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Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double‐stranded DNA without sequence specificity by placing its triple‐stranded β‐sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single‐point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double‐ and single‐stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.

CB2, a CNBr peptide of 36 residues from type I collagen α1(I) chain has been studied by NMR spectroscopy as a function of temperature. At low temperature, the guanidinium protons of Arg 9showed sharp 1:1:1 NMR triplets around 6.95 ppm, characteristic of 14N coupled protons ( 1JNH=52 Hz) when the quadrupolar relaxation rate is drastically reduced. These spectral characteristics and the low temperature coefficient of the 1:1:1 triplets (Δδ/Δ Tof −3.6 ppb/°C) suggest that the H atoms of the protonated guanidinium moiety of Arg 9in the triple helix are slowly exchanging with bulk water, most likely involved in hydrogen bonds. On the basis of conformational energy computations on a model segment of type I collagen (Vitagliano, L., Némethy, G., Zagari, A. and Scheraga, H.A. (1993) Biochemistry 32, 7354–7359), similar to CB2, our data could indicate that the guanidinium group of Arg 9form hydrogen bonds with a backbone carbonyl of an adjacent chain probably by using the N ϵhydrogen, leaving the four N ηhydrogens bound to water molecules that must be in slow exchange with bulk water and that could therefore be considered structural elements of the trimeric α1(I) CB2 triple helix. The behaviour of Arg 9has been investigated also in terms of equilibrium between random monomer and helical trimer conformations controlled by temperature. The thermal unfolding process was found to be reversible and the melting point resulted to be 17°C.

Investigations were performed on recombinant ribonuclease P2 from Sulfolobus solfataricus, previously cloned and expressed in Escherichia coli[Fusi, P., Grisa, M., Mombelli, E., Consonni, R., Tortora, P. and Vanoni, M. (1995) Gene 154, 99–103]. NMR and photo-CIDNP spectroscopies showed that the enzyme possesses an aromatic cluster constisting of Phe 5, Tyr 7, Phe 31and Tyr 33while Trp 23is fully exposed to solvent. Phe 31, Tyr 33and Trp 23are located within a triple stranded antiparallel β-sheet, each one being part of an amino acid stretch matching consensus sequences for RNA binding. Phe 31and Trp 23are exposed to and specifically interact with a flavin dye used as a model ligand, with a topology reminiscent of that found in several eubacterial and eukariotic RNA-binding proteins.