Biophysics of Bacteriophage Infection

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Biophysics of Bacteriophage ? Infection
Evilevitch, A.

• Li Tai Fang
• Gelbart / Knobler Group
• Department of Chemistry and Biochemistry

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Phage ? Infection
Capsid
Phage DNA
LamB
cell membrane
condemned E. coli cell
Note figure not drawn to scale
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Phage ? Infection
Capsid
Phage DNA
LamB
cell membrane
condemned E. coli cell
dead E. coli walking
Note figure not drawn to scale
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Bacteriophage ?

• dsDNA
• ? 50 nm
• L 16,500 nm (330 ?)
• Rg 380 nm
• Rcapsid 28 nm

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Driving Force of Infection

• DNA is confined at close-packed density
• Crowding
• configurational entropy due to confinement
• Rg, DNA gtgt Rcapsid
• Bending
• stiffness of dsDNA
• ?DNA Rcapsid
• Electrostatic self-repulsion
• negative charges repel each other
• affected by solution conditions, e.g. ambient salt

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Pressure vs. Genome Length Inside
Tzlil, Kindt, Gelbart, Ben-Shaul, Biophys. J. 84,
1616 (2003)
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External pressure inhibits ejection
Incubation
Centrifugation
PEG 8000 Outside
UV260
DNase
Note figure not drawn to scale
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nucleotides in solution
with LamB
weight percent of PEG8000
Evilevitch, Lavelle, Raspaud, Knobler, and
Gelbart, PNAS, 100, (2003)
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genome ejection vs. osmotic pressure
osmotic pressure controlled by varying PEG
concentration
Evilevitch, Lavelle, Raspaud, Knobler, and
Gelbart, PNAS, 100, (2003)
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DNA Ejection 3.5 atm osmotic pressure and Mg2
Fang, LT
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a result from simulation
Mg2
DNA
DNA
Mg2
Mg2
Mg2
Mg2
Mg2
Mg2
Mg2
Lee, K-C and Liu, A
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DNA Ejection3.5 atm osmotic pressure
phage with 48.6 kbps genome
Fang, LT
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Different Salt _at_ 4 atm
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Cell mimic

• To be able to separately control the environment
  of the cell and the capsid
• Two approaches
• Two-chamber cell construction
• Giant vesicles

Note figure not drawn to scale
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Two-chamber Cell
fluorescent probe
membrane
LamB
wafer
? phage
cover glass
channel
Note figure not drawn to scale
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Two-chamber Cell
fluorescent probe
membrane
LamB
wafer
? phage
cover glass
channel
Note figure not drawn to scale
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Giant Vesicles
Decher, G, et al, Biochim Biophy Acta, 1990
Note figure not drawn to scale
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ratcheting mechanism
Note figure not drawn to scale
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ratcheting mechanism
cell membrane
DNA binding proteins
Zandi, Reguera, Rudnick, and Gelbart, PNAS, 100,
15, 2003
Note figure not drawn to scale
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RNA polymerase
As a molecular motor 35 pN of force -- reported
by Wang, MD, et al, 1998, Science
RNA polymerase
Note figure not drawn to scale
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Summary

• Genome translocation occurs due to stored energy
  inside the capsid
• Counter-ions affect the magnitude of the stored
  energy by orders of magnitude
• Dynamic studies of genome ejection
• How will different molecules -- e.g., proteins,
  salt -- change the ejection kinetics?

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Acknowledgement
Committee

• Charles Knobler
• William Gelbart
• Joseph Loo
• Giovanni Zocchi
• Tom Chou

Dept. of Chemistry and Biochemistry Dept. of
Chemistry and Biochemistry Dept. of Chemistry and
Biochemistry Dept. of Physics and Astronomy Dept.
of Biomathematics