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      A strategy for analyzing bond strength and interaction kinetics between Pleckstrin homology domains and PI(4,5)P2 phospholipids using force distance spectroscopy and surface plasmon resonance.

      1 , , ,
      The Analyst
      Royal Society of Chemistry (RSC)

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          Abstract

          Phospholipids are important membrane components involved in diverse biological activities ranging from cell signaling to infection by viral particles. A thorough understanding of protein-phospholipid interaction dynamics is thus crucial for deciphering basic cellular processes as well as for targeted drug discovery. For any specific phospholipid-protein binding experiment, various groups have reported different binding constants, which are strongly dependent on applied conditions of interactions. Here, we report a method for accurate determination of the binding affinity and specificity between proteins and phospholipids using a model interaction between PLC-δ1/PH and phosphoinositide phospholipid PtdIns(4,5)P2. We developed an accurate Force Distance Spectroscopy (FDS)-based assay and have attempted to resolve the problem of variation in the observed binding constant by directly measuring the bond force. We confirm the FDS findings of a high bond strength of ∼0.19 ± 0.04 nN by Surface Plasmon Resonance (SPR) data analysis, segregating non-specific interactions, which show a significantly lower K(D) suggesting tight binding.

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          Author and article information

          Journal
          Analyst
          The Analyst
          Royal Society of Chemistry (RSC)
          1364-5528
          0003-2654
          Jul 07 2015
          : 140
          : 13
          Affiliations
          [1 ] Stanford BioADD Laboratory, Stanford, California 94305, USA. jayraja@stanford.edu.
          Article
          10.1039/c5an00498e
          26040325
          32606c48-76ab-4279-8c93-fc2c0f828eb8
          History

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