Effects of virion and salt concentrations on the Raman signatures of filamentous phages fd, Pf1, Pf3, and PH75.
Filamentous phages consist of a single-stranded DNA genome encapsidated by several thousand copies of a small alpha-helical coat protein subunit plus several copies of four minor proteins at the filament ends. The filamentous phages are important as cloning vectors, vehicles for peptide display, and substrates for macromolecular alignment. Effective use of a filamentous phage in such applications requires an understanding of experimental factors that may influence the propensity of viral filaments to laterally aggregate in solution. Because the Raman spectrum of a filamentous phage is strongly dependent on the relative orientation of the virion with respect to the polarization direction of the electromagnetic radiation employed to excite the spectrum, we have applied Raman spectroscopy to investigate lateral aggregation of phages fd, Pf1, Pf3, and PH75 in solution. The results show that lateral aggregation of the virions and anisotropic orientation of the aggregates are both disfavored by high concentrations of salt (>200 mM NaCl) in solutions containing a relatively low virion concentration (<10 mg/mL). Conversely, the formation of lateral aggregates and their anisotropic orientation are strongly favored by a low salt concentration (<0.1 mM NaCl), irrespective of the virion concentration over a wide range. The use of Raman polarization effects to distinguish isotropic and anisotropic solutions of filamentous phages is consistent with previously reported Raman analyses of virion structures in both solutions and fibers. The Raman data are supported by electron micrographs of negatively stained specimens of phage fd, which permit an independent assessment of salt effects on lateral aggregation. The present results also identify new Raman bands that serve as potential markers of subunit side-chain orientations in filamentous virus assemblies.
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas City, Missouri 64110, USA.
Stacy A
SA
Overman
David M
DM
Kristensen
Priya
P
Bondre
Barbara
B
Hewitt
George J
GJ
Thomas
D016880:Anisotropy; D025561:Bacteriophage Pf1 / Q000737:chemistry / Q000648:ultrastructure; D017912:Inovirus / Q000737:chemistry* / Q000648:ultrastructure; D017105:Pseudomonas Phages / Q000737:chemistry / Q000648:ultrastructure; D012965:Sodium Chloride / Q000737:chemistry*; D012996:Solutions; D013059:Spectrum Analysis, Raman / Q000379:methods; D016963:Thermus thermophilus / Q000737:chemistry / Q000821:virology; D014771:Virion / Q000737:chemistry* / Q000648:ultrastructure
D012996:Solutions; D012965:Sodium Chloride