Our staff maintains and operates a series of sophisticated biophysical instrumentation. Among the analytical methods available to intramural researchers are: analytical ultracentrifugation, isothermal titration calorimetry, surface plasmon resonance, differential scanning calorimetry, dynamic light scattering, circular dichroism spectroscopy, and asymmetric flow field-flow fractionation. In order to gain insight into the formation of signalling clusters in T-cell activation, our resource, in collaboration with NCI and the NIBIB, has applied analytical ultracentrifugation (AUC) and isothermal titration calorimetry (ITC) toward characterizing the binding of the SH2 domain of SLP76 to specific peptide fragments of ADAP having controlled ratios of phosphorylation at 2 distant binding-site tyrosine residues. In collaboration with the NICHD, 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. In collaboration with two different labs within the NIAID, we have utilized sedimentation equilibrium and sedimentation velocity approaches of AUC to investigate the association state of various secondary amyloidosis protein (SAA) constructs, and to probe the assembly state of malaria merozoite surface protein 3 (MSP3).
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