Conformations, interactions, and thermostabilities of RNA and proteins in bean pod mottle virus: investigation of solution and crystal structures by laser Raman spectroscopy.

Abstract

We report and interpret laser Raman spectra of the three virion components of bean pod mottle virus (BPMV). The top component of BPMV is an empty capsid; middle and bottom components package the RNA2 and RNA1 genome segments, respectively. All components were investigated as both single crystals and aqueous solutions, the latter over wide ranges of temperature and ionic strength. The isolated RNA2 molecule of BPMV middle component was also investigated in both H2O and D2O solutions. The results permit assessment of RNA and protein structures, their mutual interactions in the virions, and their conformational thermostabilities and comparison of these structural characteristics for solution and crystal states of the particles. The principal findings of this study are (i) The extent of ordered A-form backbone (74%) and of base pairing (38% AU + 22% GC) in unpackaged (aqueous) RNA2 are significantly altered by packaging. The A-form secondary structure of RNA2 is increased by 12 +/- 4%, and guanine base interactions are also substantially increased with packaging. (ii) The thermostability of Raman-monitored secondary structure of unpackaged RNA2 (Tm approximately 43 degrees C) is greatly increased in the packaged state (Tm approximately 53 degrees C). This increase corresponds to a stabilization of the A-form backbone geometry in 15 +/- 5% of genome nucleotides. (iii) Packaging of RNA2 in the middle component stabilizes subunit-subunit interactions of the capsid, as evidenced by a thermal denaturation temperature Td approximately 65 degrees C for the virion, compared with Td approximately 55 degrees C for the empty capsid. (iv) Raman marker-band shifts implicate the purine 7N sites of RNA2 and aromatic side chains of subunits as the principal targets for RNA-subunit interaction. (v) At the conditions of the present experiments (8 degrees C, pH approximately 7, moderate ionic strength), the subunit secondary structures observed for solutions of the top, middle, and bottom components are indistinguishable by Raman spectroscopy from secondary structures observed for corresponding crystalline samples. (vi) On the other hand, side chains of subunits in the top component (empty capsid) yield significantly different Raman intensities in crystalline and solution states. These differences are interpreted as the result of changes in a small number of side-chain environments between crystal and solution. (vii) Similarly, small differences exist between RNA Raman markers of crystalline and aqueous virions, which are attributed to altered environments of nucleotide residues and to a small increase in the amount of A-form backbone geometry upon going from the crystal to the solution.(ABSTRACT TRUNCATED AT 400 WORDS)