Raman dynamic probe of hydrogen exchange in bean pod mottle virus: base-specific retardation of exchange in packaged ssRNA

We describe a novel approach to investigating exchange kinetics in biological assemblies. The method makes use of a Raman multichannel analyzer coupled with a dialysis flow cell. We employ this methodology to determine exchange rates of labile hydrogens in both the packaged RNA genome and protein subunits of bean pod mottle virus (BPMV). In the BPMV assembly, which is similar to human picornaviruses, the x-ray structure indicates that about 20% of the ssRNA chain is ordered at the threefold vertices of the icosahedral capsid, although the nucleotide bases in the ordered segments are not known (Chen et al., 1989). Here, we compare exchange profiles of the native virus with those of the empty capsid, model nucleic acids and aqueous solvent to reveal the following exchange characteristics of BPMV RNA and protein: (i) Base-specific retardation of exchange is observed in the packaged RNA. (ii) Retardation is greatest for uracil residues, for which the first-order exchange rate constant (kU = 0.18 +/- 0.02 min-1) is 40% lower than that of either the H2O solvent or adenine or cytosine groups of RNA (ksolv approximately kA approximately kC = 0.30 +/- 0.02 min-1). (iii) Retardation of exchange is also observed for the guanine residues of packaged RNA. (iv) No appreciable exchange of amide NH groups of capsid subunits occurs within the time of complete exchange (t approximately 10 min) of packaged RNA or bulk solvent. Thus, the present results identify sites in both the protein subunits (amide NH) and RNA nucleotides (amino NH2 and imino NH) which are resistant to solvent-catalyzed hydrogen exchange. We propose that retardation of exchange of labile sites of the RNA nucleotides is a consequence of the organization of the RNA chromosome within the virion. Our findings support a model for BPMV in which surface and buried domains of capsid subunits are extensively and rigidly hydrogen-bonded, and in which uracil and guanine exocyclic donor groups of packaged RNA are the principal targets for subunit interaction at the threefold vertices of the capsid.