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Locked nucleic acids (LNAs) are formed by bicyclic ribonucleotides where the O2′ and C4′ atoms are linked through a methylene bridge and the sugar is blocked in a 3′-endoconformation. They represent a promising tool for therapeutic and diagnostic applications and are characterized by higher thermal stability and nuclease resistance with respect to their natural counterparts. However, structural descriptions of LNA-containing quadruplexes are rather limited, since few NMR models have been reported in the literature. Here, the first crystallographically derived model of an all-LNA-substituted quadruplex-forming sequence 5′-TGGGT-3′ is presented refined at 1.7 Å resolution. This high-resolution crystallographic analysis reveals a regular parallel G-quadruplex arrangement terminating in a well defined thymine tetrad at the 3′-end. The detailed picture of the hydration pattern reveals LNA-specific features in the solvent distribution. Interestingly, two closely packed quadruplexes are present in the asymmetric unit. They face one another with their 3′-ends giving rise to a compact higher-order structure. This new assembly suggests a possible way in which sequential quadruplexes can be disposed in the crowded cell environment. Furthermore, as the formation of ordered structures by molecular self-assembly is an effective strategy to obtain nanostructures, this study could open the way to the design of a new class of LNA-based building blocks for nanotechnology.

The [beta][gamma]-crystallin superfamily includes highly diverse proteins belonging to all of the kingdoms of life. Based on structural topology, these proteins are considered to be evolutionarily related to the long-lived [beta][gamma]-crystallins that constitute the vertebrate eye lens. This study reports the crystallographic structure at 0.99 Å resolution of the two-domain [beta][gamma]-crystallin (geodin) from the sponge Geodia cydonium. This is the most ancient member of the [beta][gamma]-crystallin superfamily in metazoans. The X-ray structure shows that the geodin domains adopt the typical [beta][gamma]-crystallin fold with a paired Greek-key motif, thus confirming the hypothesis that the crystallin-type scaffold used in the evolution of bacteria and moulds was recruited very early in metazoans. As a significant new structural feature, the sponge protein possesses a unique interdomain interface made up by pairing between the second motif of the first domain and the first motif of the second domain. The atomic resolution also allowed a detailed analysis of the calcium-binding site of the protein.

The domain swapping is a phenomenon, which is observed in an increasing number of proteins. Among the swapped proteins, bovine seminal ribonuclease (BS-RNase) represents a unique example, as the process occurs in the native mixture between two dimeric forms, MxM (swapped) and M=M (unswapped), held together by the additional constraints of two inter-chain disulphide bridges. This peculiarity offers the opportunity to study the effects of selected mutations at the O-interface on the M=M / MxM equilibrium, within a substantially invariant quaternary assembly. Two variants, having Pro 19 (P19A) or the whole sequence of the hinge peptide 16-22 replaced by the corresponding residues of RNase A (BS-hinge-A), show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. On the contrary, mutation of L28 (L28Q, P19A/L28Q) significantly affects the swapping processes. The X-ray structures of the MxM forms of P19A and BS-hinge-A, and the MxM and M=M forms of the double mutant P19A/L28Q have been determined. The structural effects of the mutations are discussed on the basis of the MxM/M=M equilibrium data measured in solution. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement P19A, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. On the other hand, the substitution in position 28 points to a crucial role of the interface residues in the swapping of BS-RNase. Keywords: swapping , ribonuclease mutations, structural

The Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high catalytic activity even at low temperature. The structure, stability and dynamics of PhSOD have been determined and compared with those of its mesophilic counterpart from E. coli (EcSOD). PhSOD was found to have structure and stability very similar to Ec-SOD. However, the psychrophilic protein shows an increased flexibility of the active site with respect to its mesophilic homologue. Two PhSOD mutants (C57S and C57R) have been also characterized. The C57R mutation significantly alters the half-denaturation temperature of the protein. The structural and dynamic changes induced by this mutation with respect to the C57S and wild-type structure were correlated with modifications in the thermal stability of the mutant. Altogether these data illustrate how evolution can adjust psychrophilic enzyme sequences to alter the flexibility, without compromising the overall protein structure.

Ribonucleases catalyze RNA cleavage reactions. A specialized class of RNases shows a high cytotoxicity toward tumor cell lines, critically dependent on the ability of these molecules to evade the action of the cytosolic ribonuclease inhibitor (RI). Here, we report the X-ray structure of two dimeric variants of RNase1: HHP2-RNase1 and des(16-20)RNase1. HHP2-RNase1 is a covalent dimeric protein, cytotoxic for several malignant cell lines, in which RNase1 has been engineered to reproduce the sequence of bovine seminal ribonuclease helix-II and to eliminate a negative charge on the surface. Des(16-20)RNase1 is a highly stable domain swapped dimer constituted by chains in which five residues in the loop linking the N-terminal helix of RNase1 to the rest of the protein have been deleted. The analysis of HHP2-RNase crystals indicates structural details which can explain the high antitumor activity of the protein. The structure of des(16-20)RNase1 shows a tetrameric association of two swapped dimers, that suggests a pathway of large-scale oligomerization of the protein.

L-~t-Aspartiumylglycylglycine fl-benzyl methyl ester trifluoroacetate, C16H22N30+.C2F302, M r =465.39, orthorhombic, P2,212 ~, a=5.425 (2), b = 12.159 (1), c= 32.925 (8))k, V= 2171 (1)A 3, Z = 4, D x = 1.42 Mg m -a, /(Cu K~t) = 1.5418 A, Iz(Cu Ka) = 1.1 mm -1, F(000) = 968, room tem- perature, final R -- 0.071 for 1550 observed reflections. The title compound consists of the triftuoroacetate anion and the peptide with the amino terminus protonated. The conformation of the peptide linkages is trans and the main-chain conformation of the tripeptide is not fully extended. The packing is determined by extensive hydrogen bonding with every available donor atom participating. Introduction. Peptides containing a t-blocked aspartyl residue easily cyclize forming a substituted succinimide (Bodansky & Kwei, 1978). From a structural point of view the formation of this cycle requires that the dihedral angle ~, of the aspartyl residue (N~Ci-- C/----Ni+x) assumes a value close to -120 °, which falls in a disallowed region of the Ramachandran map for an L residue. It has been suggested that the deamidation of an asparaginyl side chain in proteins may occur through the formation of the cyclic imide as intermedi- ate (Aswad, 1984). As a part of our ongoing research into structural properties of t-blocked aspartyl peptides and aminosuccinyl peptides (Capasso, Mattia, Maz- zarella & Zagari, 1984a,b; Capasso, Mazzarella, Sica & Zagari, 1984), we have synthesized and studied, by X-ray analysis, the tripeptide L-Asp(fl-Bzl)-Gly-Gly- OMe.Tfa.