Measurement of coiled-coil stability by fluorescence resonance energy transfer

POSTER

Abstract

Alpha helical coiled-coils are common structural motifs by which proteins assemble into larger complexes. The leucine zipper-like oligomerization domain of heterogeneous nuclear ribonucleoprotein C (CLZ) assembles into an anti-parallel tetramer with a coiled-coil fold. Fluorescently-tagged CLZ peptides were designed and characterized as appropriate to perform measurements using fluorescence resonance energy transfer (FRET) to study the overall stability of the assembly. Experiments revealed that the appearance of FRET in a sample was time dependent and thus reflective of the kinetics of the tetramer's formation. Furthermore, FRET assays using varying concentrations of labeled peptides have allowed the first measurement of the equilibrium dissociation constant (K$_{D})$ of the CLZ tetramer, which is representative of the strength of specific molecular interactions within the oligomer. The method can be used to study effects of mutations in this coiled coil on the thermodynamic stability of the complex.

Authors

  • James Pino

    Departments of Chemistry and Physics, University of Tennessee at Chattanooga

  • Chris Neu

    Florida State University, NHMFL, Institut Neel, Institute of Semiconductors, Chinese Academy of Sciences, Beijing, China, Florida State University, Department of Physics, Florida State University, Tallahassee, FL, Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, Florida State University, National High Magnetic Field Laboratory, Institute of Physics, Chinese Academy of Sciences, China, Austin Peay State University, University of Pardubice, Davidson College, Covenant College, Lookout Mtn, GA 30750, Russian Academy of Sciences, Moscow, RU, University of Tennessee Space Institute, Tullahoma, TN, USA, Sandia National Laboratories,* Albuquerque, NM, Arkansas State University, Jonesboro, AK, None, Mississippi State University, Mississippi State University Department of Physics and Astronomy, Florida State University and the National High Magnetic Field Laboratory, Faculte des Sciences et Techniques and Universite Aix-Marseille, MIT Haystack Observatory, Departments of Chemistry and Physics, University of Tennessee of Chattanooga, Departments of Chemistry and Physics, University of Tennessee at Chattanooga, Western Kentucky University, Thomas Jefferson National Accelerator Facility, Newport News, VA, Fermilab, University of Virginia and Fermilab, Indiana University Bloomington, Indiana University Health Proton Therapy Center, University of North Florida, National Superconducting Cyclotron Laboratory, Michigan State University, Flordia State University, Louisiana State University, Washington University, Argonne National Laboratory, Lawrence Berkeley National Laboratory, Department of Physics, Florida State University., JINR, Tsinghua University, LBNL, LBNL/Vanderbilt University, Vanderbilt University, Florida A\&M University, Georgia Institute of Technology, Department of Physics, Virginia Tech, Gatton Academy, Exeter University, Florida International University, Office of Research, University of North Florida, Physics Department, University of North Florida, NSCL, Michigan State University, Physics Department, Florida State University, University of Tennessee-Knoxville, Neel Institut, Kazan Federal University, Bielefeld University, Ben Gurion University, LCIB - CEA, University of Georgia, Lawrence Livermore National Laboratory, Livermore, CA 94550, NHMFL, Tallahassee, USA, Department of Chemistry, University of Waterloo, Ontario, Canada, NHMFL, Tallahassee FL 32310 USA, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany, Wigner Research Center for Physics, Budapest, Hungary, Experimental physics II, University of Augsburg, Germany, Materials Science Division, Argonne National Laboratory, Argonne, Illinois 60439, National High Magnetic Field Laboratory Florida State University, Tallahassee, Florida 32310, IM2NP-CNRS (UMR 7334) and Universite Aix-Marseille, National High Magnetic Field Laboratory Applied Superconductivity Center, Heifei National Lab for Physical Science at Microscale, USTC, Institute of Physics, Chinese Academy of Sciences, Department of Electrical Engineering, Rice University, National High Magnetic Field Laboratory, National High Magnetic Field Laboratory/FSU, University of Virginia

  • Chris Neu

    Florida State University, NHMFL, Institut Neel, Institute of Semiconductors, Chinese Academy of Sciences, Beijing, China, Florida State University, Department of Physics, Florida State University, Tallahassee, FL, Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, Florida State University, National High Magnetic Field Laboratory, Institute of Physics, Chinese Academy of Sciences, China, Austin Peay State University, University of Pardubice, Davidson College, Covenant College, Lookout Mtn, GA 30750, Russian Academy of Sciences, Moscow, RU, University of Tennessee Space Institute, Tullahoma, TN, USA, Sandia National Laboratories,* Albuquerque, NM, Arkansas State University, Jonesboro, AK, None, Mississippi State University, Mississippi State University Department of Physics and Astronomy, Florida State University and the National High Magnetic Field Laboratory, Faculte des Sciences et Techniques and Universite Aix-Marseille, MIT Haystack Observatory, Departments of Chemistry and Physics, University of Tennessee of Chattanooga, Departments of Chemistry and Physics, University of Tennessee at Chattanooga, Western Kentucky University, Thomas Jefferson National Accelerator Facility, Newport News, VA, Fermilab, University of Virginia and Fermilab, Indiana University Bloomington, Indiana University Health Proton Therapy Center, University of North Florida, National Superconducting Cyclotron Laboratory, Michigan State University, Flordia State University, Louisiana State University, Washington University, Argonne National Laboratory, Lawrence Berkeley National Laboratory, Department of Physics, Florida State University., JINR, Tsinghua University, LBNL, LBNL/Vanderbilt University, Vanderbilt University, Florida A\&M University, Georgia Institute of Technology, Department of Physics, Virginia Tech, Gatton Academy, Exeter University, Florida International University, Office of Research, University of North Florida, Physics Department, University of North Florida, NSCL, Michigan State University, Physics Department, Florida State University, University of Tennessee-Knoxville, Neel Institut, Kazan Federal University, Bielefeld University, Ben Gurion University, LCIB - CEA, University of Georgia, Lawrence Livermore National Laboratory, Livermore, CA 94550, NHMFL, Tallahassee, USA, Department of Chemistry, University of Waterloo, Ontario, Canada, NHMFL, Tallahassee FL 32310 USA, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany, Wigner Research Center for Physics, Budapest, Hungary, Experimental physics II, University of Augsburg, Germany, Materials Science Division, Argonne National Laboratory, Argonne, Illinois 60439, National High Magnetic Field Laboratory Florida State University, Tallahassee, Florida 32310, IM2NP-CNRS (UMR 7334) and Universite Aix-Marseille, National High Magnetic Field Laboratory Applied Superconductivity Center, Heifei National Lab for Physical Science at Microscale, USTC, Institute of Physics, Chinese Academy of Sciences, Department of Electrical Engineering, Rice University, National High Magnetic Field Laboratory, National High Magnetic Field Laboratory/FSU, University of Virginia

  • Chris Neu

    Florida State University, NHMFL, Institut Neel, Institute of Semiconductors, Chinese Academy of Sciences, Beijing, China, Florida State University, Department of Physics, Florida State University, Tallahassee, FL, Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, Florida State University, National High Magnetic Field Laboratory, Institute of Physics, Chinese Academy of Sciences, China, Austin Peay State University, University of Pardubice, Davidson College, Covenant College, Lookout Mtn, GA 30750, Russian Academy of Sciences, Moscow, RU, University of Tennessee Space Institute, Tullahoma, TN, USA, Sandia National Laboratories,* Albuquerque, NM, Arkansas State University, Jonesboro, AK, None, Mississippi State University, Mississippi State University Department of Physics and Astronomy, Florida State University and the National High Magnetic Field Laboratory, Faculte des Sciences et Techniques and Universite Aix-Marseille, MIT Haystack Observatory, Departments of Chemistry and Physics, University of Tennessee of Chattanooga, Departments of Chemistry and Physics, University of Tennessee at Chattanooga, Western Kentucky University, Thomas Jefferson National Accelerator Facility, Newport News, VA, Fermilab, University of Virginia and Fermilab, Indiana University Bloomington, Indiana University Health Proton Therapy Center, University of North Florida, National Superconducting Cyclotron Laboratory, Michigan State University, Flordia State University, Louisiana State University, Washington University, Argonne National Laboratory, Lawrence Berkeley National Laboratory, Department of Physics, Florida State University., JINR, Tsinghua University, LBNL, LBNL/Vanderbilt University, Vanderbilt University, Florida A\&M University, Georgia Institute of Technology, Department of Physics, Virginia Tech, Gatton Academy, Exeter University, Florida International University, Office of Research, University of North Florida, Physics Department, University of North Florida, NSCL, Michigan State University, Physics Department, Florida State University, University of Tennessee-Knoxville, Neel Institut, Kazan Federal University, Bielefeld University, Ben Gurion University, LCIB - CEA, University of Georgia, Lawrence Livermore National Laboratory, Livermore, CA 94550, NHMFL, Tallahassee, USA, Department of Chemistry, University of Waterloo, Ontario, Canada, NHMFL, Tallahassee FL 32310 USA, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany, Wigner Research Center for Physics, Budapest, Hungary, Experimental physics II, University of Augsburg, Germany, Materials Science Division, Argonne National Laboratory, Argonne, Illinois 60439, National High Magnetic Field Laboratory Florida State University, Tallahassee, Florida 32310, IM2NP-CNRS (UMR 7334) and Universite Aix-Marseille, National High Magnetic Field Laboratory Applied Superconductivity Center, Heifei National Lab for Physical Science at Microscale, USTC, Institute of Physics, Chinese Academy of Sciences, Department of Electrical Engineering, Rice University, National High Magnetic Field Laboratory, National High Magnetic Field Laboratory/FSU, University of Virginia

  • Kristin Whitson

    Departments of Chemistry and Physics, University of Tennessee of Chattanooga, Departments of Chemistry and Physics, University of Tennessee at Chattanooga