Our staff maintains and operates a battery of sophisticated solution-based biophysical instrumentation. Among the analytical methods available to intramural researchers are: analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), differential scanning calorimetry (DSC), dynamic light scattering (DLS), circular dichroism spectroscopy (CD), and asymmetric flow field-flow fractionation (AF4). In collaboration with the NICHD and investigators within the NIBIB, analytical ultracentrifugation sedimentation velocity has been used to quantify the strength of high-affinity (low nanomolar Kd) self-association for the amino terminal domain (ATD) of AMPA subtype glutamate receptor ion channels, major mediators of fast excitatory synaptic transmission in the human brain. This work was published in the Journal of General Physiology. Ultra-small gold nanoparticles are especially attractive in applications requiring delivery to crowded intracellular spaces in the cytosol and nucleus of cells. In collaboration with the Laboratory of Cellular Imaging and Macromolecular Biophysics of the NIBIB, we have used AUC and DLS to characterize the size-distribution of MBA and GSH stabilized ultrasmall gold particles. This work was published in the journal Small. Avastin (Bevacizumab) is a monocolnal antibody and the first clinically available angiogenesis inhibitor in the US. First approved in 2004 by the U.S. Food and Drug Administration (FDA) for metastatic colon cancer and non-small cell lung cancer, and is also often used to treat wet AMD. Many AMD patients treated with Avastin experience increased ocular pressure and pain post-injection. Current research by many groups suggests that antibody aggregation is responsible and the groups are exploring methods for quantification of trace amounts of aggregates as well as potential causes. In a project with investigators within the NEI, we have employed AUC and dynamic light scattering to detect and quantify trace aggregates present in several Avastin samples that have been handled in various ways to look for sources of aggregation. In collaboration with investigators within the NIAID, the QMMI unit was able to confirm the predicted secondary structure of a synthetic peptide fragment of gp120. We were further able to measure interactions of this fragment with soluble CD4 construct. The work work has application in the treatment of HIV patients and an article is in preparation.

Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2012
Total Cost
$245,709
Indirect Cost
Name
National Institute of Biomedical Imaging and Bioengineering
Department
Type
DUNS #
City
State
Country
Zip Code
Jensen, Jaime L; Balbo, Andrea; Neau, David B et al. (2015) Mechanistic Implications of the Unique Structural Features and Dimerization of the Cytoplasmic Domain of the Pseudomonas Sigma Regulator, PupR. Biochemistry 54:5867-77
Zhao, Huaying; Balbo, Andrea; Metger, Howard et al. (2014) Improved measurement of the rotor temperature in analytical ultracentrifugation. Anal Biochem 451:69-75
May, Nathan A; Wang, Qiuhong; Balbo, Andrea et al. (2014) Human herpesvirus 7 U21 tetramerizes to associate with class I major histocompatibility complex molecules. J Virol 88:3298-308
Ghirlando, Rodolfo; Zhao, Huaying; Balbo, Andrea et al. (2014) Measurement of the temperature of the resting rotor in analytical ultracentrifugation. Anal Biochem 458:37-9
Zhao, Huaying; Berger, Anthony J; Brown, Patrick H et al. (2013) Analysis of high-affinity assembly for AMPA receptor amino-terminal domains. J Gen Physiol 141:747-9
Ghirlando, Rodolfo; Balbo, Andrea; Piszczek, Grzegorz et al. (2013) Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references. Anal Biochem 440:81-95
Coussens, Nathan P; Hayashi, Ryo; Brown, Patrick H et al. (2013) Multipoint binding of the SLP-76 SH2 domain to ADAP is critical for oligomerization of SLP-76 signaling complexes in stimulated T cells. Mol Cell Biol 33:4140-51
Sousa, Alioscka A; Morgan, Jeffrey T; Brown, Patrick H et al. (2012) Synthesis, characterization, and direct intracellular imaging of ultrasmall and uniform glutathione-coated gold nanoparticles. Small 8:2277-86
Zhao, Huaying; Berger, Anthony J; Brown, Patrick H et al. (2012) Analysis of high-affinity assembly for AMPA receptor amino-terminal domains. J Gen Physiol 139:371-88
Zhao, Huaying; Brown, Patrick H; Schuck, Peter (2011) On the distribution of protein refractive index increments. Biophys J 100:2309-17

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