Lelio Mazzarella

The deletion of five residues in the loop connecting the N-terminal helix to the core of monomeric human pancreatic ribonuclease leads to the formation of an enzymatically active domain-swapped dimer (desHP). The crystal structure of desHP reveals the generation of an intriguing fibril-like aggregate of desHP molecules that extends along theccrystallographic axis. Dimers are formed by three-dimensional domain swapping. Tetramers are formed by the aggregation of swapped dimers with slightly different quaternary structures. The tetramers interact in such a way as to form an infinite rod-like structure that propagates throughout the crystal. The observed supramolecular assembly captured in the crystal predicts that desHP fibrils could form in solution; this has been confirmed by atomic force microscopy. These results provide new evidence that three-dimensional domain swapping can be a mechanism for the formation of elaborate large assemblies in which the protein, apart from the swapping, retains its original fold.

Multiwavelength anomalous dispersion (MAD) is the most widespread approach in structural biology for the determination of the crystal structure of a novel protein. Mass spectrometry is currently used to evaluate the selenomethionine (SeMet) content in solution, but excluding fluorescence spectroscopy at the absorption edge, no other routine method to check the SeMet incorporation and storage in the crystal state is yet available. Raman microscopy is an increasingly popular tool in physical biochemistry, with applications ranging from studies of ligand binding to secondary-structure analysis. Here, a novel methodological development is presented for the analysis via Raman microscopy of SeMet-labelled protein crystals to be used for MAD crystallography. The method is described and supported by validation and application to two novel proteins (a betagamma-crystallin-like protein and a DNA-binding protein). Markers of the SeMet residues are in the range 570-600 cm(-1), where proteins do not usually show Raman bands.

The Root effect is a widespread property in fish hemoglobins (Hbs) that produces a drastic reduction of cooperativity and oxygen‐binding ability at acidic pH. Here, we report the high‐resolution structure of the deoxy form of Hb isolated from the Antarctic fish Trematomus bernacchii (HbTb) crystallized at pH 6.2 and 8.4. The structure at acidic pH has been previously determined at a moderate resolution (Ito et al., J Mol Biol 1995;250:648–658). Our results provide a clear picture of the events occurring upon the pH increase from 6.2 to 8.4, observed within a practically unchanged crystal environment. In particular, at pH 8.4, the interaspartic hydrogen bond at the α₁β₂ interface is partially broken, suggesting a pK_a close to 8.4 for Asp95α. In addition, a detailed survey of the histidine modifications, caused by the change in pH, also indicates that at least three hot regions of the molecule are modified (Eβ helix, Cβ‐tail, CDα corner) and can be considered to be involved at various levels in the release of the Root protons. Most importantly, at the CDα corner, the break of the salt bridge Asp48α–His55α allows us to describe a detailed mechanism that transmits the modification from the CDα corner far to the α heme. More generally, the results shed light on the role played by the histidine residues in modulating the strength of the Root effect and also support the emerging idea that the structural determinants, at least for a part of the Root effect, are specific of each Hb endowed with this property. Proteins 2006.

The cathodic hemoglobin component of the Antarctic fish Trematomus newnesi (HbCTn) is a Root‐effect protein. The interpretation of its functional properties in relation to its sequence is puzzling. Indeed, HbCTn sequence is characterized by an extremely low histidyl content, and in particular by the lack of His146β and His69β, which are believed to be important in Bohr and Root effects, respectively. Furthermore, previous analyses suggested that the local environment of Asp95α, Asp99β, and Asp101β should not be appropriate for the formation of Asp–Asp interactions, which are important for the Root effect. Here, we report the high‐resolution crystal structure of the deoxy form of HbCTn. Our data provide a structural interpretation for the very low oxygen affinity of the protein and insights into the structural determinants of the Root effect protein. The structure demonstrates that the presence of Ile41α and Ser97α at the α₁β₂ interface does not prevent the formation of the inter‐Asp interactions in HbCTn, as previous studies had suggested. The present data indicate that the hydrogen bond formed between Asp95α and Asp101β, which is stabilized by Asp99β, is per se sufficient to generate the Root effect, and it is the minimal structural requirement needed for the design of Root‐effect Hbs. Proteins 2006.