We present the spatial structure of binase, a small extracellular ribonuclease, derived from I H-NMR* data in aqueous solution. The total of 20 structures were obtained via torsion angle dynamics using DYANA program with experimental NOE and hydrogen bond distance constraints and P P and M M I dihedral angle constraints. The final structures were energy minimised with ECEPP/2 potential in FANTOM program. Binase consists of three K K-helices in N-terminal part (residues 61 6, 26^32 and 41^44), five-stranded antiparallel L L-sheet in Cterminal part (residues 51^55, 70^75, 86^90, 94^99 and 1041 08) and two-stranded parallel L L-sheet (residues 22^24 and 495 1). Three loops (residues 36^39, 56^67 and 76^83), which play significant role in biological functioning of binase, are flexible in solution. The differences between binase and barnase spatial structures in solution explain the differences in thermostability of binase, barnase and their hybrids.z 1998 Federation of European Biochemical Societies.

The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys-40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys-82-Ala mutant is described by two 2-state transitions. The lower-temperature one corresponds to barstar denaturation and the higher-temperature transition to ribonuclease melting. The barstar mutation Cys-40-Ala, which is within the principal barnase-binding region of barstar, simplifies the melting to a single 2-state transition. The presence of residue Cys-40 in barstar results in additional stabilization of ribonuclease in the complex.z 1999 Federation of European Biochemical Societies.

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