Mammalian heterogeneous ribonucleoprotein A1 and its constituent domains. Nucleic acid interaction, structural stability and self-association.

Abstract With a view toward further understanding the structure-function relationships of the eukaryotic heterogeneous ribonucleoprotein (hnRNP) A1, and in particular its multiplicity of nucleic acid-interactive domains, we have studied the nucleic acid binding properties of the globular N-domain (UP1) and sequence-repetitive, flexible C-domain, the thermal denaturation of UP1 and the concomitant effects of binding polynucleotide,and the self-associative properties of the full-length protein. Utilizing protein tryptophan fluorescence as a probe, polynucleotide binding was shown to stabilize UP1 against thermal unfolding. The denaturation profile of UP1-poly(thymidylic acid)complexes was biphasic, suggesting that unfolding of the two subdomains of UP1 can occur independently. This is in agreement with a previously proposed structure in which only one of the two UP1 subdomains binds the nucleic acid. The subdomains of UP1 can be prepared by controlled proteolysis of A1, further indicating that these two globular segments within A1 are connected by an exposed, flexible linkage. Circular dichroism measurements on UP1 confirm previous data that this portion of A1 binds single-stranded nucleic acids non-co-operatively. UP1 clearly shows a preference for single-stranded nucleic acids with a 2′-OH, since its affinity for poly(U) is three times higher than for poly(dU), and five times higher than its affinity for poly(2′-OCH 3 U). The nucleic acid-interactive properties of the C-domain were further examined by preparing a synthetic peptide polymer ( M r ≈ 12,000) containing about seven repeats of a 16-residue sequence, GNFGGGRGGNYGGSRG, which in turn comprises two copies of the C-terminal consensus, GN(F/Y)GG(G/S)RG. The polymer of this sequence exhibited significant affinity for the fluorescent polyribonucleotide, poly(ethenoadenylic acid), binding stoichiometrically at ≤ 0·2 M-Na + . Complex formation was accompanied by an increase in aggregate formation, as indicated by the appearance of scattering. For purposes of comparison, the data were analyzed via , the linear co-operative model of McGhee and von Hippel, though this model may not be fully descriptive of the protein-nucleic acid complex(es) formed in this case. In contrast to the non-co-operative binding mode of the UP1 domain, the C-polymer exhibited moderate co-operativity, comparable to that seen with full-length A1. Although addition of sufficient NaCl reversed the interaction, a sigmoidal binding isotherm could still be observe (with sufficient added polymer) at 0·8 M-NaCl. This suggests that non-electrostatic interactions contribute significantly to the free energy of binding. The peptide polymer did not exhibit any apparent specificity for single-stranded versus double-stranded and ribonucleic versus deoxyriblonucleic acids. The polymer catalyzed DNA interstrand annealing, an activity previously observed in full-length A1 and A1-derived C-terminal fragment. A non-repetitive synthetic peptide, containing only one copy of the 16-residues sequence, showed neither nucleic acid binding nor annealing activity. This suggests that the repetition of the consensus sequence is required for theses properties. Self-association of intact A1 was observed in the presence or absence of nucleic acids, and may be mediated by hydrophobic interactions residing in its C-terminal domain.