Single-molecule nanopositioning: Structural transitions of a helicase-DNA complex during ATP hydrolysis

Hamza Balci, Sinan Arslan, Sua Myong, Timothy M. Lohman, Taekjip Ha

Research output: Contribution to journalArticlepeer-review

Abstract

The conformational states of Escherichia coli Rep helicase undergoing ATP hydrolysis while bound to a partialduplex DNA (pdDNA) were studied using single-molecule FRET. Crystallographic studies showed that Rep bound to singlestranded DNA can exist in open and closed conformations that differ in the orientation of the 2B subdomain. FRET measurements between eight Rep mutants donor-labeled at different residues and pdDNA acceptor-labeled at the junction were conducted at each of the four nucleotide states. The positions of donor-labeled residues, based on crystal structure, and FRET measurements between these donor molecules and the acceptor fluorophore at the DNA junction were used to predict the most likely position for the DNA junction using a triangulation algorithm. These predicted junction positions are compared with the crystal structure to determine whether the open or closed conformation is more consistent with the FRET data. Our data revealed that there are two distinct Rep-pdDNA conformations in the ATPgS and ADP states, an unexpected finding. The primary conformation is similar to that observed in nucleotide-free and ADP.Pi states, and the secondary conformation is a novel conformation where the duplex DNA and 2B subdomain moved as a unit by 13 Å relative to the rest of the protein. The primary conformation found in all nucleotide states is consistent with the closed conformation of the crystal structure however; the secondary conformation is a new conformation that has not been observed before. We discuss the possible implications of this newly observed conformation.

Original languageEnglish (US)
Pages (from-to)976-984
Number of pages9
JournalBiophysical journal
Volume101
Issue number4
DOIs
StatePublished - Aug 17 2011
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics

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