R44GM130201

A Reliable Switched Angle Spinning (SAS) Probe with Gradients (PFG) for Proteins in Solid-State NMR AbstractSolid-state NMR (ssNMR) biotechnology is emerging as a method of choice for high-resolution structure determination for integral membrane proteins (IMPs). ssNMR provides a unique platform to investigate protein dynamics and functional studies of a wide range of biomolecules in their supramolecular assemblies. While there exists a suite of magic angle spinning (MAS) and oriented sample (OS) solid state NMR experiments for structural characterization of small- and medium-sized proteins, these methods face several challenges in larger systems. Central to the challenges are NMR sensitivity and resolution. Fast MAS and 1H detected experiments improve sensitivity but are limited by sample volume and relatively poor resolution over small isotropic chemical shift dispersion. Additionally, the efficiency of MAS experiments depends largely on through-bond and through-space coupling constants, solvent suppression, and coherence pathways selection during rotor synchronized multi-pulse applications. They also suffer from sensitivity loss due to local and global motions in proteins. On the other hand, static OS NMR experiments in membrane proteins improve resolution by measuring anisotropic shifts and heteronuclear dipolar couplings but are limited to dilute spins and low gamma 15N detection only. It has long been realized that unification of MAS and OS has the ability to widen the spectroscopic applications to large globular and membrane proteins.Switched angle spinning (SAS) probes unify MAS, dynamic angle spinning (DAS) and variable angle spinning (VAS) techniques in ssNMR, and potentially correlate isotropic and anisotropic shifts/couplings in more than one Fourier dimension. Such powerful techniques are still far from practical use, because SAS probes in the past have suffered from the lack of reliability due to hardware failures such as the survival of multi-channel rf-leads, rf coil performance including B1 field strength and homogeneity, spinning stability, and lastly rapid reorientation and accurate angle reproducibility. Technical difficulties and engineering challenges thus far have limited the probe technology to only two frequency channels.This proposal seeks Phase-II funding for the continued development of a reliable switched angle spinning probe devoid of previously encountered hardware related issues and compatible with high power pulsed-field gradients. The Phase-I probe demonstrated feasibility with fixed tuning frequencies for 1H, 13C, and 15N nuclei at 11.7 T for biological applications. The phase-II probe will advance the technology by extending the tuning capabilities in two versions, an H/X/Y SAS-PFG probe with two broad-band low-frequency channels, and a 1H/19F/X SAS-PFG probe. Additionally, these triple-channel probes will be compatible with a commercially available three-axis gradient coil in order to enable gradient enhanced spectroscopic methods, diffusion NMR, and micro-imaging capabilities in solid state. The advent of such a probe will enhance our ability to develop novel methods for NMR study of proteins and screening of therapeutic drugs.A Reliable Switched Angle Spinning (SAS) Probe with Gradients (PFG) for Proteins in Solid-State NMR Narrative: Worldwide thousands of laboratories in academia and industry use solid-state NMR, with a majority of the applications driven by the need for structure and function determination in biological macromolecules. The availability of a highly novel add-on accessory (an SAS-PFG probe) for existing ssNMR systems that can dramatically improve the quality of the needed information, and obtain that information quickly, can be transformative in certain areas of health-related research, including development of drugs for Alzheimer’s Disease. The instrument that will come from this proposed effort is expected to provide a valuable business opportunity for the company, with ripple effects extending throughout its community.