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An intramolecular FRET system monitors fingers subdomain opening in Klentaq1
Author(s) -
Allen William J.,
Rothwell Paul J.,
Waksman Gabriel
Publication year - 2008
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.073309208
Subject(s) - förster resonance energy transfer , single molecule fret , biophysics , nucleotide , dna , base pair , polymerase , polymerization , chemistry , biology , biochemistry , fluorescence , physics , polymer , gene , organic chemistry , quantum mechanics
A major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating DNA base is selected and delivered to the polymerase active site. Structural evidence suggests a large open‐to‐closed conformational change affecting the fingers subdomain as being crucial to the process. We previously designed a FRET system capable of measuring the rate of fingers subdomain closure in the presence of correct nucleotide. However, this FRET system was limited in that it could not directly measure the rate of fingers subdomain opening by FRET after polymerization or in the absence of DNA. Here we report the development of a new system capable of measuring both fingers subdomain closure and reopening by FRET, and show that the rate of fingers subdomain opening is limited only by the rate of polymerization. We anticipate that this system will scale down to the single molecule level, allowing measurement of fingers subdomain movements in the presence of incorrect nucleotide and in the absence of DNA.