are otherwise inaccessible to be studied by conventional NMR. This includes, membrane proteins involved in the communication between cells and environment. In particular, a membrane protein that is involved in the stress response of cells will be measured with the requested cryoprobe. The proposed structural studies of this membrane protein will deepen our physical understanding of signaling through the membrane and the stress response of a cell. Similarly, the function of a potassium channel, which controls the flux of potassium through the membrane, will be studied. Potassium channels play a fundamental role in maintaining membrane excitability and in regulation of intracellular ion concentrations. Furthermore, multi-domain RNA-binding proteins involved in protein-engineering and protein regulation and their interaction with RNA will be studied to elucidate the mechanism of action of this class of proteins. In addition, amyloid proteins will be studied by NMR. Amyloid proteins are associated with neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease and it is hoped that structural studies of this class of proteins will help in the elucidation of the mechanism of toxicity of these diseases. In general, the structural studies of proteins and other biomolecules undertaken on the 900 MHz spectrometer will result in a detailed physical understanding of the function and properties thereof, which is a basis for translational and pharmaceutical research.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Associated projects

Number Title Start Funding scheme
117845 Structural Studies of Aggregates and Membrane Proteins 01.01.2008 Project funding
118118 NMR structure determination of protein-RNA complexes involved in pre-mRNA editing and translation regulation 01.10.2007 Project funding
113730 NMR in structural biology: protein structure determination and studies of protein-protein interactions 01.01.2007 Project funding

Abstract

The requested equipment is a low-temperature NMR triple-resonance probe with cryo-platform, that enhances the signal-to-noise up to a factor of 3 on the 900 MHz NMR spectrometer at the ETH. This increase in signal-to-noise is achieved by cryo-cooling of the receiver coils and preamplifier(s) by cold He gas (20 to 30 K), thereby reducing the thermal noise. The development of this so-called CryoProbe (a trademark of the company Bruker), which is available up to a magnetic field of 900 MHz 1H frequency, delivers the single largest increase in NMR sensitivity in the last few decades. This jump in sensitivity enables (i) to increase sample throughput up to one order of magnitude, (ii) data collection of sample amounts that were considered too small, and (iii) the structural and dynamical analysis of very large biomolecules and biomolecular complexes that were considered too big for NMR just a few years ago. Once in the cold state the long-lived CryoProbe can virtually be used like a conventional probe with the only drawback of considerable loss in signal-to-noise with samples of high salt-content. Signal-to-noise combined with spectral resolution is the holy grail of solution-state NMR. The NMR study of the structure, dynamics and function of biomolecules involves the measurement of hundreds or thousands of resonances of nuclear spins and their interactions. Unfortunately, many spins have similar chemical shifts yielding chemical shift degeneracy and peak overlap in the NMR spectra. Since with increasing molecular weight of the protein of interest the number of resonance lines and concomitantly the chemical shift degeneracy increases, NMR studies were limited to very small biological systems (< 10 kDa) until 15 years ago. The recent developments of spin _ labeling procedures, heteronuclear multidimensional experiments and the TROSY concept (at least for some nuclei) in combination with NMR spectrometers of higher magnetic fields resulted in a significant reduction of chemical shift degeneracy that enabled the study of large and complex biomolecular systems by NMR. The purchase at the ETH of the high-end Bruker spectrometer with 900 MHz 1H frequency must be seen in this context (note: to date the highest, commercially available NMR spectrometer runs at 950 MHz). When compared to a 600 MHz high-resolution NMR spectrometer, the 900 MHz NMR spectrometer has double the sensitivity, 50% increase in resolution along every spectral dimension (for many experiments) and an optimal use of the TROSY effect.Here, we request a triple-resonance cryoprobe for the 900 MHz NMR instrument at the ETH to achieve the highest possible sensitivity in combination with the highest resolution. The requested triple-resonance cryoprobe will be applicable to the most important nuclei for biomolecular applications (i.e. 1H, 2H, 15N, 13C). In the following we summarize the immediate benefits and major drawback of the requested cryoprobe:(i) The installation of the cryoprobe would increase the sensitivity for most samples by a factor of 2-3 and hence would reduce the time required for a particular experiment by a factor of about 4-9. With other words the purchase of the cryoprobe with a price of around 1/10 of the spectrometer itself would have a similar measurement capacity as four-nine 900 MHz spectrometers with conventional probes. In regard to the three young NMR groups with predicted increasing number of group members and the heavily booked and often over-booked 900 MHz spectrometer, the installation of the cryoprobe would be highly advantages (note: the NMR spectrometer is running non stop 24 hours 365 days a year). Furthermore, knowing that the 900 MHz NMR instrument is the only one in Switzerland, the installation of the cryoprobe would make some NMR time available for outside users in Switzerland.(ii) The anticipated increase of a factor of 2-3 in sensitivity with the cryoprobe makes it possible to measure biomolecular systems difficult to prepare at high concentrations or to produce in large quantities. Typically, on conventional probes at 900 MHz 1H frequency most NMR experiments can be carried out at concentrations in the range of 100-600 micromolar (with a volume of around 300 microliter). Thus with a cryoprobe on the 900 MHz spectrometer, highly demanding NMR experiments can be measured at concentrations of 30-200 micromolar. Less protein concentration-demanding NMR experiments (i.e. [15N,1H]- and [13C,1H]-correlation experiments) can be carried out at concentrations of a few micromolar.(iii) The NMR study of large biomolecular systems requests highest sensitivity combined with the highest magnetic field available. Because slow rotational tumbling of large biomolecular systems causes fast transverse relaxation dramatic signal loss occurs during the NMR experiments. It is therefore evident that a signal increase of a factor of 2-3 is thereby often essential for the acquisition of good quality NMR spectra. (iv) The requested cryoprobe has in addition to the cryo-cooled 1H receiver coil and preamplifier also a cryo-cooled 13C receiver coil and preamplifier making 13C direct detection possible. Although for many experiments direct 13C-detection has a lower sensitivity as compared to the equivalent 1H detection, 13C detection is advantageous in certain experiments and systems. In particular, 13C-detected experiments have been designed (for example 13C-13C-TOCSY and 13C-13C-NOESY) to study 13C nuclei without a 1H nuclei attached, which includes 13C nuclei of deuterated proteins, and to overcome fast relaxation properties of NMR experiments relying on 1H detection when studying high molecular weight proteins, proteins with paramagnetic centers, or proteins with conformational exchange dynamics. These experiments are nowadays feasible because cryogenic probes have brought 13C-detected experiments into an experimentally useful sensitivity range even for biomolecular applications. In addition, 13C-detected experiments are less sensitive to the salt concentration of the sample solution than 1H-detected experiments. (v) The main drawback of the requested cryoprobe is the inherent problem that the resistance of a salty sample is the major determinant of the signal-to-noise ratio. Several approaches have been proposed to reduce the resistance contribution of the sample: These includes the encapsulation of proteins in a water cavity formed by reverse micelles in low viscosity fluids, the optimal selection of low mobility, low conductivity buffer ions including the use of dipolar ions such as Gly and Arg, and the use of 3 mm tubes or rectangular sample tube designed to take advantage of an optimized electric field distribution inside the sample. For optimal sensitivity, we will use the rectangular tubes as well as tubes with smaller diameters in combination with a screening of the sample condition towards low conductivity buffers. Because the exchange from a cryoprope to a conventional probe and vice versa decreases the life time of the cryoprobe and takes one day of work, only in very special cases the cryoprobe will be exchanged to the conventional probe. Hence, if high conductivity/mobility buffers are unavoidable for a biomolecule of interest and it can not be concentrated enough to be measured beneficially in a 3 mm tube, the 600 or/and 750 MHz spectrometers at the ETH operating with conventional probes will be used. The 600 MHz spectrometers will also be used for the measurement of less common spin _ nuclei such as Phosphorous, Fluorine, Xenon, Thallium, for which special probes are available at the ETH. In addition, for most of these nuclei the field of 600 MHz 1H frequency is superior to 900 MHz because of the magnetic-field dependent chemical shift anisotropy. In summary, we state that the installation of a cryoprobe on the 900 MHz spectrometer at the ETH is mandatory for the continuation of a competitive research in structural biology using NMR.
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