Martino Bolognesi

Expression, purification and crystallisation of neuro- and cytoglobins

Neuroglobin and cytoglobin, members of the globin family, are present in vertebrate cells at very low concentrations. As the function of both proteins is still a matter of debate, it is very important to be able to produce and purify these proteins, and in general all members of the globin family, to homogeneity. For this purpose, this chapter describes the expression of neuro- and cytoglobin by E. coli and its preparative purification. These proteins are then used in crystallization experiments. Also an analytical purification strategy is discussed in detail.

Inhibition of serine proteinases by p-carbethoxyphenyl esters of ε-guanidino- and ε-amino caproic acid: Thermodynamic and molecular modeling study

AbstractThe inhibitory effect of the clinically used p-carbethoxyphenyl ester of ϵ-guanidino-caproic acid metha-nesulphonate (ϵ-GCA-CEP) on the catalytic properties of human LYS77-plasmin (EC 3.4.21.7), bovine factor Xa (EC 3.4.21.6), bovine α-thrombin (EC 3.4.21.5), ancrod (EC 3.4.21.28), crotalase (EC 3.4.21.30), bovine β-trypsin (EC 3.4.21.4), porcine pancreatic β-kallikrein-B (EC 3.4.21.39, human urinary kallikrein (EC 3.4.21.35) and the Mr 54,000 species of human urokinase (EC 3.4.21.31) was investigated (between pH 2.0 and 8.5, I = 0.1 M;T = 21 ϵ 0.5ϵC), and analyzed in parallel with that of the homologous derivative p-carbethoxyphenyl ϵ-amino-caproate hydro chloride (ϵ-ACA-CEP). On lowering the pH from 5.5 to 3.0, values of the apparent dissociation inhibition constant (Ki) for ϵ-GCA. CEP and ϵ-ACA-CEP interaction with the serine proteinases considered increase, reflecting the acidic pK-shift upon inhibitor binding of a single ionizing group. Over the whole pH range explored, (i) ϵ-GCA-CEP interact...

Mapping protein matrix cavities in human cytoglobin through Xe atom binding: a crystallographic investigation

Cytoglobin is the fourth recognized globin type, almost ubiquitously distributed in human tissues; its function is still poorly understood. Cytoglobin displays a core region of about 150 residues, structurally related to hemoglobin and myoglobin, and two extra segments, about 20 residues each, at the N- and C-termini. The core region hosts a large apolar cavity, held to provide a ligand diffusion pathway to/from the heme, and/or ligand temporary docking sites. Here we report the crystal structure (2.4A resolution, R-factor 19.1%) of a human cytoglobin mutant bearing the CysB2(38) --> Ser and CysE9(83) --> Ser substitutions (CYGB*), treated under pressurized xenon. Three Xe atoms bind to the heme distal site region of CYGB* mapping the protein matrix apolar cavity. Despite the conserved globin fold, the cavity found in CYGB* is structured differently from those recognized to play a functional role in myoglobin, neuroglobin, truncated hemoglobins, and Cerebratulus lacteus mini-hemoglobin.