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). Accuracy and precision in AUC are critical to properly characterize protein samples. Our collaboration with Peter Schuck and Huaying Zhao resulted in the development of new devices to monitor temperature measurements of the rotor and to make accurate determinations of the radial magnification of the optic systems. 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 continue to use AUC and DLS techniques to characterize the size-distribution of MBA and GSH stabilized ultrasmall gold particles in buffers with and without Fetal Bovine Serum (FBS). Using AUC, DLS and ITC, we continue to explore the size, shape and self association properties of various protein systems. Collaborative, ongoing projects with NIAID led to exploratory AUC work on oligomeric states of wild type and mutant polypeptide constructs of NSP5, helped characterize individual proteins such as DBL2x, PfCSA-pcm2 and their mixtures to answer questions regarding high affinity binding between these species and enabled continued investigations into the study of the different oligormeric properties of several forms of serum amyloid A (SAA). In collaboration with NHGRI, ITC experiments will help elucidate the binding and dissociation properties of a small molecule, Ro5-3335 to various protein complexes. Once these conditions are worked out, several analogs of Ro5-3335we will be studied. Affinity measurements by SPR using the Biacore 3000 are in progress to study binding epitopes and affinities between the homologous and wild type HBV-core antigen produced from HBV -ALF (Hepatitis B virus-associated acute liver failure) and germline anti-core IgM and IgG antibodies that may play a major role in the pathogenesis of HBV-associated ALF.

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Project End
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Budget End
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Biomedical Imaging & Bioengineering
Department
Type
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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; Magone, M Teresa et al. (2011) The molecular refractive function of lens ?-Crystallins. J Mol Biol 411:680-99

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