SBIR-STTR Award

A Reliable Switched Angle Spinning (SAS) Probe with Gradients (PFG) for Proteins in Solid-State NMR : Solid-state NMR (ssNMR) biotechnology is emerging as an alternative 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 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 funding for the 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 will have fixed tuning frequencies for 1H, 13C, and 15N nuclei at 11.7 T for biological applications only. The phase-II probe will advance the technology with two broad-band low-frequency channels to accommodate other functional elements important in biology and chemistry. Additionally, the triple-channel probe will be compatible with standard three-axis gradient coils 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.

Public Health Relevance Statement:
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.

Project Terms:
Alzheimer's Disease; senile dementia of the Alzheimer type; primary degenerative dementia; dementia of the Alzheimer type; Primary Senile Degenerative Dementia; Alzheimers disease; Alzheimers Dementia; Alzheimer's; Alzheimer syndrome; Alzheimer sclerosis; Alzheimer disease; Alzheimer Type Dementia; Alzheimer; Biology; Biophysics; biophysical sciences; biophysical principles; biophysical foundation; Biotechnology; Biotech; Malignant Neoplasms; neoplasm/cancer; malignancy; Malignant Tumor; Cancers; Catalysis; Cell Nucleus; Nucleus; Chemistry; Communicable Diseases; Infectious Disorder; Infectious Diseases; Infectious Disease Pathway; Communities; Crystallization; Diabetes Mellitus; diabetes; Diffusion; Disease; Disorder; Pharmaceutical Preparations; drug/agent; Pharmaceutic Preparations; Medication; Drugs; Elements; Engineering; Environment; Face; facial; faces; Goals; Health; Industry; Integral Membrane Protein; Transmembrane Protein Gene; Transmembrane Protein; Intrinsic Membrane Protein; Laboratories; Magic; Membrane Proteins; Surface Proteins; Membrane-Associated Proteins; Membrane Protein Gene; Metabolism; Metabolic Processes; Intermediary Metabolism; Methods; Modernization; Motion; Nuclear Magnetic Resonance; Peptides; Play; Proteins; Protons; Hydrogen Ions; H+ element; Research; Research Proposals; Role; social role; Rotation; Signal Transduction; biological signal transduction; Signaling; Signal Transduction Systems; Intracellular Communication and Signaling; Cell Signaling; Cell Communication and Signaling; Solvents; Technology; Time; Amyloid beta-Protein; soluble amyloid precursor protein; beta amyloid fibril; amyloid-b protein; amyloid beta; abeta; a beta peptide; Aß; Amyloid ß-Protein; Amyloid ß-Peptide; Amyloid ß; Amyloid Protein A4; Amyloid Beta-Peptide; Amyloid Alzeheimer's Dementia Amyloid Protein; Alzheimer's amyloid; Alzheimer's Amyloid beta-Protein; Alzheimer beta-Protein; Measures; Businesses; Protein Domains; Peptide Domain; Tertiary Protein Structure; globular protein; macromolecule; improved; Area; Phase; Biological; Xray Crystallography; X-Ray/Neutron Crystallography; X-Ray Diffraction Crystallography; X Ray Crystallographies; Single Crystal Diffraction; X-Ray Crystallography; Chemicals; Failure; data base; Data Bases; Databases; Funding; Biological Function; Biological Process; Therapeutic; instrument; Mechanics; mechanical; Physiologic pulse; Pulse; exhaustion; Dimensions; Frequencies; Techniques; System; Neurodegenerative Disorders; neurodegenerative illness; degenerative neurological diseases; degenerative diseases of motor and sensory neurons; Neurologic Degenerative Conditions; Neurodegenerative Diseases; Neural degenerative Disorders; Neural Degenerative Diseases; Nervous System Degenerative Diseases; Degenerative Neurologic Disorders; Degenerative Neurologic Diseases; interest; Services; Membrane; membrane structure; Performance; solid state; structural biology; Structure; novel; drug market; Cell surface; Sampling; protein protein interaction; drug development; NMR Spectroscopy; nuclear magnetic resonance spectroscopy; NMR Spectrometer; drug discovery; RF coil; Amyloid Fibrils; protein structure; Academia; Detection; Protein Dynamics; Reproducibility; Resolution; trend; developmental; Development; pathway; Pathway interactions; virtual; designing; design; Coupling; insoluble aggregate; protein aggregate; combat; solid state NMR; SSNMR; solid state nuclear magnetic resonance; drug candidate; screening; biophysical parameters; biophysical measurement; biophysical characterization; biophysical characteristics; biophysical properties; materials science; experimental research; experiment; experimental study; imaging capabilities

A Reliable Switched Angle Spinning (SAS) Probe with Gradients (PFG) for Proteins in Solid-State NMR Solid-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.

Public Health Relevance Statement:
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.

Project Terms:
Alzheimer's Disease; AD dementia; Alzheimer; Alzheimer Type Dementia; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer's; Alzheimer's disease dementia; Alzheimers Dementia; Alzheimers disease; Primary Senile Degenerative Dementia; dementia of the Alzheimer type; primary degenerative dementia; senile dementia of the Alzheimer type; Biophysics; biophysical foundation; biophysical principles; biophysical sciences; Biotechnology; Biotech; Cell Nucleus; Nucleus; Communities; Complement; Complement Proteins; Crystallization; Diffusion; Disease; Disorder; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Elements; Engineering; Environment; Face; faces; facial; Goals; Health; Industry; Integral Membrane Protein; Intrinsic Membrane Protein; Transmembrane Protein; Transmembrane Protein Gene; Laboratories; Magic; Magnetic Resonance Imaging; MR Imaging; MR Tomography; MRI; Medical Imaging, Magnetic Resonance / Nuclear Magnetic Resonance; NMR Imaging; NMR Tomography; Nuclear Magnetic Resonance Imaging; Zeugmatography; Membrane Proteins; Membrane Protein Gene; Membrane-Associated Proteins; Surface Proteins; Methods; Motion; Peptide Hydrolases; Esteroproteases; Peptidases; Protease Gene; Proteases; Proteinases; Proteolytic Enzymes; Peptides; Phospholipids; Phosphatides; Play; Proteins; Research; Research Personnel; Investigators; Researchers; Research Proposals; Role; social role; Solvents; Technology; Temperature; Time; Roentgen Rays; X-Radiation; X-Ray Radiation; X-ray; Xray; 2,4-Dinitrophenol; 2,4-DNP; DNP; Amyloid beta-Protein; Alzheimer beta-Protein; Alzheimer's Amyloid beta-Protein; Alzheimer's amyloid; Amyloid Alzheimer's Dementia Amyloid Protein; Amyloid Beta-Peptide; Amyloid Protein A4; Amyloid ß; Amyloid ß-Peptide; Amyloid ß-Protein; Aß; a beta peptide; abeta; amyloid beta; amyloid-b protein; beta amyloid fibril; soluble amyloid precursor protein; Measures; Businesses; Tertiary Protein Structure; Peptide Domain; Protein Domains; globular protein; macromolecule; improved; Area; Solid; Phase; Biological; Physiological; Physiologic; Chemicals; Chemical Structure; Failure; Funding; Biological Process; Biological Function; Therapeutic; instrument; mechanical; Mechanics; Pulse; Physiologic pulse; Dimensions; Frequencies; Complex; Techniques; System; 3-D; 3D; three dimensional; 3-Dimensional; interest; membrane structure; Membrane; Performance; solid state; structural biology; E protein; Speed; Structure; novel; drug market; Cell surface; Modeling; Sampling; protein protein interaction; drug development; drug discovery; RF coil; Academia; Detection; device development; instrument development; Device or Instrument Development; Drug Binding Site; Motor; Protein Dynamics; Reproducibility; Resolution; Transmembrane Domain; TM Domain; Transmembrane Region; trend; Preparation; Process; Development; developmental; Pathway interactions; pathway; virtual; protein aggregation; insoluble aggregate; protein aggregate; Coupled; Coupling; solid state nuclear magnetic resonance; SSNMR; solid state NMR; drug candidate; flexibility; flexible; operation; screening; biophysical properties; biophysical characteristics; biophysical characterization; biophysical measurement; biophysical parameters; Structural Protein; experimental study; experiment; experimental research; imaging capabilities; 2019-nCoV; 2019 novel corona virus; 2019 novel coronavirus; COVID-19 virus; COVID19 virus; CoV-2; CoV2; SARS corona virus 2; SARS-CoV-2; SARS-CoV2; SARS-associated corona virus 2; SARS-associated coronavirus 2; SARS-coronavirus-2; SARS-related corona virus 2; SARS-related coronavirus 2; SARSCoV2; Severe Acute Respiratory Distress Syndrome CoV 2; Severe Acute Respiratory Distress Syndrome Corona Virus 2; Severe Acute Respiratory Distress Syndrome Coronavirus 2; Severe Acute Respiratory Syndrome CoV 2; Severe Acute Respiratory Syndrome-associated coronavirus 2; Severe Acute Respiratory Syndrome-related coronavirus 2; Severe acute respiratory syndrome associated corona virus 2; Severe acute respiratory syndrome corona virus 2; Severe acute respiratory syndrome coronavirus 2; Severe acute respiratory syndrome related corona virus 2; Wuhan coronavirus; coronavirus disease 2019 virus; hCoV19; nCoV2