Fluorescence microscopy of single viral capsids

Shijun Huang, Shirley J. Hayes, Philip Serwer

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

To build a foundation for the single-molecule fluorescence microscopy of protein complexes, the present study achieved fluorescence microscopy of single, nucleic acid-free protein capsids of bacteriophage T7. The capsids were stained with Alexa 488 (green emission). Manipulation of the capsids' thermal motion was achieved in three dimensions. The procedure for manipulation included embedding the capsids in an agarose gel. The data indicate that the thermal motion of capsids is reduced by the sieving of the gel. The thermal motion can be reduced to any desired level. A semilogarithmic plot of an effective diffusion constant as a function of gel concentration is linear. Single, diffusing T7 capsids were also visualized in the presence of single DNA molecules that had been both stretched and immobilized by gel-embedding. The DNA molecules were stained with ethidium (orange emission). This study shows that single-molecule (protein and DNA) analysis is possible for both packaging of DNA in a bacteriophage capsid and other events of DNA metabolism. The major problem is the maintenance of biochemical activity.

Original languageEnglish (US)
Pages (from-to)270-280
Number of pages11
JournalJournal of Structural Biology
Volume135
Issue number3
DOIs
StatePublished - Jan 1 2001

Keywords

  • Bacteriophage T7
  • DNA, metabolism of
  • Fluorescent staining, protein-specific
  • Gel, agarose
  • Gel, sieving by

ASJC Scopus subject areas

  • Structural Biology

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