The RAS Initiative Biochemistry and Biophysics Research Team uses a variety of biochemical and biophysical assays to characterize the interaction between RAS proteins and their effectors in solution or on the membrane. In addition, our researchers develop assays to identity small molecules inhibitors of RAS.

Analyzing interactions between RAS and effectors

In our previous work we have used a combination of NMR, neutron reflectivity, protein foot printing and SPR to determine the interaction of KRAS with the domains of RAF and other effector proteins on membrane mimetics.

Currently we have assays that measure the interaction between RAS and effectors (RAF, PI3K, RAL GDS), GTPase Activating Proteins and Guanine Nucleotide Exchange Factors. In addition, we use SPR and MST to validate the binding of small molecule inhibitors of RAS.

Model of KRAS fly casting
KRAS fly-casting

Uncovering a membrane-distal conformation of KRAS

Study shows a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.
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What is this? Model of KRAS fly-casting, with the G-domain as bait for the RAF1-RBD, as a mechanism to recruit RAF to the membrane. Credit: Adapted from Van et al.
Information

Our capabilities and specializations

We are constantly evaluating new approaches to target KRAS and working with our colleagues to screen new molecules for their inhibitory activity towards KRAS.

In addition, we are focused on understanding how RAS binding and membrane engagement mediate the activation of RAF. We are using a combination of biophysical methods and computational simulations to shed light on the process of RAS mediated RAF activation.

Measuring changes in biophysical properties

With our specialized tools, we measure changes in the biophysical properties (fluorescence, temperature, refractive index, and electro-magnetic) of recombinant proteins to quantitate with interaction with small molecules or other proteins.

  • Surface Plasmon Resonance Spectroscopy
  • Proximity assays: Alpha and HTRF
  • Fluorescence-based activity assays
  • Thermal shift assays
  • Microscale Thermophoresis
  • Solution state NMR
  • Neutron reflectivity

Collaborators

We collaborate with investigators who provide expertise in biophysical methodologies such as NMR (Marco Tonelli), EPR (Jason Sidabras), neutron reflectometry (Frank Heinrich), neutron scattering (Oak Ridge National Laboratory) and protein:membrane interactions (Stephen Sligar). In addition, we complement our experimental measurements with molecular dynamics simulations of KRAS and effector interaction on membranes (Lawrence Livermore National Laboratory and Los Alamos National Laboratory).

  • Theras
  • Sanofi
  • Frank Heinrich, NIST Center for Neutron Science
  • Marco Tonelli, National Magnetic Resonance Facility at Maddison
  • Jason Sidabras, Medical College of Wisconsin
  • Stephen Sligar, University of Illinois Champagne Urbana
  • Lawrence Livermore National Laboratory
  • Los Alamos National Laboratory
  • Oak Ridge National Laboratory