A series of related nucleotide analogues that aids optimization of fluorescence signals in probing the mechanism of P-loop ATPases, such as actomyosin

Biochemistry. 2004 Nov 16;43(45):14463-71. doi: 10.1021/bi0486334.


We have synthesized a set of ATP and ADP analogues that have a fluorophore linked to the nucleotide via the 3'-position of the ribose moiety. Combinations of three different coumarins are each attached via different length linkers. A linker based on propylenediamine increases the separation between the nucleotide and fluorophore relative to that of the previously reported ethylenediamine-linked coumarin nucleotides [Webb, M. R., and Corrie, J. E. T. (2001) Biophys. J. 81, 1562-1569]. A synthesis of 3'-amino-3'-deoxyATP is described using a combination of chemical and enzymatic procedures, mostly from published methods for synthesis of this compound but with some modifications that improved the convenience of the experimental procedures. This compound is used as a basis of a series of analogues with effectively a zero-length linker. Fluorescence properties of all these analogues are described, together with the kinetics of their interaction with rabbit skeletal myosin subfragment 1 in the presence and absence of actin. One particular analogue, deac-aminoATP [3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine 5'-triphosphate], shows a 17-fold enhancement of fluorescence upon binding to this (skeletal) myosin II. As the diphosphate, it exhibits a large signal change upon dissociation from the actomyosin, with kinetics similar to those of natural ADP. The ability of this set of analogues to produce large signals indicated potential uses when scarce proteins are studied in small amounts.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actomyosin / chemistry*
  • Actomyosin / metabolism
  • Adenosine Diphosphate / analogs & derivatives*
  • Adenosine Diphosphate / chemical synthesis
  • Adenosine Diphosphate / metabolism*
  • Adenosine Triphosphatases / chemistry*
  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / analogs & derivatives*
  • Adenosine Triphosphate / chemical synthesis
  • Adenosine Triphosphate / metabolism*
  • Animals
  • Azides / chemical synthesis
  • Azides / metabolism
  • Coumarins / chemical synthesis
  • Coumarins / metabolism
  • Deoxyadenine Nucleotides / chemical synthesis
  • Deoxyadenine Nucleotides / metabolism
  • Deoxyadenosines / chemical synthesis
  • Deoxyadenosines / metabolism
  • Dideoxynucleotides
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / metabolism*
  • Fluorometry
  • Myosin Subfragments / chemistry
  • Myosin Subfragments / metabolism
  • Rabbits
  • Spectrometry, Fluorescence / methods
  • Spectrophotometry


  • Azides
  • Coumarins
  • Deoxyadenine Nucleotides
  • Deoxyadenosines
  • Dideoxynucleotides
  • Fluorescent Dyes
  • Myosin Subfragments
  • 2',3'-dideoxyadenosine triphosphate
  • 3'-azido-3'-deoxyadenosine
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • Actomyosin
  • coumarin
  • Adenosine Triphosphatases