Structure of the full-length TRPV2 channel by cryo-EM.

Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels

Purification, crystallization and structure determination of native GroEL from Escherichia coli lacking bound potassium ions

A 3.02 Å crystal structure of native GroEL from E. coli is presented

Evaluating mass spectrometry based hydroxyl radical protein foot-printing of a benchtop Flash oxidation system against a synchrotron X-ray beamline.

Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation and mass spectrometry is a well-validated structural biology method that is providing critical insights into macromolecular dynamics. Numerous alternative sources for HRPF such as laser photolysis and plasma irradiation complement synchrotron-based HRPF. A recently developed commercially available instrument based on flash lamp photolysis, the Fox® system, enables access to laboratory benchtop HRPF. Here, we evaluate the feasibility of standardizing HRPF experiments in-house with a benchtop Fox® instrument as a precursor to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase enzyme (LOx) as a model system, we carried out hydroxyl radical (•OH) labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed with high glucose concentrations to mimic highly scavenging conditions in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the Fox® and XFP HRPF methods was compared, and we found that tuning •OH dosage enabled an optimum labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of Fox® and XFP labeling approaches, showing that benchtop instruments such as Fox® photolysis system can increase throughput and accessibility of HRPF technology

Structure of the full-length TRPV2 channel by cryo-EM.

Transient receptor potential (TRP) proteins form a superfamily Ca(2+)-permeable cation channels regulated by a range of chemical and physical stimuli. Structural analysis of a 'minimal' TRP vanilloid subtype 1 (TRPV1) elucidated a mechanism of channel activation by agonists through changes in its outer pore region. Though homologous to TRPV1, other TRPV channels (TRPV2-6) are insensitive to TRPV1 activators including heat and vanilloids. To further understand the structural basis of TRPV channel function, we determined the structure of full-length TRPV2 at ∼5 Å resolution by cryo-electron microscopy. Like TRPV1, TRPV2 contains two constrictions, one each in the pore-forming upper and lower gates. The agonist-free full-length TRPV2 has wider upper and lower gates compared with closed and agonist-activated TRPV1. We propose these newly revealed TRPV2 structural features contribute to diversity of TRPV channels

Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanisms

Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanism

Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X‑ray Beamline

Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation (XFP) and mass spectrometry is a well-validated structural biology method that provides critical insights into macromolecular structural dynamics, such as determining binding sites, measuring affinity, and mapping epitopes. Numerous alternative sources for generating the hydroxyl radicals (•OH) needed for HRPF, such as laser photolysis and plasma irradiation, complement synchrotron-based HRPF, and a recently developed commercially available instrument based on flash lamp photolysis, the FOX system, enables access to laboratory benchtop HRPF. Here, we evaluate performing HRPF experiments in-house with a benchtop FOX instrument compared to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase (LOx) as a model system, we carried out •OH labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed under high glucose concentrations to mimic the highly scavenging conditions present in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the FOX and XFP HRPF methods was compared, and we found that tuning the •OH dosage enabled optimal labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of FOX and XFP labeling approaches, demonstrating that benchtop instruments such as the FOX photolysis system can increase both the throughput and the accessibility of the HRPF technique

A Novel Molecular Diagnostic of Glioblastomas: Detection of an Extracellular Fragment of Protein Tyrosine Phosphatase µ12

We recently found that normal human brain and low-grade astrocytomas express the receptor protein tyrosine phosphatase mu (PTPµ) and that the more invasive astrocytomas, glioblastoma multiforme (GBM), downregulate full-length PTPµ expression. Loss of PTPµ expression in GBMs is due to proteolytic cleavage that generates an intracellular and potentially a cleaved and released extracellular fragment of PTPµ. Here, we identify that a cleaved extracellular fragment containing the domains required for PTPµ-mediated adhesion remains associated with GBM tumor tissue. We hypothesized that detection of this fragment would make an excellent diagnostic tool for the localization of tumor tissue within the brain. To this end, we generated a series of fluorescently tagged peptide probes that bind the PTPµ fragment. The peptide probes specifically recognize GBM cells in tissue sections of surgically resected human tumors. To test whether the peptide probes are able to detect GBM tumors in vivo, the PTPµ peptide probes were tested in both mouse flank and intracranial xenograft human glioblastoma tumor model systems. The glial tumors were molecularly labeled with the PTPµ peptide probes within minutes of tail vein injection using the Maestro FLEX In Vivo Imaging System. The label was stable for at least 3 hours. Together, these results indicate that peptide recognition of the PTPµ extracellular fragment provides a novel molecular diagnostic tool for detection of human glioblastomas. Such a tool has clear translational applications and may lead to improved surgical resections and prognosis for patients with this devastating disease