Biophysics of Bacteriophage λ Infection

1. Biophysics ofBacteriophage λ Infection Li Tai Fang Gelbart / Knobler Group Department of Chemistry and Biochemistry Evilevitch, A.

2. Phage λ Infection Capsid Phage DNA LamB cell membrane condemned E. coli cell Note: figure not drawn to scale

3. Phage λ Infection Capsid Phage DNA LamB cell membrane condemned E. coli cell “dead E. coli walking” Note: figure not drawn to scale

4. tangent vector s+ds s ri rj center of mass T2 phage osmotically shocked by salt Kleinschmidt, A. 1962 Bacteriophage λ • dsDNA ξ= 50 nm L = 16,500 nm (330 ξ) Rg= 380 nm • Rcapsid = 28 nm

5. Driving Force of Infection DNA is confined at close-packed density: • Crowding • configurational entropy due to confinement • Rg, DNA >> Rcapsid • Bending • stiffness of dsDNA • ξDNA≈ Rcapsid • Electrostatic self-repulsion • negative charges repel each other • affected by solution conditions, e.g. ambient salt

6. Pressure vs. Genome Length Inside Tzlil, Kindt, Gelbart, Ben-Shaul, Biophys. J. 84, 1616 (2003)

7. External pressure inhibits ejection Incubation Centrifugation PEG 8000 Outside UV260 DNase Note: figure not drawn to scale

8. nucleotides in solution with LamB weight percent of PEG8000 Evilevitch, Lavelle, Raspaud, Knobler, and Gelbart, PNAS, 100, (2003)

9. genome ejection vs. osmotic pressure osmotic pressure controlled by varying PEG concentration Evilevitch, Lavelle, Raspaud, Knobler, and Gelbart, PNAS, 100, (2003)

10. DNA Ejection3.5 atm osmotic pressure and Mg2+ Fang, LT

11. Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ a result from simulation Mg2+ DNA DNA Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Mg2+ Lee, K-C and Liu, A

12. DNA Ejection3.5 atm osmotic pressure phage with 48.6 kbps genome Fang, LT

13. Different Salt @ 4 atm

14. 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

15. Two-chamber Cell fluorescent probe membrane LamB wafer λ phage cover glass channel Note: figure not drawn to scale

16. Two-chamber Cell fluorescent probe membrane LamB wafer λ phage cover glass channel Note: figure not drawn to scale

17. Giant Vesicles Decher, G, et al, Biochim Biophy Acta, 1990 Note: figure not drawn to scale

18. ratcheting mechanism Note: figure not drawn to scale

19. ratcheting mechanism cell membrane : DNA binding proteins Zandi, Reguera, Rudnick, and Gelbart, PNAS, 100, 15, 2003 Note: figure not drawn to scale

20. cell membrane 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

21. 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?

22. 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