Biomolecular Interactions and Solution Dynamics

 Biomolecular Interactions

The Biomolecular Research Facility has a BIACore 3000   instrument to measure interactions between molecules using Surface Plasmon Resonance. In this technique, a protein or other molecule is bound to a modified gold surface. The protein or other molecule which may bind to the immobilized protein is passed over the immobilized protein. If the two molecules bind, the optical properties of the gold surface change.

This instrument can be used to

  • determine if proteins bind to each other
  • determine which domains of proteins are involved in binding
  • measure affinities and rates of binding between a protein and other proteins, DNA, lipids, small molecules
  • isolate a protein binding partner which is then identified by mass spectrometry
  • epitope mapping
  • determine the concentration of protein with binding activity

Surface plasmon resonance studies of protein binding allow use of native proteins, avoiding the use of radioisotopes or other labels. The sensitivity of the technique is greater than immune precipitation and can study weak interactions, including fast dissociations. Biacore provides an explanation of the technique on their web site at:
http://www.biacore.com/lifesciences/technology/introduction/data_interaction/index.html

Some data obtained here:

  • The binding constant between an avian leukosis virus protein and its cellular receptor was measured at 3 pM. Because of the tight binding, standard conditions had to be modified to give an accurate reading. See: Sue E. Delos, Jesse A. Godby, and Judith M. White. Receptor-Induced Conformational Changes in the SU Subunit of the Avian Sarcoma/Leukosis Virus A Envelope Protein: Implications for Fusion Activation. J Virol. 2005 March; 79(6) : 3488–3499.
  • Rate of binding and desorption between lipids and annexin.
  • Binding kinetics between antibody and protein
  • Isolation of protein from mouse egg cells for identification by mass spectrometry
  • Binding rates and affinity between zizimin and FLG protein

 Finding suitable conditions observe interactions may require considerable investigation prior to the actual experiment. Contact Dr. John Shannon ( 243-9399) about experiments using this instrument. Successful experiments require homogeneous proteins and considerable planning. For background information on the technique, go to the BiaCore home page.

Circular dichroism

The facility has a AVIV 215 spectropolarimeter for spectral investigations of protein tertiary structure. This instrument is available for use by investigators, with limited support by the facility.

Dynamic light scattering

The facility has a Protein Solutions dynamic light scattering instrument for estimating solution molecular weights in the range 14,000 to over 150,000.

Dr. John Shannon (phone 3-9399 jds1c@virginia.edu) is in charge of the instruments listed above.

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