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 hydro gen isotope exchange dynamics of nucleic acid and protein constituents in a double-stranded DNA virus, the icosahedral bacteriophage P22. Th e Raman dynamic method employs a dialysis flow cell to control D2O eff lux into an H2O solution of the virus sample while the rates of deuter ium exchange of protons in the viral nucleic acid and protein molecule s are measured spectrophotometrically in real time. The method provide s structural and kinetic information about three different and distinc t classes of exchangable protons of the native virion: (1) labile imin o (NH) and amino (NH2) protons of the bases which participate in Watso n-Crick hydrogen bonding in the packaged genome; (2) pseudolabile puri nic (8CH) protons that line the major groove of packaged P22 DNA; and (3) main-chain amide (NH) protons of viral subunits comprising the she ll 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 exchan ge rapidly, most amide protons in capsid subunits are resistant to sol vent-catalyzed exchange. Further, stereospecific retardation of exchan ge is observed for major-groove protons of the packaged P22 genome. Th e quantitative measurements can be summarized and interpreted as follo ws. (1) Imino and amino protons of all bases in packaged P22 DNA excha nge more rapidly (approximately 2-fold faster) than the corresponding protons in unpackaged P22 DNA. Remarkably, packaging actually accelera tes labile imino and amino hydrogen exchanges of the viral DNA, an eff ect which can be attributed to selective stabilization in the packaged chromosome of a base-pair open state (breathing model). (2) Conversel y, purine 8CH exchange rates in packaged P22 DNA are significantly ret arded in comparison to those of unpackaged P22 DNA. The observed 8CH e xchange retardation effects are similar for both adenine and guanine r esidues, indicating that they do not originate from purine-specific in teractions but probably reflect steric shielding of the major groove o f packaged DNA from free access to solvent. This effect is likely dist ributed throughout the 43,400 base-pair genome. (3) Only a small popul ation (approximate to 15 to 20%) of subunit amide protons exchanges wi thin the time frame of complete exchange of all protons of packaged P2 2 DNA. Complete exchange of the capsid is not achieved even after seve ral months of incubation at 40 degrees C. The viral capsid thus repres ents a largely non-exchangable shell which packages a highly exchangab le double-stranded DNA molecule. The present results confirm that the B form secondary structure of P22 DNA is essentially fully conserved w ith packaging. The exchange dynamics of P22 DNA and capsid molecules a re considered in the light of available structural data and models pro posed for condensation of double-stranded B DNA in icosahedral viruses .