Cryoprobe Upgrades for 500 &600 MHz NMR Spectrometers

King, Glenn

Abstract

The NMR structural biology program at UConn Health Center (UCHC) is undergoing a major expansion of its user base. In order to sustain this expansion we need to solve two major problems: (i) time on the instruments is now at a premium and projects are often delayed because of limited spectrometer time; (ii) as users pursue more cutting-edge projects, the instruments are being challenged with macromolecules that are larger, more insoluble, or in other ways intractable with conventional probe technology. In order to ameliorate these problems, we are requesting cryoprobe upgrades for the 500 and 600 MHz spectrometers in the NMR Structural Biology Facility at UCHC. The NMR Structural Biology Facility at UCHC comprises 500 and 600 MHz Varian spectrometers as well as an associated Computational Facility with a bank of Linux computers containing a suite of software for NMR data analysis and calculation/analysis of macromolecular structures. The Facility is supported by funds from UCHC and by user fees, and is maintained by a full-time Facility Manager. There are currently eight major users who are listed here as participating investigators. However, there is a queue of new investigators wanting to undertake structure determinations, and each new user will require 6-8 weeks of spectrometer time per project. While the UCHC spectrometers are superbly maintained and have excellent signal-to-noise (S/N) for their vintage, they are now eight years old and badly in need of a cryoprobe upgrade in order to sustain cutting-edge structural biology research programs. Cryoprobe technology, which affords a 5-fold increase in S/N compared to conventional probes, will alleviate many of the problems that UConn investigators currently face: (i) large macromolecules that are poorly soluble or tend to aggregate will be amenable to study, since the enhanced sensitivity of cryoprobes allows for data collection at concentrations as low as 0.15 mM (c.f. ? 0.8 mM on conventional probes); (ii) several UConn groups study protein folding. Cryoprobes allow partially folded proteins that aggregate at high concentration to be studied at concentrations as low as 0.05 mM; (iii) proteins available in only small quantities, such as those isolated from natural sources, can be structurally characterized; (iv) the time savings afforded by cryoprobes will make possible the study of several proteins with limited stability; (v) for proteins that are soluble at reasonable concentrations (> 0.8 mM), data can be acquired much faster on a cryoprobe, thus allowing a larger group of users to regularly access the facility. Thus, addition of cryoprobes to the UCHC NMR Facility would facilitate continued expansion of the user base and allow users to pursue a range of cutting-edge research programs that would be impossible using conventional probes.