Applications of NMR spectroscopy to biological to biological macromolecules are limited by the low sensitivity that derives from the very small degree of polarization of spins states obtainable with current generation superconducting magnets and from the noise figures of receiver coils and pre-amplifiers. Thus, coiling both the receiving coil and pre-amplifier has long been recognized as promising increases in sensitivity, provided that the necessary cooling to approximately 20 K could be achieved without unacceptable losses in filling factor of the probe. (due to the requirement that the sample be maintained at room temperature). So-called cryogenic or cooled metal probes have in the last year become commercially available in the configurations necessary for high resolution solution NMR spectroscopy of biological macromolecules. These probes provide a three to four-fold increase in sensitivity in sensitivity compared with conventional probes for biological samples. A group of established and new investigators at Columbia University and Albert Einstein College of Medicine request funding for a Bruker Instruments CryoProbe Accessory to be installed on a shared Bruker DRX600 NMR spectrometer housed in the Columbia Presbyterian Medical Center NMR facility. Funding also is requested for a refrigerator recirculator (process chiller) necessary to cool the CryoProbe Accessory. High resolution solution NMR spectroscopy conventionally requires high sample concentrations, approaching 1 mM; isotropic enrichment of rare spins such as 13C and 15N to facilitate heteronuclear double and triple resonance experiments; and lengthy signal acquisition times, ranging from hours to approximately one week. While many applications of NMR to biological systems will benefit from the increased sensitivity of the CryoProbe accessory (resulting primarily in reduced data acquisition times or reduced amounts of sample), the present grant proposal emphasizes applications to important biological systems that are intractable without the increased sensitivity of cryoprobe because sample concentrations cannot be increased arbitrarily (membrane proteins embedded in micelles and soluble proteins encapsulated in reverse micelles), isotopic enrichment is not feasible (chemically synthesized proteins and nucleic acids) or for which temporal sample stability is limiting (enzyme systems). In each of these cases, sufficient sensitivity cannot be obtained simply by increasing acquisition times, due to practical limitations on total experimental durations.
| Valentine, Elizabeth R; Palmer 3rd, Arthur G (2005) Microsecond-to-millisecond conformational dynamics demarcate the GluR2 glutamate receptor bound to agonists glutamate, quisqualate, and AMPA. Biochemistry 44:3410-7 |
