HYDROGEN-EXCHANGE DYNAMICS OF THE P22 VIRION DETERMINED BY TIME-RESOLVED RAMAN-SPECTROSCOPY - EFFECTS OF CHROMOSOME PACKAGING ON THE KINETICS OF NUCLEOTIDE EXCHANGES

We describe the application of laser Raman spectroscopy to probe hydrogen isotope exchange dynamics of nucleic acid and protein constituents in a double-stranded DNA virus, the icosahedral bacteriophage P22. The Raman dynamic method employs a dialysis flow cell to control D2O efflux into an H2O solution of the virus sample while the rates of deuterium exchange of protons in the viral nucleic acid and protein molecules are measured spectrophotometrically in real time. The method provides structural and kinetic information about three different and distinct classes of exchangable protons of the native virion: (1) labile imino (NH) and amino (NH2) protons of the bases which participate in Watson-Crick hydrogen bonding in the packaged genome; (2) pseudolabile purinic (8CH) protons that line the major groove of packaged P22 DNA; and (3) main-chain amide (NH) protons of viral subunits comprising the shell that encapsidates the DNA. The results obtained on P22 demonstrate that interchange of aqueous solvent with the virion interior is rapid and complete. We find that while labile protons of packaged DNA exchange rapidly, most amide protons in capsid subunits are resistant to solvent-catalyzed exchange. Further, stereospecific retardation of exchange is observed for major-groove protons of the packaged P22 genome. The quantitative measurements can be summarized and interpreted as follows. (1) Imino and amino protons of all bases in packaged P22 DNA exchange more rapidly (approximately 2-fold faster) than the corresponding protons in unpackaged P22 DNA. Remarkably, packaging actually accelerates labile imino and amino hydrogen exchanges of the viral DNA, an effect which can be attributed to selective stabilization in the packaged chromosome of a base-pair open state (breathing model). (2) Conversely, purine 8CH exchange rates in packaged P22 DNA are significantly retarded in comparison to those of unpackaged P22 DNA. The observed 8CH exchange retardation effects are similar for both adenine and guanine residues, indicating that they do not originate from purine-specific interactions but probably reflect steric shielding of the major groove of packaged DNA from free access to solvent. This effect is likely distributed throughout the 43,400 base-pair genome. (3) Only a small population (approximate to 15 to 20%) of subunit amide protons exchanges within the time frame of complete exchange of all protons of packaged P22 DNA. Complete exchange of the capsid is not achieved even after several months of incubation at 40 degrees C. The viral capsid thus represents a largely non-exchangable shell which packages a highly exchangable double-stranded DNA molecule. The present results confirm that the B form secondary structure of P22 DNA is essentially fully conserved with packaging. The exchange dynamics of P22 DNA and capsid molecules are considered in the light of available structural data and models proposed for condensation of double-stranded B DNA in icosahedral viruses.