Oral Presentation The 3rd Prato Conference on Pore Forming Proteins 2015

Pore-forming proteins: Simultaneous structure-function methods & potential applications (#5)

Joseph W.F. Robertson 1 , Arvind K. Balijepalli 1 , Joseph E. Reiner 2 , Sina Bavari 3 , Rekha Panchal 3 , Jingyue Ju 4 , Minchen Chen 4 , Shiv Kumar 4 , Carl Fuller 5 , Jason Campbell 1 , Jason Ryan 1 , Pragya Shrestha 1 , Vasileia Georgiou 1 , Ji-Hong Kim 1 , Charles Cheung 1 , John Kasianowicz 1
  1. NIST, Gaithersburg, MD, USA
  2. Physics, Virginia Commonwealth University, Richmond, VA, USA
  3. USAMRIID, Fort Detrick, MD, USA
  4. Chemical Engineerign, Columbia University, New York, NY, USA
  5. Genia Technologies, LLC, Mountain View, CA, USA

Pore-forming proteins have long been a major focal point in membrane biophysics.  In addition to providing the molecular basis of nerve and muscle activity, some act as toxic agents in many bacterial-induced diseases.  Recently, pore-forming proteins have proven to be useful as single molecule sensors.  Our research follows two tightly correlated lines of study for the development and improvement of this technology.  We utilize nanopores as sensitive biomarker detectors and develop new structural methods to determine the structure of functioning proteins.  Specifically, we will discuss the development of the alpha-hemolysin nanopore for single molecule detection and its use as a calibration tool in membrane protein structural studies.  We will show that the exquisite detail revealed by electronic data demand accurate structural information of fully functional pore-forming proteins.  Because transmembrane protein structure determination via x-ray crystallography has proven difficult, and that method cannot ensure that the putative structures are of functional molecules, we are developing other structural methods.  These techniques make use of a surface-tethered model membrane system, which enables simultaneous electrochemical impedance spectroscopy and surface plasmon resonance spectroscopy to interrogate the binding efficiency and functional integrity of the pores.  In addition, we demonstrated that neutron reflectometry can be used on this system to provide high resolution structural information in the plane perpendicular to the membrane.  We are also developing a novel version of electron spin resonance spectroscopy to bridge the gap between common structural techniques and functional assays such as classical electrophysiology.  We will discuss the use of these methods for practical applications (e.g., DNA sequencing and determining the size and charge on single polymers to better than monomer resolution), to the elucidation of structural elements of proteins that resist crystallization such as the anthrax protective antigen channel.