Designing a nine cysteine-less DNA packaging motor from bacteriophage T4 reveals new insights into ATPase structure and function

Kiran Kondabagil, Li Dai, Reza Vafabakhsh, Taekjip Ha, Bonnie Draper, Venigalla B. Rao

Research output: Contribution to journalArticle

Abstract

The packaging motor of bacteriophage T4 translocates DNA into the capsid at a rate of up to 2000bp/s. Such a high rate would require coordination of motor movements at millisecond timescale. Designing a cysteine-less gp17 is essential to generate fluorescently labeled motors and measure distance changes between motor domains by FRET analyses. Here, by using sequence alignments, structural modeling, combinatorial mutagenesis, and recombinational rescue, we replaced all nine cysteines of gp17 and introduced single cysteines at defined positions. These mutant motors retained in vitro DNA packaging activity. Single mutant motors translocated DNA molecules in real time as imaged by total internal reflection fluorescence microscopy. We discovered, unexpectedly, that a hydrophobic or nonpolar amino acid next to Walker B motif is essential for motor function, probably for efficient generation of OH- nucleophile. The ATPase Walker B motif, thus, may be redefined as "β-strand (4-6 hydrophobic-rich amino acids)-DE-hydrophobic/nonpolar amino acid".

Original languageEnglish (US)
Pages (from-to)660-668
Number of pages9
JournalVirology
Volume468
DOIs
StatePublished - Nov 1 2014
Externally publishedYes

Fingerprint

DNA Packaging
Bacteriophage T4
Cysteine
Adenosine Triphosphatases
Amino Acids
Sequence Alignment
Capsid
DNA
Product Packaging
Fluorescence Microscopy
Mutagenesis

Keywords

  • ATPase
  • Bacteriophage T4
  • DNA packaging
  • Molecular motor
  • Virus assembly
  • Walker B motif

ASJC Scopus subject areas

  • Virology
  • Medicine(all)

Cite this

Designing a nine cysteine-less DNA packaging motor from bacteriophage T4 reveals new insights into ATPase structure and function. / Kondabagil, Kiran; Dai, Li; Vafabakhsh, Reza; Ha, Taekjip; Draper, Bonnie; Rao, Venigalla B.

In: Virology, Vol. 468, 01.11.2014, p. 660-668.

Research output: Contribution to journalArticle

Kondabagil, Kiran ; Dai, Li ; Vafabakhsh, Reza ; Ha, Taekjip ; Draper, Bonnie ; Rao, Venigalla B. / Designing a nine cysteine-less DNA packaging motor from bacteriophage T4 reveals new insights into ATPase structure and function. In: Virology. 2014 ; Vol. 468. pp. 660-668.
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